No title

Advances in

BOTANICAL RESEARCH VOLUME 18

Advances in

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

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

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

Kings College, London, England University of Reading, Reading, England Rothamsted Experimental Station, England University of Shefield, Shefield, England University College, London, England Waite Agricultural Research Institute,.Australia

a

BOTANICAL RESEARCH Edited by

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

VOLUME 18

1991

ACADEMIC PRESS Harcourt Brace Jovanovich, 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 01991 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.

British Library Cataloguing in Publication Data Advances in botanical research.-Vol. 18 1. Botany-Periodicals 581’.05 QK1 ISBN 0-12-005918-5

Typeset by Phoenix Photosetting, Chatham, Kent Printed by Galliard Printers Ltd, Great Yarmouth, Norfolk

CONTRIBUTORS TO VOLUME 18

D. BUTTRY, Chemistry Department, University of Wyoming, Laramie, WY82071, USA G. CHEN, Chemistry Department, University of Wyoming, Laramie, W Y 82071, USA J. GRACE, University of Edinburgh, Institute of Ecology and Resource Management, Darwin Buildings, King’s Buildings, Mayfield Road, Edinburgh EH9 3JU, U K G. MARTIN, Botany Department, P. 0. Box 3165, University of Wyoming, Laramie, W Y 82071, USA H. SAXE, Ministry of the Environment, National Environmental Research Institute, Division of Terrestrial Ecology, Vejlsavej 11, DK-8600 Silkeborg, Denmark J. G. STREETER, Department of Agronomy, Ohio State University and Ohio Agricultural Research and Development Center, Wooster, OH 44691, U S A P. VAN GARDINGEN, University of Edinburgh, Institute of Ecology and Resource Management, Darwin Building, The Kings Buildings, Mayfield Road, Edinburgh EH93JU, U K T. C. VOGELMANN, Botany Department, P.O. Box 3165, University of Wyoming, Laramie, W Y 82071, USA

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PREFACE

In this volume of Advances in Botanical Research we start with a very comprehensive survey by Saxe of the physiological effects of various atmospheric pollutants, SOz, NO, and 03,alone, or in combination with each other, in both short- and long-term exposures. The author also considers the various fumigation methods and experimental strategies used to explore pollutant effects. The chapter concludes with an evaluation of the use of physiological and biochemical responses to pollutants as diagnostic tools for predicting injury caused by pollutants. Enormous advances have been made in the study of Rhizobiurnllegume interactions over the last decade. The approaches of molecular genetics have perhaps captured most of the attention but progress has also been made on the more physiological and metabolic aspects. The chapter by Streeter is concerned with the coordinated regulation of the separate metabolic systems of plant cell and bacterial endosymbiont and considers aspects of metabolite transfer. The major section of the chapter is concerned with the transfer and processing of carbon with emphasis on the role of 4C organic acids in providing reducing equivalents in the bacteroid. The chapter ‘Plants and Wind’ by van Gardingen and Grace, first considers conceptual and physical aspects of the relationship. The general limitations of classical micrometeorological techniques as applied to heterogenous vegetation are outlined and more advanced approaches defined. A major section of the chapter is concerned with the effect of wind on various aspects of energy transfer between the plant and its environment and a PASCAL computer program is provided for calculating the effect of wind speed on water use and surface temperatures under a range of conditions. The authors then consider biological responses to wind. Some of these, such as thigmomorphogenesis, are quite well defined, but the basis of perception and response is poorly understood although some advances are being made at the molecular level. Improved understanding will eventually permit quantitative predictions to be made about the influence of, for example, shelter on plant growth or agronomic yield. Knowledge of the light regime that exists inside plant tissues is important to the understanding of photosynthetic performance in intact leaves and the use of light as an environmental cue in various developmental processes. It is vii

viii only with the relatively recent development of fibre optic microprobes, with tip diameters as small as 2 bm that botanists have been able to explore the internal optical properties of leaf tissue, and the chapter by Vogelmann et al. describes how such probes may be made and the associated instrumentation necessary, before examining examples of their application to various physiological problems such as the focusing and propagation of light within leaf tissues in relation to light harvesting in photosynthesis. Finally, as usual I would like to pay my thanks to each of the contributors in the present volume for their patience with the editor and their efforts to make his task easier. J. A. Callow

CONTENTS

CONTRIBUTORS TO VOLUME 18

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

PREFACE . . . . . . . . . . . . . . . . . . . . . . . .

V

vii

Photosynthesis and Stomatal Responses to Polluted Air. and the Use of Physiological and Biochemical Responses for Early Detection and Diagnostic Tools H . SAXE I . General Introduction

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

2

I1. Methods of Air Pollution Exposure of Plants and Physiological Measurements . . . . . . . . . . . . . . . . . . . . . A . Chamberless Exposure . . . . . . . . . . . . . . . . . B . Open-top and Closed-topField Chambers . . . . . . . . C . Laboratory Exposure . . . . . . . . . . . . . . . . . I11. Response of Photosynthesis and Diffusive Resistance to SO2 A . Introduction . . . . . . . . . . . . . . . . . . . . . B . Response to Short-term SO2 Exposure . . . . . . . . C. Response to Long-term SO2 Exposure . . . . . . . .

. .

. . . .

. IV . Response of Photosynthesis and Diffusive Resistance to NO. A . Introduction . . . . . . . . . . . . . . . . . . . . . B . Response to Short-termNO. Exposure . . . . . . . . . C. Response to Long-term NO. Exposure . . . . . . . . . V. Response of Photosynthesis and Diffusive Resistance to SO2 NO2 . . . . . . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . B . Response to Short-term SO2 NO2 Exposure . . . . . . C. ResponsetoLong-termS02+NOzExposure . . . . . .

. .

.

+

+

ix

. .

4 4 5 6

7 7 17 26 34 34 35 39 43 43 43 48

CONTENTS

X

VI . Response of Photosynthesis and Diffusive Resistance to O3 . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . B . Response to Short-term O3Exposure C. Response to Long-term O3Exposure

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

VII . Response of Photosynthesis and Diffusive Resistance to O3+ SO2 . . . . . . . . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . B . Response of Photosynthesis and Stomata1 Resistance to Short-term O3 SO2Exposure . . . . . . . . . . C. Response of Photosynthesis and Diffusive Resistance to Long-term O3 SO2Exposure . . . . . . . . . . D . Summary of the Response to Short- and Long-term O3 SO2 Exposure . . . . . . . . . . . . . . . .

49 49 51 62 69 69

+

.

70

+

.

75

.

79

VIII . Response of Photosynthesis and Diffusive Resistance to 0 3 +Acid Precipitation . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . B . Physiological Response . . . . . . . . . . . . . . . . .

79 79 81

+

IX . Response of Photosynthesis and Diffusive Resistance to Other Air Pollution Combinations . . . . . . . . . . . . . . . . . . A . Response of Photosynthesis and Transpiration to O3 NO2 Exposure . . . . . . . . . . . . . . . . . B . Response of Photosynthesis to SO2 NO2 + O3Exposure . .

+

+

X . Diagnostic Methods for Predicting Air-pollution and Stress Injury to Plants . . . . . . . . . . . . . . . . . . . . . . . . A . General . . . . . . . . . . . . . . . . . . . . . . . B . Indicator Plants . . . . . . . . . . . . . . . . . . . . C . Bioindication . . . . . . . . . . . . . . . . . . . . . D . Bioindications for Early Detection of Novel Forest Decline . E . Bioindication as a Diagnostic Tool for Selecting Plants Resistant to Novel Forest Decline and Specific Air Pollutants . . . . . . . . . . . . . . . . . . . . F. Conclusion . . . . . . . . . . . . . . . . . . . . . .

84 84 85 85 85 86 101 104 105

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105

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

105

Acknowledgements References

84

Transport and Metabolism of Carbon and Nitrogen in Legume Nodules J . G . STREETER I . Introduction

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

I1. Nodule Anatomy and Terminology

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130 131

xi

CONTENTS

A . Tissues and Cell Types . . . . . . . . . . . . . . . . . B . Organization in Infected Cells . . . . . . . . . . . . . . C . Bacteroids . . . . . . . . . . . . . . . . . . . . . . 111. Carbon Processing

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

131 134 139

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

141 141 146

IV . Nitrogen Processing . . . . . . . . . . . . . . . . . . . . A . Bacteroid Functions . . . . . . . . . . . . . . . . . . B . Host Functions . . . . . . . . . . . . . . . . . . . .

153 153 154

V . Restrictions Imposed by Microaerobic Conditions . . . . . . . A . The Oxygen Regulation System . . . . . . . . . . . . . B . Impact of Low O2on Metabolism . . . . . . . . . . . .

161 161 161

A . Host Functions . . B . Bacteroid Functions

VI . Summary and Suggestionsfor Future Work

163

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

165

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

165

Acknowledgements References

. . . . . . . .

Plants and Wind P . V A N GARDINGEN and J . GRACE I . Introduction

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

192

11. Wind Regimes Around Plants and their Role in Transport

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

193 193 199

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

208 208 210 215 217 218 221 223

.

224

. .

231 232 235 237

A . TheClassicalMicrometeorologicalApproach . . B . What Classical Micrometeorologyis Unable to Do

111. Wind and Energy Transfer

A. B. C. D.

E.

F. G. H.

Energy Balance Equation . . . . . . . . . . . . . . Boundary Layer Conductance . . . . . . . . . . . . Convective Energy Flux . . . . . . . . . . . . . . . Temperature . . . . . . . . . . . . . . . . . . . . . Transpiration . . . . . . . . . . . . . . . . . . . . . Stomata1Conductance . . . . . . . . . . . . . . . . Cuticular Conductance . . . . . . . . . . . . . . . . Solving the Energy Balance Equation for Transpiration and Surface Temperature . . . . . . . . . . . . . . .

IV . Facts. Fallacies and Mysteries . . . . . . . . . . . . . . . . A . Thigmomorphogenesis . . . . . . . . . . . . . . . . B . Abrasion . . . . . . . . . . . . . . . . . . . . . . . C. Ecological Phenomena . . . . . . . . . . . . . . . .

V . Conclusions

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.

239

xii

CONTENTS

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

240

Appendix I . . . . . . . . . . . . . . . . . . . . . . Appendix I1 . . . . . . . . . . . . . . . . . . . . . .

246 248

Fibre Optic Microprobes and Measurement of the Light Microenvironment within Plant Tissues T. C . VOGELMANN. G . MARTIN. G . CHEN and D . BU'ITRY I . Introduction

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

I1. Optical Fibre . . . . . . . . . . . . . . . . . . A . General Characteristics of Optical Fibre . . . B . Types of Optical Fibre . . . . . . . . . . . C . Transmission Characteristics . . . . . . . . 111. Microprobe Fabrication

256

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

. . . . . .

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

A . Heating and Stretching versus Chemical Etching . . . . . . B . Sputter Coating followed by Truncation of the Probe Tip . . C . Grinding and Polishing the Probe Tip followed by Coating with Evaporated Metal . . . . . . . . . . . . . . . . D . Measurement of Probe Sensitivity and Acceptance Angle . . E . Factors that Affect the Optical Properties of Probes . . . .

IV . Experimental Apparatus V . Terminology VI .

VII .

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

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

Experimental Measurements . . . . . . . . . . . . . . . A . Effect of Probe Orientation on Light Measurements within Thick Samples . . . . . . . . . . . . . . . . . . B . Effect of Probe Acceptance Width on Light Measurements C. Strongly versus Weakly Absorbed Wavelengths of Light . D . Isotropy of Scattered Light . . . . . . . . . . . . . . E . Tissue Effects . . . . . . . . . . . . . . . . . . . . . F. Signal Interpretation: Reality or Artifact? . . . . . . . . Prognosis and Future Applications

AUTHOR INDEX

260 260 262 263 266 268 270 272

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273

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273 277 277 278 283 283

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289

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292

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293

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297

Acknowledgements References

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

257 257 258 259

SUBJECTINDEX . . . . . . . . . . . . . . . . . . . . . . . .

313

Photosynthesis and Stornatal Responses to Polluted Air. and the Use of Physiological and Biochemical Responses for Early Detection and Diagnostic Tools

H . SAXE Ministry of the Environment. National Environmental Research Institute. Division of Terrestrial Ecology. VejlsQvej11. DK-8600 Silkeborg. Denmark

I . General Introduction . . . . . . . . . . . . . . . . . . . I1.

Methods of Air Pollution Exposure of Plants and Measurements . . . . . . . . . . . . . . A . Chamberless Exposure . . . . . . . . . B . Open-top and Closed-top Field Chambers C . Laboratory Exposure . . . . . . . . .

2

Physiological

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

. . . . . . . .

4 4 5 6

I11. Response of Photosynthesis and Diffusive Resistance to SO2 . . A . Introduction . . . . . . . . . . . . . . . . . . . . . B . Response to Short-term SO2Exposure . . . . . . . . . . C. Response to Long-term SO2 Exposure . . . . . . . . . .

7 7 17 26

IV .

Response of Photosynthesis and Diffusive Resistance toNO. . . A . Introduction . . . . . . . . . . . . . . . . . . . . . B . Response to Short-term NO. Exposure . . . . . . . . . . C . Response to Long-term NO. Exposure . . . . . . . . . .

34 34 35 39

Response of Photosynthesis and Diffusive Resistance to SO2 + NO2 . . . . . . . . . . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . B . Response to Short-term SO2 NO2 Exposure . . C. Response to Long-term SO2 + NO2 Exposure . .

43 43 43 48

V.

+

. . . . . . . .

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

VI .

Response of Photosynthesis and Diffusive Resistance to 0 3 . . . 49 A . Introduction . . . . . . . . . . . . . . . . . . . . . 49 B . Response to Short-term 0 3 Exposure . . . . . . . . . . 51 C . Response to Long-term 0 3 Exposure . . . . . . . . . . . 62 Copyright 01991 Academic Press Limited Advances in Botanical Research Vol . 18 ISBN &12-0059115

All rights of reproduction in any form reserved

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VII.

Response of Photosynthesis and Diffusive Resistance to 0 3 so2 . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . B. Response of Photosynthesis and Stomata1 Resistance to Short-term O3+ SO2Exposure . . . . . . . . . . . C. Response of Photosynthesis and Diffusive Resistance to Long-term O3 SOzExposure . . . . . . . . . . . D. Summary of the Response to Short- and Long-term O3+ SOzExposure . . . . . . . . . . . . . . . . .

+

+

VIII.

IX.

Response of Photosynthesis and Diffusive Resistance to 0 3 Acid Precipitation . . . . . . . . . . . . . A. Introduction . . . . . . , . . . . . . . . . . . . . . B. Physiological Response . . . . . . . . . . . . . . . . .

+

Response of Photosynthesis and Diffusive Resistance to Other Air Pollution Combinations . . . . , . . . . . . . . . . . . . A. Response of Photosynthesis and Transpiration to O3+ NO2 Exposure . . . . . . . . . . . . . . . . . B. Response of Photosynthesis to SO2 + NO2 0 3 Exposure . .

+

X.

Diagnostic Methods for Predicting Air-pollution and Stress Injury to Plants . . . . . . . . . . . . . . . . . . . . . . . . A. General . . . . . . . . . . . . . . . . . . . . . . . B. Indicator Plants . . . . . . . . . . . . . . . . . . . . C. Bioindication . . . . . . . , . . . . . . . , . . . . . D. Bioindications for Early Detection of Novel Forest Decline . E. Bioindication as a Diagnostic Tool for Selecting Plants Resistant to Novel Forest Decline and Specific Air Pollutants . . . . . . . . . . . . . . . . . . . . F. Conclusion . . . . . . . . . . . . . . . . . . . . . . Acknowledgements References . .

. .

. .

. .

. . .

,

. . .

. . .

.

.

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.

. . .

. . . . .

.

.

69 69

70 75

79 79 79 81 84 84 84 85 85 85 86 101 104 105 105

. .

. .

.

105

I. GENERAL INTRODUCTION Important physiological processes like photosynthesis, respiration, the stomatal mechanism, phloem loading and carbon allocation, are all known to be affected by air pollution (Darrall, 1989). A wide range in sensitivity in physiological responses to SO*, NO,, 0 3 , acid precipitation and HF is evident from the literature. Some of this variation is clearly due to genetic factors, though much of it is in response to differences in environmental conditions both prior to, during and after pollutant exposure. Recognizing the importance of the environment, the present review opens with an over-

PHOTOSYNTHESIS AND STOMATAL RESPONSES

3

view of different fumigation methods, and a brief discussion of their relevance. The main substance of the present review, however, deals with the voluminous literature on direct effects of SO*, NO,, 0 3 and acid precipitation on photosynthesis, respiration and stomata, under the influence of climate and exposure strategies: short-term, long-term, single gas and mixed gases. The present paper does not, therefore, include an overview of indirect effects of air pollution, or of carbon allocation, nor does it treat the numerous mechanistic explanations for the observed effects in great detail. The latter have recently been reviewed by Heath (1988), Jager etal. (1986) Koziol and Whatley (1984), Schulte-Holstede et al. (1988) and Wellburn (1988). Since the physiological and biochemical responses, are key-factors to the health of plants, they may be used as diagnostic tools for predicting injury by air pollution and general stress to individual species and the decline of whole populations. The present chapter is concluded with an evaluation of such methods of bioindication, since they could be of great relevance to the future and ongoing battle to diminish the deterioration of our physical and biological environment. Even though a decline in net photosynthesis is one of the most straightforward and intuitively logical explanations for a decline in growth, Noyes has (1980) found that another physiological process, loading of sieve-tubes, declined to 60%, 55% and 35% relative to a control, while photosynthesis declined to only loo%, 88% and 30% when exposed briefly to 100,1000 and 3000 ppb SO2respectively. If the results of Noyes (1980) are also true under long-term, low SO2 concentrations, sieve-tube loading and eventual carbon allocation (Darrall, 1989) could be as limiting to the growth and development of plants as direct effects on photosynthesis and stomates. Changes in carbon allocation usually favour leaf development over root growth, which can compensate for a decline in net assimilation rate up to a certain point. But it also limits water and mineral uptake from soils with low moisture content (Cooley and Manning, 1987; Darrall, 1989). Very low concentrations of gaseous air pollutants often cause stornatal opening, while higher, but realistic concentrations induce a decline in photosynthesis, a stimulation of dark respiration and a closure of stomata. Though the diffusive resistance or conductance or the transpiration may not always be accurate estimates of the degree of stornatal opening when affected by gaseous pollutants (Omasa et al., 1985), I have maintained the stornatal “analogy”, and often talk of stornatal opening and closure to make the “direction” of a response clear. Exposure of plants to mixtures of pollutants often amplifies the physiological responses, and generally cause less predictable responses. Future research into physiological effects of air pollutants should integrate measurements of key physiological parameters with the study of

4

H. SAXE

growth parameters, as affected by realistic mixtures of pollutants. In this way, the underlying mechanisms of changes in growth and development will be more fully understood.

11. METHODS OF AIR POLLUTION EXPOSURE OF PLANTS AND PHYSIOLOGICAL MEASUREMENTS Several experimental methods have been used to determine the physiological responses and the yield loss (or gain) of agricultural crops and trees due to pollutants present in ambient air (Ormrod et af., 1988). Environmental conditions such as light (photon fluence density, light period and spectral distribution), temperature (soil, air and plant), relative humidity, wind speed and direction, and water and nutrient availability in the soil, affect plant responses to gaseous pollutants, as do the exposure kinetics (McLaughlin et af., 1979; Temple et af., 1985a). Exposure and measurements in the open field give the most realistic results, while those in a controlled environment in a laboratory phytotron give the most reproducible results. The latter is of importance when studying the mechanisms of pollutant injuries and the influence of the environment. The use of open-top and closed-top field chambers is in effect, intermediate between these two approaches. A. CHAMBERLESS EXPOSURE

While pollution exposures of plants in the open field, with no modifications of the environment by chambers, represent the most realistic situation, they make physiological assessment rather tedious, since measurements have to be carried out on individual plants or leaves, and because the natural environment is unstable. It also implies that each plant or branch being tested has to be contained in a small, transparent chamber during measurements (Caput and Belot, 1978); Air temperature and humidity in these cuvettes may be regulated to be identical to the conditions outside, but changes in light and other environmental parameters will inevitably induce chamber effects, though the measurements may be completed before they become too apparent. On a large scale, vegetation or ecosystem responses (Sigal and Suter, 1987; Winner and Mooney, 1985; McLeod et af.,1988; McLeod and Baker, 1988), are examined along pollutant gradients in specific geographic regions in the vicinity of defined emitters, such as industry, cities, highways or volcanos. On the smaller scale of experimental field systems the gaseous pollutants are either emitted at a low flow in the so-called ZAPS (Laurence etaf.,1982) through a number of nozzles regularly placed on the perimeter of a circular

PHOTOSYNTHESIS AND STOMATAL RESPONSES

5

exposure field, where only up-wind nozzles are active, or at a high flow in the so-called air-exclusion systems. The latter include particulate and activated charcoal filters, pressure-type blowers, a mixing manifold, and perforated inflatable ducts positioned between rows of plants, and have the advantage over ZAPS, in that they can produce a nearly pollution-free environment. Olszyk et al. (1986) compared the physical environment in an airexclusion system with that of open-top and closed-top field chambers, and found that soil and air temperatures, light intensity and dew formation were closer to open-field conditions than either open-top or closed-top chambers. Wind speed, however, was consistently higher in the air-exclusion system than in chambers or in ZAPS, if air exclusion was to be as efficient as in open-top chambers. A research alternative to air-exclusion systems, ZAPS or ecological studies along existing pollution gradients is the use of chemical protectants (“chemical exclusion”). Such fungicides as benomyl, ethylene diurea and triazol as well as polyamines have been applied as foliar spray, as soil drench or as a seed treatment to prevent O3effects on crop plants and trees (Hofstra et al., 1983; Musselman, 1985; Beckerson and Ormrod, 1986; Mackay et al., 1987; Smith et al., 1987). Other biotic effects besides the protection from ozone, however, tend to confound the usefulness of this type of chamberless exposure. Rain exclusion may be obtained with rolling glasshouses, as pioneered by the Brookhaven National Laboratory in New York (Evans and Thompson, 1984), but then it is not really a chamberless exposure any longer. B. OPEN-TOP AND CLOSED-TOP FIELD CHAMBERS

1. Construction materials Field chambers must be adequately anchored and be made of materials that resist wind pressure. As in all fumigation chambers, the surfaces exposed to polluted air must not absorb the administered (or naturally occurring) pollutants. Stainless steel, glass and PTFE (polytetrafluoroethylene) teflon (sheets and tubing) meet these demands. PTFE, however, is slightly permeable to a few gases, for example CO2. PVDF (polyvinylidene fluoride) could be an alternative. 2. Open-top chambers Open-top chambers (OTCs) represent the most widely used outdoor exposure design, being in function at dozens of places in the USA, all over Europe, and in some Third-World research facilities. The general design includes particulate and activated charcoal filters, pressure-type blowers, a mixing manifold, and perforated inflatable ducts positioned in the perimeter of a cylindrical exposure chamber, which is open at the top. The specific design, however, varies both in material, size, forced air flow, and in the

6

H. SAXE

shape of the top rim, which excludes a proportion of the air intruding through the top. Rain may be excluded with automatic “umbrellas” (Mandl et al., 1988). The design and performance of open-top chambers were originally described by Heagle et al. (1973), Heck et al. (1979) and Davis and Rogers (1980). Contemporary OTCs are computerized, as described by Nystrom et al. (1982). Again, physiological measurements are rather tedious, since they have to be carried out on individual plants or leaves as in the chamberless exposures. The weight lysimeter was designed as an experimental apparatus for automating the assessment of the long-term effects of gaseous pollutants and acidic precipitation on trees in chamberless or OTC exposure systems (Mandl er al., 1988). 3. Closed-top chambers Closed field chambers come in several designs. Some look much like opentop chambers with a transparent top (Musselman et al., 1986), while others are designed for an improved air flow (Ashenden et al., 1982; Lucas et al., 1987). Closed-top chambers allow better control of atmospheric conditions than open-top chambers, but differ more from outdoor light and precipitation conditions and, when not air conditioned, also in temperature and humidity. Physiological measurements may be carried out on a chamber-canopy basis, though large variations within the chamber and in the external climate complicate detailed mechanistic studies. C. LABORATORY EXPOSURE

Phytorrons (Downs, 1980) are series of chambers with a highly controlled environment for plant growth. When used for air pollutant exposures, e.g. by placing a number of smaller exposure chambers in the controlled environment, conditions are optimal for reproducible studies of the mechanisms behind various responses. The Agricultural Research Service in the USA has developed a design (conrinuously stirred tank reactors CSTR) (Heck et al., 1978), which is today the most commonly used exposure system in laboratories in North America and Western Europe. The original CSTR design was not conceived for simultaneous physiological measurements, but has since been considerably refined by several laboratories (e.g. McFarlane and Pfleeger, 1987). There are, however, several other old and new designs, e.g. those by Darrall (1986), Gmur et al. (1983), Lockyer et al. (1976), Mortensen (1982a,b), Saxe (1983), Saxe (1987a), and Saxe and Murali (1989a). Unfortunately, results with the same species may vary from one chamber system to the next. Koziol (1980) concluded, after investigating such conflicting

PHOTOSYNTHESIS AND STOMATAL RESPONSES

7

results, that part of the discrepancy was due to differing plant density and leaf boundary layer resistance, R,. By rule of thumb, chambers should have an air exchange rate of one exchange per minute in order to minimize gas adsorption to chamber walls. If, however, precise, simultaneous measurements of gas exchange are desired during pollutant exposures, restrictions are imposed on the maximum air exchange rate in the chambers, as for example with a low growth rate of the plants, due to the choice of species or the environment. R , should be measured, as it is one of the potentially important parameters in gas exchange. With a fan keeping a low R,, the exposure concentration is better estimated by the chamber outlet concentration, rather than the inlet concentration, since healthy plants take up substantial amounts of the gaseous pollutants at low air exchange rates (Koziol, 1980). Most phytotron exposure systems today, at least those with simultaneous physiological measurements, are computer controlled. The system developed by Saxe and Murali (1989a) is a contemporary example. The complete software of this system for control, calculations and plotting is now available from the author.

111.

RESPONSE OF PHOTOSYNTHESIS AND DIFFUSIVE RESISTANCE TO SO:! A . INTRODUCTION

Sulphur dioxide concentrations as averages of European rural areas (the EMEP monitoring network) ranged between 2 and 13 ppb SO2 as monthly averages during 1979-1986, with the higher concentrations in the winter and in Central Europe (Eliassen et al., 1988). With daily or hourly averages, however, the concentrations were higher, and in urban areas and surroundings, SO2 concentrations reached 10-50 times the rural levels. The response of photosynthesis and diffusive resistance to air pollutants depends for a given plant and environment, both on the pollutant concentration(s), and the exposure time(s). These are combined in the concept of dosage (Fowler and Cape, 1982). The external dose is defined as the exposure concentration multiplied by the exposure time, and the effective dose as the external dose multiplied by the intake rate of the plant (Darrall, 1989). The latter varies with species, cultivar, ecotype, age and the environmental conditions. However, even plant response relative to effective dose is ambiguous, since a short exposure at high concentrations was more injurious than the same quantity given as a longer exposure at a lower concentration (McLaughlin et al., 1979; Temple er al., 1985a). Ideally, therefore, the effects of exposures should be analysed in terms of both

8

H. SAXE

dose-response functions, with a range of exposure periods, and timeresponse functions with a range of concentrations (e.g. Saxe and Murali, 1989a,b,c; Saxe, 1989). Published data reflect a wide range in the sensitivity of photosynthesis, diffusive resistance and respiration to SO2upon exposure to short-term (< 1 day) and long-term (> 1 day) episodes (Fig. 1, Table I). The responses are very dependent on the species and the environment. Data for one species and climate, therefore, cannot be generalized, and combined exposures with SO1 and other gases further complicate the situation. The most common

100 80

s 60 i !!40

40

20

Corn OX 0

1

2

3

Cucumber OX 4

60

Lucern 60%

Broid born 701 0

1

2

3

4

0

Time, h

I 2 3 4 Time, h

Fig. 1. Transpiration response of 16 plant species to short-term (up to 4h), high level (2ppm) SO2 exposure, and percentage leaf necrosis. (Adapted from Furukawa ef al. (1979a).)

TABLE I Changes in net photosynthesis (PS), “stomata1opening” (ST) and respiration (R) in short- and long-term SO2 fumigations‘ Reference

SHORT TERM Biscoe et al. (1973) Unsworth et al. (1972) Black and Unsworth (1980) Black and Unsworth (1979) Natori and Totsuka (1984a) L’Hirondelle and Addison (1985)

Species and cultivar

Vicia faba (broad bean) cv. “Great Green Longpod” Vicia faba (broad bean) Zea mays (maize, corn) Vicia faba (broad bean), cv. “Dylan” Vicia faba (broad bean), cv. “Dylan” Euonymous japonica Pinus banksiana (Jack pine)

SO2 Duration Concentration (PPb)

22

5G500 50-500 17.5

2h 2h 2h 2h 6h lh 2h lh 2h 15 min lh 2h 3h lh Ih 3h 1h

Glycine max (soybean) strain T219

Noyes (1980)

Phaseolus vulgaris cv. “Black Valentine” Betula pendula (European white birch)

1000 300

Betula lufea (yellow birch)

300 900 300 300 300

Betula populifolia (gray birch)

2h

35 50 95 950 950 250 250 500 500

Takemoto and Noble (1982)

Biggs and Davis (1980)

20 min 30 min 30 min

300

PS-response ST-response (% control) (% control)

R-responseb (% control)

TABLE I-contd. Reference

Species and cultivar

so2

Duration

Concentration (PPb) Muller et al. (1979) White er al. (1974) Black and Black (1979) Carlson (1983a) Darrall (1986) Sisson et al. (1981) Benett and Hill (1973)

Oshima et al. (1973) Darrall(l986)

Taylor et al. (1986)

Glycine mar (soybean) cv. “Wells” 2 h 20 min later Medicago sativa (alfalfa), cv. “Ranger” Vicia faba (broad bean), adaxial cv. “Dylan”, abaxial Glycine max (soybean) cv. “Wayne” Lolium perenne (ryegrass), cv. “S23” Carya illinoensis (pecan) Hordeum vulgare (barley) cv. “Trebi” Medicago sativa (alfalfa), cv. “Ranger” Helianthus annuus (sunflower) cv. “Russian Mammoth” Hordeum vulgare (barley) cv. “Sonja” Vicia faba (field bean), cv. “Three Fold White” cv. “Blaze” Geranium carolinianum Resistant Sensitive

PS-response ST-response (% control) (% control)

79 405

4h 3h 40 min

137 83 120

350 200 200 200 450 200

lh 2h 2h 2h 2h 2h

97

975

30 rnin

81

250

2h

90

400

2h

81

200

3h

92

300

2h

92

300 300

2h 2h

86 81

300 300

3h 3h

90

-

92 73 84

70

R-responseh (% control)

Barton ef al. (1980) Furukawa et al. (1979b) Matsuoka (1978) Saxe and Murali (1989a) Saxe (1989) Alscher et al. (1987)

Phaseolus vulgaris, 33% RH cv. “Red Kidney”, 71% RH Populus euramericana (poplar) C V . “1-214” Oryza (rice) Picea abies (Norway spruce), 9 half-siblings after 4 years in a protected environment As above + winter in extreme frost Pisum sativum (pea) cv. “Progress” cv. “Nugget” cv. “Pinto”

Helianthus annuus cv. “Russian Mammoth” Winner and Mooney (1980~) Atriplex triangularis

Furukawa et al. (1980)

Sij and Swanson (1974)

Caput and Belot (1978) Bonte et al. (1977) Hallgren and Gezelius (1982)

Atriplex sabulosa Phaseolus vulgaris (bean) cv. “Pinto” Zea mays (corn) Pinus nigra Pinus pinea Pelargonium x hortorum Pinus silvestris (Scots pine)

1000 1000

20 min-1 h 20 rnin-1 h

50-85 41- 6

800 170

1h 25 rnin Sh

85 95

790

4h

80

810

4h

93

800 800 1000 1000 3000

1h 15 rnin 1h 15 min lh 4h lh

24 46 88 86

1500 500 500 SO0 1000 1000 1000 1000 1000 1000

30 rnin 2h 7h lh lh 4h lh 4h lh lh lh 4-6h 14 h 5.5 h 12h 3h

27 110 100 112 86 86 79 76

1000 280 280 750 750 1425

40

-

100 60 75

60 71

91-102 (TR) 114- 76 (TR)

-

-

701100 (DRPR) -

TABLE I-contd. Reference

Species and cultivar

so2

Duration

Concentration (ppb) Glycine max (soybean) cv. “Essex” Pisum sativum (pea) Rao et al. (1983) cv. “Little Marvel” Helianthus annuus, Omasa et al. (1985) visible injury cv. “Russian Mammoth”, no visible injury Kimmerer and Kozlowski Populus tremuloides, (1981) sensitive clone resistant clone Kropff (1987) Vicia faba (bean) Winner and Mooney (1980a) Diplacus aurantiacus Sorghum bicolor (sorghum) Ushijima and Tazaki (1977) Zea mays cv. “Golden Cross Bantam” Phaseolus vulgaris cv. “Pinto” f i s t and Davis (1979) 32°C 21°C 80% R H 60% RH Pinus silvestris Katainen et al. (1987) Majernik and Mansfield (1971) Vicia faba (bean) cv. “Winsor Harlington” Winner and Mooney (1980a) Heteromeles arbutifolia Larrea tridentata Olszyk et al. (1987)

PS-response ST-response (% control) (% control)

R-responseb (% control)

Chevone and Yang (1985)

700

2h

500

3h

1500

lh

1500

lh

200 200 250 250

8h 8h 2h 9h 10h

250

10h

900

4h 4h 4h 4h 5h 10h 10h 8h 2h

800

900

900 900 870 250 lo00 420 2000

NS

-

85 92 78

120 (DR) -

76

126 (DR)

-

Saxe (1983)

Phaseolus vulgaris cv. “Processor” Hordeum vulgare (six cultivars) Ashmore and Onal (1984) Majernik and Mansfield (1972) Vicia faba (bean) cv. eG‘WiniorHarlington” Amundson and Weinstein Glycine max (soybean) cv. “Beeson”, sensitive (1981) cv. “Hark”, resistant Furukawa et al. (1979a) 16 species (Fig. 1) Brenninger and Tranquillini Fraxinus excelsior (1983) Picea abies Abies alba LONG-TERM Ashenden (1979)

Katainen et al. (1987)

Maas et al. (1987) Hallgren and Gezelius (1982) L‘Hirondelle and Addison (1985) Cowling and Koziol (1978) Rao et al. (1983) Saxe (1983)

Phaseolus vulgaris cv. “Canadian Wonder” Pinus silvestris L.

Spinacia oleracea, day cv. “Monosa”, night Pinus sylvestris (Scots pine) Pinus banksiana (Jack pine) Lolium perenne L. cv. “S23” Pisum sativum (pea) cv. “Little Marvel” Phaseolus vulgaris (kidney bean) cv. “Processor”

355 950

12h 6h

-

95

95 NS (CJ

-

700

10h

-

Max. 300 (C,)

-

1950 1950 2000 1875 1875 1875

4h 4h 4h 6h 6h

143 (RI) 228 (RI) 80-20 (TR)

-

-

450 (RI) 143 (RI)

-

100 100 100 11 34 79 120 150 25 25 75 150 95

1 day 3 days 5 days 30 days 30 days 30 days 30 days 30 days 14 days 14 days 5 days 5 days 4 days

19 150 200 500 25 100 100

22-29 days 22-29 days 3 days 3 days 25 days 10 days 25 days

6h

-

38 87 70 -

106 85 78 75 93

-

83 52

-

NS NS NS 45 97 91 84

120 (TR) 118 (TR) NS (TR)

-

-

NS (TR) 144 (TR) 85 (Rs) 107 (Rs) NS (GI

NS (TR) NS (TR) NS (Cd 70 (CS) 97 (TR) 89 (TR) 75 (TR)

-

-

123 (DR) 127 (DR) 131 (DR) 145 (DR) 134 (DR) -

NS (DR) -

NS (DR) NS (DR) -

NS (DR) NS (DR) 92 (DR)

TABLE I-contd. Reference

so2

Species and cultivar

Duration

Concentration (PPb) Bytnerowicz ef al. (1987)

Triticum aestivum (winter wheat) Beckerson and Hofstra (1979a) Phaseolus vulgaris (cv. “Sanilac”) Abies alba (silver fir) Keller (1978) Spring

30

22 days

-

150

4 days

-

50

14 days/ 70 days 14 days/ 70 days 14 days/ 70 days 14 days/ 70 days 14 days/ 70 days 14 days/ 70 days

65/71

100 Summer

50 100

Autumn

50 100

Picea excelsa (spruce) Spring

50 100

Summer

50 100

Autumn

PS-response ST-response (% control) (% control)

50

100

14 days/ 42 days 14 days/ 70 days 14 days/ 70 days 14 days/ 70 days 14 days/ 70 days 14 days/ 70 days

54/45 96/84 106173 78/72 75/44 77/80 97/85 102197 88/78 115/113 92/92

R-responseb (% control)

Beckerson and Hofstra (1979b) Raphanus sativus (radish) Cucumis sativus (cucumber) Glycine mux (soybean) Norby and Kozlowski (1982) Betula papyrifera (paper birch) High humiditv Lo” humidit; Raphanus sativus cv. “Comet” Sat0 et al. (1979) Lolium perenne, Koziol et al. (1986) (perennial rye grass) Resistant Sensitive L’Hirondelle et al. (1986) Pinus banksiana (Jack pine) Freer-Smith (1985) Klein et al. (1978) Murray (1985)

Shimizu et al. (1980) Olszyk et al. (1987) Comic (1987) Bell et al. (1979) Lorenc-Plucinska (1982)

Betula pendula Pisum sativum (pea) Zea mays (corn) Medicago sativa L. (alfalfa) cv. “CUF 101” Helianthus annuus (sunflower) cv. “Russian Mammoth” Larrea tridentata Picea abies L. (Norway spruce) Last 5 days with drought Rewatered on day 48 Lolium perenne (ryegrass) cv. “S23” Pinus sylvestris (Scots pine) Resistant Sensitive

150 150 150

5 days 5 days 5 days

-

200 200 40

4 days 4 days 23 days

-

50 50 300 300 55 100 100 30 30 63 63

28 days 28 days 5 days 35 days 28 days 17 days 17 days ca. 60 days ca. 133 days ca. 60 days ca. 130 days

100 113 108 65

50 200 75 75 75 16 25 159

42 days 13 days 35 days 40 days 52 days 173 days 144 days 108 days

81 67 NS 31 59 65 NS 69

lo00 lo00

3 days 3 days

59 35

-

NS -

-

65 88 NS -

-

-

173169 (DR/PR) 113147 (DFUPR)

TABLE I - c o n t d . ~

~~

Reference

Species and cultivar

so2

Duration

Concentration (PPb) Mooney et al. (1988) Biggs and Davis (1982) Keller and Hasler (1986) Jones and Mansfield (1982) Atkinson et al. (1988a) Garsed et al. (1979) Jensen (1981) Taylor et al. (1986) Farrar et al. (1977)

Houpis and Helms (1985)

Preston (1988)

Raphanus sativus (radish) cu. “Cherry Bell” Populus maximowizii Picea abies (Norway spruce) Phleurn pratense (Timothy grass) Raphanus sativus L. cu. “Cherry Belle” Betula pendula (European white birch) Populus deltoides x trichocarpa Geranium carolinianum Resistant Sensitive Pinus sylvestris (origin north Scotland) High light Low light Pinus ponderosa Summer Winter Late spring Salvia mellifera (shrub)

240 250 25 120

14 days

18 days

PS-response ST-response (% control) (% control)

77

-

200 57

ca. 180 days 40 days 25 days 37 days 98 days

85 124 82 NS

250

25 days

68

450 450

16 days 16 days

128 75

200

60 60

238 days 81 (NS) 238 days 140 (NS)

75 75

275 days

53

400 days

33 63

75

96170

550 days 25 years

R-responseb (% control)

-

a When several concentrations and exposure periods were applied in one experiment, only the lowest or the few lowest concentrations, and in long-term exposures only the shortest periods with an effect are quoted. C,,leaf diffusive conductance to H2O (cm s-’); C, stomatal conductance to H20 (cm s-’); R,, stomatal resistance to H 2 0 (s cm-’); R I ,leaf resistance to H 2 0 (scm-’); W,, width x length of stomatal pore; PR, photorespiration; DR, dark respiration; TR, transpiration; RH, relative humidity; NS, non-significant; % control, activity relative to a control during and/or before exposure; -, not measured or not given. References are listed according to the lowest applied external dose. Where two figures are given separated by a solidus, this refers to DR and PR respectively.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

17

condition in the ambient environment includesfluctuating pollutant concentrations; this was only rarely simulated in laboratory exposures with mixed gases (Mueller and Garsed, 1984). B. RESPONSE TO SHORT-TERM SO2 EXPOSURE

Since short-term fumigations with SO;?,often performed with plants grown in protected environments, do not necessarily reflect the reactions of the plants under chronic exposures to mixed gaseous pollutants in the complex stress of the open field, the advantage of observing short-term responses to acute SO2 largely rests with three aspects: 1. it simulates a common situation in urban areas where SO2 peaks may reach several hundred parts per billion (Wentzel, 1985); 2. their use in simple mechanistic studies of interactions between photosynthesis, the stomata1 mechanism and uptake, since these are fundamentally the same with chronic exposures; and 3. their use as diagnostic tools, when these have been documented to reflect a situation in the “real world” (e.g. Saxe and Murali, 1989a,b,c; Saxe 1989). Use of short-term exposures to elucidate more complex causal mechanisms (e. g. photochemical and biochemical responses) are only of suggestive value in relation to most ambient situations, and should be re-evaluated in long-term studies to determine their realistic value.

I.

Photosynthesis response to short-term SO2 exposure

Typical responses. In the studies quoted in Table I, photosynthesis inhibition was normally only found in short-term exposures when the concentration multiplied by duration was above 400ppb h, and at the lower concentrations only after 2 h or more of exposure. Exceptions were given by Black and Unsworth (1979) who also found inhibitions at lower exposure concentrations and durations, and by a few other authors (Muller et al., 1979; Winner and Mooney, 1980c; Takemoto and Noble, 1982), who found a stimulation of photosynthesis at low exposure levels. A t higher concentrations and durations, however, the stimulation always turned to inhibition (Takemoto and Noble, 1982). It is possible, however, that most authors never looked for effects of lower SO2 levels, or that their techniques did not provide statistical significance for effects emerging at the lower levels (Darrall, 1986). Typically, the photosynthetic activity declined linearly in response to the acute SO2 exposures for one or a few hours, after which it reached a constant level (Bennett and Hill, 1973; Sij and Swanson, 1974; Black and Unsworth,

18

H. SAXE

1979; Furukawa et al., 1979b; Taylor et al., 1986; Kropff, 1987). This creates problems in calculating and comparing dose-response effects, since the dose-relative response varies with time after the steady-state level of inhibition is reached, i.e. larger apparent effects are seen with short/high doses. The apparent exceptions seen by Winner and Mooney (1980a,c), who found a continuous photosynthetic decline for several hours, may be explained by the fact that they used chamber input concentration to represent the chamber concentration. With a constant input concentration, the chamber concentration increases with time (Koziol, 1980; Saxe, 1983) (see Fig. 4),due to saturation of plant absorption. The exposure chamber used by Winner and Mooney (1980a) did not absorb SO2 at relative humidities (RHs) of < 90%. Response mechanisms. Short-term exposures to SO2 may cause photosynthesis to decline (or increase) both in response to direct effects on chloroplast structure and function, and indirectly in response to effects on stomata (see Fig. 3) and other factors with a secondary influence on photosynthesis. Barton et al. (1980), Taylor et al. (1986) and Sison et al. (1981) all found residual (mesophyll) resistance to be much more important than stomatal resistance in SO2 inhibition of photosynthesis. Barton et al. (1980) found that mesophyll resistance in bean leaves was 99% of the total leaf resistance at high humidity and 89% with low humidity. It was increased 25 times more by SO2 under high humidity than under low humidity conditions. Taylor et al. (1986) found both residual and stomatal resistance to be a little larger and to increase a little more with SO2 exposure in the more resistant of two Geranium cultivars (Table I). Concluding from results of less injury on soybean when stomatal opening was diminished by increased COz levels, Carlson (1983a) supported the view that stomata play a protective role even when their resistance to pollutant uptake was smaller than the residual resistance. Carbon dioxide, however, could have protected photosynthesis against SO2 in other ways than by stomatal closure (Furukawa et al. 1979b). Brenninger and Tranquillini (1983), Hunt and Black (1988), Winner and Mooney (1980b) and several others, showed for a number of species (Table I), that inhibition of photosynthesis by acute SO2 exposures could at the most be partly explained by stomatal closure. Alscher et al. (1987) found that photosynthesis in pea recovered as usual when SO2 exposure was terminated, while leaf conductance did not. Furthermore, in one cultivar (“Progress”) photosynthesis declined during SO2 exposure, while leaf conductance did not. Clearly, in this case a stomatal response was not involved in the photosynthesis response to SO2. Alscher et al. (1987) also measured changes in sulphite accumulation, reduced glutathione, 1,6-bisphosphatase, glyceraldehyde-3-phosphate dehydrogenase, and electron transport in isolated thylakoids. They interpreted their results to express an inhibition by SO2 of photosynthesis at a site within the chloroplast which was

PHOTOSYNTHESIS AND STOMATAL RESPONSES

19

not associated with electron transport and which was more sensitive to SO;?/sulphitethan was the inactivation of various light modulated enzyme reactions (Alscher, 1984). One likely candidate for this site would be ribulose-l,5-bis-phosphate-carboxylase (RuBPC), and another would be the transport of intermediates across the chloroplast envelope. It was Ziegler (1972) who originally demonstrated that sulphite could inhibit RuBPC in vitro by competing with HC03-, enough to explain in vitro inhibition of photosynthesis by sulphite (Silvius et al., 1975). Alscher et al. (1987) also concluded that the H 2 0 2 scavenging system is a plausible candidate for determining photosynthetic sensitivity to SO;?. Studies prior to those of Alscher et al. (1987) on the effects of SO2 and other gaseous pollutants on the thylakoid electron transport processes had already demonstrated that it took relatively high concentrations of gaseous pollutants to induce effects (Sugahara, 1984, Shimazaki, 1988). Adams etal. (1989) found effects of SO;? on photosystem I1 to be a secondary effect. It has even been found that low SOzconcentrations induced a stimulation of electron transport in cereals, in accordance with the increase sometimes observed in photosynthesis and transpiration with low pollutant exposures (Saxe, unpublished data). Furukawa et al. (1979b) found that increased C02 levels protected photosynthesis against SO2 in poplar, with no effect by CO;?on SO2 sorption. The COz protection in this case was, therefore, suggested to work through an improved competition of CO;? and SO3’- (a hydration product of SO;?)in reacting with RuBPC, or through an accelerated detoxification of S 0 3 2 - (to SO4’-), by increasing photosynthates and/or enzymes. Gezelius and Hallgren (1980), however, did not find S 0 3 2 - in pine seedlings to compete with c02. Kropff (1987) confirmed for Vicia faba that SO2 did not affect stomata (except indirectly through the feedback loop between net photosynthesis and internal CO;?concentration, Fig. 3), indicating that SO2 only inhibited photosynthesis through increased residual (mesophyll) resistance. He suggested changes in the affinity of ribulose-1,5-bis-phosphate-carboxylaseoxidase (RuBPCO) towards oxygen. RuBPCO would thus be relatively inhibited in its CO;?scavenging function. Several other workers have reported SO;?inhibition of RuBPC, e.g. Caemmerer and Farquhar (1981), Cerovic et a1. (1982), and Winner and Mooney (1980b). In work with isolated barley protoplasts, Pfanz et al. (1987b) supported the fact that SO2can inhibit photosynthesis by different mechanisms at low and high cellular concentrations of SO2 and its anions. At very low concentrations, they found inhibition by SO2 to be very low, but when acidification could not be compensated by p H stabilizing cellular mechanisms, it was the major element in SO;?toxicity. At higher levels of SO;?,anion toxicity and/or radical formation during oxidation of SO;?to sulphate was thought to play the major role in inhibition.

20

H. SAXE

There are also several older in vitro studies which have shown effects of anions of SO2 (i.e. HS03-, S03;!- and S04;!-) besides the quoted effects on RuBPC, e.g. non-specific effects by sulphite on membranes (Luttge et al., 1972) and proteins (Cecil and Wake, 1962). These effects were mainly caused by the sulphite reaction with disulphide bonds according to the equation: RS-SR

+ S032-

c*

RS-

+ RS-S03-

Among the consequences of SO2 injured membranes were effects on frost sensitivity in trees (e.g. Feiler, 1985; Davison et al., 1988), structural changes in thylakoid membranes (Huttunen and Soikkeli, 1984) and altered stomatal function (described below). Anderson et al. (1988) reviewed the effects of SO2 and sulphite on stromal metabolism.

Influence of the environment. Darrall (1986) found a surprisingly high threshold level for SO2 injury to photosynthesis in winter barley, winter wheat and oil-seed rape, and suggested that growing these three plants outdoors had increased their hardiness, compared to plants grown in the protected environment of a greenhouse. Inhibition of photosynthesis by SO;!in poplar and pine decreased with increasing quantum flux density (Katainen et al., 1987; Adams et al., 1989). Hunt and Black (1988) demonstrated the importance of the adaptation of Vicia faba to light and temperature for just 1 week prior to short-term exposures to 100-600 ppb SO2. Net photosynthesis of plants exposed to optimal pretreatment conditions (150 W m-2, 20°C) exhibited a marked reduction on exposure to SO2. However, in plants pretreated with low light (60 W mP2) or low temperature (10OC) this inhibition was significantly reduced (Fig. 2). Saxe (1989) showed that exposure of young Norway spruce, previously grown in a protected environment, to cold outdoor winter conditions, made the photosynthesis of the young trees less sensitive to acute SO2 exposures. This could be explained by either adaptation and/or natural selection, since only half of the spruce trees survived the severe winter. Brenninger and Tranquillini (1983) found that acute SO;! exposures depress the relative photosynthesis in several forest trees more during summer than during winter conditions. Atkinson et al. (1988b) expanded earlier work on the desert shrubs Diplacus aurantiacus and Heteromeles arbutifolia (Table I) (Winner and Mooney, 1980a,b) to show that irrigation increased stomatal conductance and, thereby, the capacity to absorb SO;!,and the ability of this to inhibit photosynthesis (and transpiration). This is analogous to the responses of another shrub (Larrea) studied by Olszyk et al. (1987). Taylor et al. (1986) suggested that different SO2 susceptibility resulting from ecotypic differentiation was caused by different sensitivity of photo-

PHOTOSYNTHESIS AND STOMATAL RESPONSES

21

60.r( v)

5

5

2 s

high

111

sa 40Y

u c

.”g Y

;E;

3 a

20-

Y

.p

loo

0

00

-

‘0.0

0 0

0

OJ

0

012

d3

d4

65

oh

synthesis and different “repair” rates. At least in the case of the studied Geranium ecotypes, the plants had become genetically different through “environmental pressure”. Reversibility of photosynthesis and visible symptoms. The decline of photosynthesis in short-term exposures was generally not related to visible scorching (i.e. loss of photosynthetic tissue) at the lowest or medium concentrations. Even after several hours of exposure to several hundred parts per billion SOz, recovery of low or moderately affected photosynthesis typically began instantly (e.g. Bennett and Hill, 1973;Sisson etal., 1981; Darrall, 1986), and was complete within relatively few hours. The recovery sometimes performed an overshoot (Muller et a f . , 1979). After a severe inhibition (i.e. more than 25-30%), it often took longer for photosynthesis to recover (e.g. Bennett and Hill, 1973).

22

H. SAXE

2. Response of diffusive resistance to short-term SO2 exposure Typical responses. As for the photosynthetic response, the stomatal response to SO2was often particularly sensitive in plants grown in protected environments (e.g. Unsworth et al., 1972; Biscoe et al., 1973; L'Hirondelle and Addison, 1985). Frequently, the response to low doses was initial opening (Majernik and Mansfield, 1971, 1972; Caput and Belot, 1978; Furukawa et al., 1979b; Biggs and Davis, 1980; Winner and Mooney, 1980c; Takemoto and Noble, 1982; Natori and Totsuka, 1984a). However at 500600ppb h and with exposures of 2 h or longer, the stomata typically closed, whatever the reaction at lower doses, although there were a few exceptions to this rule (Majernik and Mansfield, 1971). The results obtained by Furukawa et al. (1979a) illustrate the large variability in the stomatal response to acute SO2 exposure (Fig. 1). Fumigations with 2ppm SO2 induced the rapid decline of the transpiration rate of tomato, rice and peanut. In other plant species, the transpiration gradually decreased with or without an initial increase. Species with a rapid decline in the transpiration rate were generally more tolerant to SO2, estimated as percentage leaf necrosis, than species with a gradual decrease. However, the Ginkgo plant was an exception; in this case an increased transpiration rate prevailed for about 3 h with no visible injury. This proved once more that stomatal avoidance of SO2 uptake is not the only mechanism by which a plant may protect itself against SO2 injury. Biscoe et al. (1973) found short-term exposure of Viciafaba to 22-540 ppb SO2 to open stomata and, although stomatal resistance generally increased with leaf age, the opening response to SO2was larger and more rapid in the older leaves. A brief review of the response of diffusive resistance to SO2 (and 0 3 was ) recently given by Winner et al. (1988). Response mechanisms. Short-term exposures to SO2 may cause stomata to close (or open) both in response to direct effects on their function, and indirectly in response to the described inhibiting or stimulating effects of SO2 on photosynthesis (Fig. 3) and other factors with a secondary influence on stomata. The opening of stomata in Vicia fuba by low SO2 concentrations was suggested to be caused by structural injury to surrounding epidermal cells before injury to the guard cells themselves (Black and Black, 1979); once guard cells were also injured, stomatal closure followed. Adaxial stomata were more sensitive than abaxial stomata. In addition, Bonte and Cormis (1977) claimed acute SO2 concentrations to have a direct effect on the stomatal metabolism in Pelargonium, since the stomates did not react to 2ppm SO2 under anaerobic conditions, either in darkness or in the light, when photosynthesis would be expected to be very

PHOTOSYNTHESIS AND STOMATAL RESPONSES

23

C 0 2 conc. Fig. 3 . The intercellular C 0 2concentration constitutes a simple link between the responses of photosynthesis and stornatal opening and closing as affected by gaseous pollutants.

effective. Sulphur dioxide must, they concluded, affect the energy-requiring (opening and) closing mechanism directly. Influence of the environment. Barton et al. (1980) found the stornatal reaction in Phaseolus to acute SO2 exposures to depend on the relative humidity as indicated in Table I. Bonte and Louget (1975) found Pelargonium plants to be more injured in humid air, since this slowed the stornatal closing response to S02. Rist and Davis (1979) found stornatal conductance to be relatively more inhibited at 21°C than at 32"C, and relatively more inhibited at 80% R H than at 60% RH. However, the absolute stornatal conductance was greater at both the high humidity and the high temperature, both with or without SO*. Furthermore, since it is this absolute conductance that determines uptake, it is not surprising that leaf injury was far more severe at both high temperatures and at high relative humidities. Black and Unsworth (1980) demonstrated that the effect on stornatal conductance of 35 ppb SO2 depended on both the species and on the vapour pressure deficit (measured in kilopascal (kPa) which translates into RH%). During 27 h of continuous 17.5 ppb or 87.5 ppb SO2 exposures, stornatal conductance increased within 1 h, and then remained relatively stable throughout the exposure period (except for night closure); recovery of normal (lower) stornatal opening was not complete even a day and a half later. Majernik and Mansfield (1972) found that both light and C02 affect the stomatal reaction in Vicia faba to SOZ. At low light, 700ppb SO2 stimulated opening as much as a doubling of the light intensity (Table I); at low C02 concentrations, SO2 stimulated opening of stomata, while at high COz concentrations it tended to close stomata. While all these studies demonstrated the influence of climate on stornatal responses to SO2 during

24

H. SAXE

SO2 exposure, the results by Hunt and Black (1988) and others demonstrated that the stornatal response to SO2 also depends on the climate prior to exposure. Under optimum environmental conditions, Hunt and Black (1988) found stornatal opening in Vicia faba in response to SO2 concentrations below 400ppb, while closure occurred above this level. However, under low light intensities, this stomatal response was reversed: closure occurred at low SO2 concentrations and opening at higher levels. After 1 week at lo”C, SO2 induced stornatal closure at all concentrations. This stornatal behaviour was reflected in the uptake of SO2, which could explain some of the observed differences in photosynthetic inhibition. However, the environmentally stressed plants appeared to be less visibly responsive to the actual amount of SO2 taken up, indicating that some internal mechanism(s) were also affected by the climate pretreatment. Stomata1 uptake of SO, and plant response. Kimmerer and Kozlowski (1981) found that resistant poplar clones maintained consistently lower daytime diffusive conductance than pollution sensitive clones. The initial uptake was, therefore, higher in sensitive clones, and their stomata were found to close during 8 h of 200 ppb S 0 2 , while stomata in resistant clones did not (Table I). The stomata of the sensitive clones were only closed by SO2 to the level of the resistant clones. This showed that stornatal conductance was important in determining the relative susceptibility of poplar clones to pollution stress. Uptake generally followed stornatal opening. This result is in agreement with several other reports (Bonte et al., 1977; Caput and Belot, 1978; Biggs and Davis, 1980; Amundson and Weinstein, 1981; Natori and Totsuka, 1984b). Amundson and Weinstein (1981) found the more sensitive cultivar of two soybean cultivars to close its stomata less, while Bonte et al. (1977) found that the more sensitive cultivars of Pelargonium were the ones to close stomata slowesr in response to acute SO2 exposures. Caput and Belot (1978) found the absorbed amount of SO2 to be proportional to the inverse of the mean stornatal resistance of exposed pine needles, and that the extent of visible injury was related to the quantity absorbed. Atkinson et al. (1988b), however, demonstrated for two shrubs that this may not always be the case; exposure to 200ppb or 600ppb SO2 could result in similar SO2 flux rates, emphasizing that plant responses to SO;?and other pollutants should be expressed not only in terms of external dose, but should also include the potential of the plants for intake of gaseous air pollutants. However, as indicated previously even stornatal uptake does not alone determine the plant response (Alscher et al., 1987; Kropff, 1987; Hunt and Black, 1988). It thus becomes evident that the difference in response of the species and cultivars listed in Table I was partly due to a difference in effective dose (the SO2 uptake) and partly to how the plants were able to “handle” the received dose.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

25

In the section on photosynthesis response mechanisms, the mesophyll resistance (to water vapour) was sometimes quoted to be higher than the stomatal resistance, and one o r the other could be the most affected by SO2 exposures, and the major determinant of SO2 flux. Olszyk and Tingey (1985a) studied the relation between stomatal and residual conductances to water vapour and SO2 in several plants, and their results support the conclusion that stomatal conductance was the major determinant of SO2 intake in Pisum, Geranium and Diplacus. Water vapour flux was confirmed generally to predict the SO2flux. In a tomato mutant, however, Olszyk and Tingey (1985a) found a “positive” residual conductance for S02. O n the whole, therefore, the factors inside leaves must be concluded potentially to play a significant role in determining the SO2 flux. Sulphur dioxide molecules experience less diffusive resistance than effluXing water molecules, because of the high water solubility of the pollutant and its unique chemical reactivity in solution (Taylor and Tingey, 1983). A carrier-mediated anion transport may contribute to the uptake of sulphur at physiological pH values, which further increases the conductance to SO2 uptake (Pfanz et al., 1987a). The carrier mechanism was found to be particularly large for transfer across the chloroplast envelope. When the divalent sulphite anion is exchanged across the chloroplast envelope, bisulphite formation results in proton uptake in the chloroplast stroma, whereas SO2 uptake into chloroplasts lowers stroma pH. Neither transpiration nor leaf or stomatal resistance or conductance, however, were meticulous expressions of the degree of stomatal opening. Stomata1 opening is best measured with a combination of microscopic techniques (Saxe, 1979). Omasa etal. (1985) found that the good correlation between stomatal conductance and the width of stomatal pores only existed until visible injury appeared. Stomata in injured inverveinal areas of sunflower leaves closed more slowly due to water soaking of the tissues, and stomatal conductance was significantly reduced relative to the size of the pores. Studies of the stomatal mechanism in leaves that become visibly injured by air pollutants are, therefore, not possible using simple porometry . 3. Respiration response to short-term SO2 exposure There have been only afew studies on respiration responses to air pollutants since Black (1984) reviewed the rather sparse literature. Acute SO2 may have no effect (Furukawa et al., 1979b, 1980; Takemoto and Noble, 1982) or cause increased dark respiration (Kropff, 1987; Hunt and Black, 1988; Saxe, 1989), and an inhibited photorespiration (Furukawa et al., 1980). Takemoto and Noble found neither inhibited photorespiration nor stimulated dark respiration to explain the observed decline of photosynthesis in soybean. The stimulated dark respiration was generally believed to reflect an increased “repair” rate.

26

H. SAXE

Hunt and Black (1988) found that dark respiration of Vicia faba was enhanced in response to SO2 under optimum environmental conditions, irrespective of the SO2concentration supplied (0-600 ppb). This respiratory stimulation did not occur, however, when plants were pretreated with low light or cold temperatures. Respiratory responses, therefore, could not explain the difference in photosynthetic inhibition in the low light and the cold treatments (Fig. 2 ) . Katainen et al. (1987), on the other hand, found SO2stimulation of dark respiration to decrease with increasing quantum flux density. 4. Summary of the responses to short-term SO2 exposure Both photosynthesis and the stomatal mechanism may be inhibited by SO2 in direct as well as indirect ways. There are several methods of evaluating stomatal versus non-stomata1 limitations to carbon assimilation (Assmann, 1988). Stomata are frequently found to open at low SO2 doses, while this condition only rarely stimulates photosynthesis. At higher doses, photosynthesis and stomatal conductance decline in a coordinated way. When SO2 was discontinued, however, photosynthesis typically recovered faster than the stomata. All studies agree that a closing of stomata protects plants against further uptake of injurious S02. Under these circumstances, however, the residual resistance is sometimes found to be larger than the stomatal resistance, thereby diminishing the relative importance of protection by stomatal closure. Several candidates for the most important factor in the SO2 affected residual resistance have been suggested. The mechanism most often proposed is inhibition of photosynthesis by sulphite, by acidification or by an effect involving the function of RuBPC. But, of course, with sufficiently high SO2 concentrations, any function in a plant will be affected. Only results of long-term, low-level SO2 exposures will reveal which of these mechanisms are of real importance for most plant environments.

C . RESPONSE TO LONG-TERM SO2 EXPOSURE

Two-thirds of the long-term SO2 exposures involving physiological measurements (Table I) were carried out in the protected environment of climate chambers or greenhouses, while only one-third were carried out in the field. In addition to this and to previously mentioned reasons for conflicting results from SO2 exposures, some of the long-term studies listed in Table I applied continuous exposures, while in others exposures were given only in certain daylight hours and sometimes only on certain week days. Where important, this is detailed in the text, rather than in the table. Furthermore, the concentrations given were sometimes averages of stable

PHOTOSYNTHESIS AND STOMATAL RESPONSES

27

levels during exposure, at other times of considerably fluctuating concentrations. Frequently seedlings rather than mature plants were used for exposures, which makes it difficult to interpret the results with respect to mature field crops and forest trees. The studies quoted in Table I, however, represent current (1989) knowledge regarding realistic evaluations of the effects of SO2 on photosynthesis, respiration and stomata. 1. Photosynthesis response to long-term SO2 exposure

Typical responses. Except for the lowest SO;! doses, photosynthesis typically declined gradually from day to day (with a fast decline the first few hours (Saxe, 1983)), with no visible leaf necrosis, and complete reversibility at the lower concentrations and durations (Hallgren and Gezelius, 1982; Rao et a f . , 1983; Saxe, 1983). Visible injury and obvious irreversibility occurred with the higher external SO2 doses. But even after visible injury had occurred, one component of the photosynthesis inhibition was stiff reversible (Fig. 4) (Saxe, 1983). The results reported by Cowling and Koziol (1978) are atypical, in that exposure of Lolium perenne for nearly 1 month to 150ppb SO2 induced visible injury, without significant effects on photosynthesis, transpiration or respiration. Some authors (e.g. L’Hirondelle et al., 1986) found that inhibition of photosynthesis by low levels of SO2 was stronger in the first week(s) than in later weeks and months, probably reflecting the fact that stomata often opened in the beginning and closed later in such exposures. Photosynthesis was sometimes stimulated early in the exposure. Atkinson et al. (1988a) found a stimulated photosynthesis after 9 and 25 days, but not after 37 days after germination of radish exposed to 200 ppb SO2 for 4 h per day, 5 days per week. At very low SO2concentrations (11 ppb), Katainen et a f . (1987) found the photosynthesis of 2-year-old Scots pine seedlings to be stimulated for at least 1 month, while higher concentrations caused a stimulation that lasted a shorter time (34ppb SO2, 10 days); at 79ppb SO2 and above, a decline was observed from the beginning. The severity of the decline increased with exposure concentrations up to 120ppb; at 150 ppb SO;?,however, the decline in pine seedling photosynthesis was less than at 34ppb (Table I). At comparable low SO2concentrations (30 and 63 ppb) for even longer exposure periods (up to 166 days) in open-top chambers, Murray (1985) found both concentrations to stimulate photosynthesis in alfalfa, except after months of exposure to the highest concentration; then the photosynthesis stimulation turned to a significant decline. Keller (1978) demonstrated for two trees (Table I) exposed in field chambers that both 50 and 100 ppb SO2 could lead to initial stimulation of photosynthesis, sometimes lasting for several weeks (Picea excelsa). Like Katainen et al., Keller (1978) showed that a higher SO2 concentration or exposure duration did not

28

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always lead to an increased inhibition of photosynthesis, though this was indeed the case in most other studies. A stimulation in photosynthesis was also found in a resistant Geranium ecotype exposed to 450ppb SO2 for 4 weeks (four 6 h exposures per week), while photosynthesis in a sensitive ecotype declined (Table I) (Taylor et al., 1986).

Response mechanisms. Hallgren and Gezelius (1982) reported an irreversible inhibition of RuBPC in pine tree seedlings after a 5-day exposure to 75-150 ppb S02, while inhibition of photosynthesis was more severe, but reversible. Clearly, the effect on RuBPC does not in this case explain all of the observed inhibition of photosynthesis. Gezelius and Hallgren (1980) had earlier concluded, however, that it takes high concentrations of S0s2to inhibit RuBPC. EXP. I

EXP. II

ref.

60

0

w)

20

30

400

10

DAYS

(AFTER

M 50 40 SEED HYDRATIOW)

Fig. 4. Long-term effects of 100ppb and 350ppb SO2 on the photosynthesis and transpiration of Phaseolus vulgaris (modified after Saxe (1983).)

PHOTOSYNTHESIS AND STOMATAL RESPONSES

29

The fact that SO2 had no effect on photosynthesis in pine seedlings at low quantum flux densities (Hallgren and Gezelius, 1982) shows that the SO2 effect is not directly connected with the primary photoreactions (i.e. the electron transport and photophosphorylation). Atkinson and Winner (1987) and Mooney et al. (1988) concluded the same, since a reduction in photosynthesis activity of fumigated relative to non-fumigated radish plants was not associated with differences in quantum yields. The principal effect of SO2 was on the leaf carboxylating capacity. The transient nature of the 25% photosynthesis depression reported for radish by Atkinson and Winner (1987) was interpreted as a reduced RuBPC activity rather than a change in amount, since the turnover rate of leaf enzymes was known to be about 10% per day. In a subsequent study, Atkinson et al. (1988a) reported that frequent fumigations with small concentrations of SO2 did not affect photosynthesis, although the carboxylation efficiency declined. The latter decline was thought to be offset by increases in RuBPC regeneration capacity. As indicated in Table I, stornatal closure did not explain the inhibition of photosynthesis observed in pine seedlings after 5 days exposure to 75150ppb SO2 (Hallgren and Gezelius, 1982). Declines in photosynthesis may thus be due to biochemical factors although biochemical responses to SO2 do not necessarily affect net photosynthesis.

Znjluence of the environment. Hallgren and Gezelius (1982) found that the inhibition of the photosynthesis of pine tree seedlings in climate chambers by 75-150ppb SO2 for 5 days depended on light; only at absorbed quantum flux densities above ca. 60 pmol m-* s-l did 75 ppb SO2 inhibit photosynthesis. Hallgren and Gezelius (1982) also exposed branches of pine trees in the field (using a small plexiglass cuvette), and found a similar inhibition and dependence on light. It was not clear, however, to what extent the inhibition of photosynthesis in the field was influenced by a chamber effect. Data obtained by Farrar et al. (1977) for field grown Pinus sylvestris suggested that it took quantum flux densities of above 400 pmol m-2 s -1 to revert a 60 ppb SO2 stimulation of photosynthesis to a decline. This dependence on light found in both studies is in accordance with the previously quoted effects of short-term exposures to S02. Carlson (1979), however, reported that the photosynthesis of white ash and sugar maple was inhibited by 1-7 days of SO2 exposure, least at intermittent light intensities, and most at low and high light intensities; but the applied SO2 concentration was very high (500ppb), which may have caused significant stornatal closure. The inhibition of photosynthesis at low light could be explained by a low “detoxification” rate, while the inhibition at high light levels could happen if light stimulation of the “detoxification processes” had less effect than the increased SO2 uptake caused by the light induced stornatal opening.

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H. SAXE

Jones and Mansfield (1982) found the growth of Timothy grass to be more inhibited by SO2 at high light levels and high temperature, and suggested that plants with a high growth rate are generally more sensitive to SO;!. Keller (1978) found that Abies alba and Picea excelsa generally react more strongly to long-term SO2 exposures (5&100ppb in field chambers) in the spring than in the summer and the autumn. Abies alba, however, was found to be nearly as sensitive in the autumn as in the spring. Data obtained by Houpis and Helms (1985) on long-term SO2 exposures of Pinus ponderosa (75 ppb in field chambers), on the other hand, indicate a large photosynthesis decline during the winter and a recovery during the spring. The seasonal dependence in the pollutant sensitivity of trees, therefore, differs with the species. Olszyk et al. (1987) found that the effects of SO2 on the photosynthesis and transpiration of a desert shrub depend on water availability in the soil; irrigation was a prerequisite €or injury. Cornic (1987), however, found photosynthesis in Norway spruce to be more sensitive to SO2during drought than after rewatering the soil. He also found greater dehydration of needle tissue in water stressed plants growing in the presence of S 0 2 . The dependence of pollutant sensitivity on water availability evidently varies with the species and the environment. Pierre and Queiroz (1988) reported on an interaction between the effects of 80 ppb SO2and water stress on enzymes and total soluble proteins. If such effects also occur in the field, this could indirectly affect the physiological state of the plant. Murray (1985) found significant 26% and 49% reductions in L-ascorbic acid in lucerne exposed several months to 30 and 63 ppb SO2, respectively, and sustained the hypothesis that this reduced the winter hardiness of the plants.

2. Response of diffusive resistance to long-term SO2 exposure Typical responses. Low concentrations of SO2 typically open stomata, while higher concentrations close stomata (Ashenden, 1979; Hallgren and Gezelius, 1982). As for photosynthesis, the decline in stomatal conductance was found typically to be manifested gradually day by day, and to retain at least part of the ability to recover even after visible injury had appeared (Fig. 4). Keller and Hasler (1986) measured the stomatal reaction at the end of a 6-month (winter + spring) exposure of Norway spruce to 25 ppb SO;! and found a 16% inhibition of transpiration in full light. However, they also found that the prolonged SOz exposure slowed the stomatal closing reaction to darkness, which increased the total transpiration on a daily basis. However, even quite high. SO;!concentrations (Biggs and Davis, 1982) (Table I) have sometimes been found to increase leaf conductance. Such an increase, though, was transient and only seen during intermittent exposures.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

31

Response mechanisms. Long-term SO2 exposures confirm what has already been described for short-term SO2 exposures, and point to only a few other mechanisms. Malate is involved in the regulation of osmotic pressure in guard cells. Rao et al. (1983) found key enzymes in malate synthesis (phosphoenolpyruvate carboxylase, NAD- and NADP-malate dehydrogenase, alanine aminotransferase) to increase in whole pea leaves in response to 3 h or 2 days of 200 or 500 ppb SO2,while the same enzymes were inhibited in the epidermis. However, since only the higher concentration (500 ppb) and longer duration (2 days) inhibited the stomatal conductance, the enzyme effects could not have been of major importance to the stornatal response. Miszalski and Lorenc-Plucinska (1988) demonstrated a K+ efflux from Vicia faba epidermal strips incubated with freshly dissolved SOz. The absence of information on the absorbed sulphur, and changes in stomatal response, and uncertainty about whether the K+ ions came from guard and subsidiary cells, however, preclude conclusions regarding a direct effect of SO2 on stornatal ion transport. Maas et al. (1987) found that only night transpiration was increased by chronic SO2 exposures, indicating that it was mainly the closing mechanism that was injured. Znfluence of the environment. Farrar et al. (1977) found leaf resistance in Pinus sylvestris to decline in response to SO2 particularly in high light. Norby and Kozlowski (1982) found transpiration in birch to be much more reduced by 200ppb SO2 under high than under low humidity. Koziol et al. (1986) found that the stomatal resistance of Lolium perenne was only affected by SO2 under conditions for slow growth (Table I), i.e. low leaf temperature (12°C) and an 8 h photoperiod. Under better conditions (21-25°C leaf temperature and a 9.5-16 h photoperiod) the stomata were not significantly affected by S02. As for the short-term exposures, these influences of the environment reflected various balances between intake of SO2 and resulting toxicity, and the detoxification processes. Stomata1 uptake of SO, and plant response. Pande (1985) screened five cultivars of spring barley and found that the stornatal diffusive resistance was highest in the most SO2 tolerant (measured by growth parameters) cultivar and lowest in the most sensitive cultivar. Pande (1985) did not find other epidermal characteristics, i.e. stomatal density and size, or structure of wax, to correlate with SO2 tolerance. Krizek et al. (1985), on the other hand, found that stomatal and trichome density as well as stomatal conductance are connected with the sensitivity of four Poinsettia cultivars. Klein et al. (1978) found no effects of 17 days of 100ppb SO2 either on the “sensitive” pea or the “resistant” corn. However, the pea took up considerably more SO2 than the corn, partly because of a lower leaf diffusion

32

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resistance in the pea. Though a higher stomatal SO2 uptake was usually associated with higher sensitivity to injury, Koziol et al. (1986) reported that the resistant one of the two cultivars of perennial ryegrass had a lower stomatal resistance when exposed to 50ppb S02. At 0 and 150ppb S02, however, the resistant cultivar had a 10% and22% higher stomatal resistance than the senstive cultivar. Under most conditions the sensitive cultivar was found to take up more sulphur than the resistant cultivar, so that the stomatal reaction in this plant did not work efficiently as an avoidance mechanism even at moderate SO2concentrations. Alternatively, Kozioletal. (1986) suggested that the relative immunity of the resistant ryegrass could be attributed to its peculiar isoenzyme of RuBPC, which shows a greater resistance to inhibition by sulphite (but also a lower basic carboxylation rate). Carlson and Bazzaz (1982) found that 2 weeks’ exposure to 250 ppb SO2 during 4 weeks of normal and elevated C02 reduced the growth of C3 species at the low C02 concentration but not the high, while the growth of C4species was stimulated at low and reduced at high C02 concentrations. They explained their results in terms of stomatal opening and response to SO2 and stomatal uptake. Hallgren et al. (1982) fumigated 15- to 20-year-old Scots pine to test the hypothesis in the field that stomatal opening regulates the uptake of sulphur. They found a significantly lower dry deposition rate than the one calculated based on stomatal conductances for water vapour. However, part of the deviation could be explained by a light-dependent re-emission of reduced sulphur compound(s) from the needles. In addition, Tschanz et al. (1986) found that SO2 could even exert a negative feedback on its own detoxification through the release of reduced sulphur compounds, as SO2 inhibited adenosine 5’-phosphosulphate sulphotransferase in spruce trees.

3. Respiration response to long-term SO2 exposure The absolute inhibition of dark respiration reported by Saxe (1983) clearly followed the reduction of leaf area caused by scorching, beginning after 2 weeks of exposure. Relative to viable leaf area he observed no changes in dark respiration. Most effects on dark respiration and photorespiration were observed with unrealistically high SO2 exposure concentrations (Lorenc-Plucinska, 1982). However, even at low and medium SO2 concentrations (11-159ppb SO2), the dark respiration of Scots pine was found to be stimulated for at least 1 month (Katainen el al., 1987). The stimulation increased linearly from the beginning of exposure, and the effect increased with SO2 exposure up to 120ppb. With 150ppb SO*, however, the stimulation was less than that at 120ppb (Table I). In general, the changes in dark respiration had little effect on the observed inhibitions of photosynthesis by low SO2 levels (Hallgren and Gezelius, 1982).

PHOTOSYNTHESIS AND STOMATAL RESPONSES

33

4. Summary of the responses to long-term SO2 exposure Stimulation of both photosynthesis and stomatal conductance by low external doses of SO2, typically turned to a reduction in photosynthesis and stomatal closure with higher concentrations and longer exposure times (Table I). In several cases, however, long-term SO2 exposures affected neither photosynthesis nor stomata significantly, indicating that SO2 exposures can be tolerated if the level and duration of the exposure is sufficiently low (Farrar et al. 1977; Garsed et al., 1979; Murray, 1985). Broadleaved tree species were found to be more tolerant of prolonged exposures than conifers (Garsed et al., 1979). However, trees live much longer than typical exposure experiments will ever last, so that even the so-called long-term experimental results easily underestimate the potential effects of S02. Furthermore, there are studies showing that very low SO2 doses could indeed affect trees and perennials within existing long-term exposures (Bell et al., 1979; Keller and Hasler, 1986). The two most likely reasons for photosynthesis decline caused by low levels of SO2 (40 ppb) seemed to be the inactivation of RuBPC (Atkinson and Winner, 1987) and stomatal closure (partly a consequence of the former), though little is yet known of what really happens at the very low levels that occur in rural areas. Mesophyll conductance may be greater than stomatal conductance because of the water solubility of SOz, its dissociation, and the carrier mediated removal of its anions from the cellular sinks. It seems likely, however, that more than a few response mechanisms are involved. The plants responded differently to SO2 according to age and genetic characters, exposure strategy, pollutant concentration and dynamics, and biogenic and non-biogenic environmental conditions. It would therefore be deceptive to classify plants according to a general physiological sensitivity, or to pinpoint a single mechanism of injury as the only important factor. All known constitutional and environmental data, however, may be fed into computer models (Luxmore, 1988) in an attempt to reach general and specific conclusions for given plants, conditions and environments. But we are still short of information. Laisk etal. (1988a,b) have recently undertaken a computer analysis of the uptake of SO2 into different cellular compartments of leaves photosynthesizing in a polluted atmosphere. The mechanistic problems which such computer models may solve are, however, outside the scope of this review.

5. Long-term ecological effects of SO2 The longest exposures were not planned experiments, but their effects have been studied in ecological and sometimes even physiological terms along long-lasting pollutant gradients (Ayazloo, 1982; Legge et al. , 1981; Winner

34

H. SAXE

and Mooney, 1985; Preston, 1988). Typically, stomatal closure was confirmed to protect resistant plants, though a faster detoxification also played a role (Ayazloo et al., 1982). An adaptation at the expense of a lowered photosynthesis rate was only partially attributable to increased leaf resistance (Legge et al., 1981). Preston (1988) found 25 years of 90-170ppb SO2 to have decreased stornatal resistance of Salvia, a perennial shrub. The author did not investigate whether this increase was an immediate response to the SO2 present, or if it involved an adaptation to the pollutant. In any case the increased stomatal opening meant increased uptake of pollutants. Though not mentioned by Preston (1988), NO, must also have been present in the emissions from the designated oil refinery. As a result of the pollution, the abundance of perennial shrub species was significantly lower and the abundance of annuals greater on the most polluted sites relative to the sites farthest away from the SO2 (and NO,) source. The influx of annuals and the decrease in the perennial cover resulted in greater species richness and a reduced shrub dominance similar to that observed in early post-fire sage scrub stands in California. Other ecological studies are mentioned in Section V1.C.

IV. RESPONSE OF PHOTOSYNTHESIS AND DIFFUSIVE RESISTANCE TO NO, A.

INTRODUCTION

In this connection NO, will be defined as N O + N 0 2 . Other species of ambient nitrogenous gases that cause direct plant responses, such as gaseous HN03, are not discussed here. Since most anthropogenic SO2 comes from combustion of fossil fuels containing sulphur, while most anthropogenic NO, is produced when air (N2 and 0 2 ) is heated during fuel combustion, it has been easier to abate SO2 than NO, pollution in ambient air, since a cleaner fuel helps to solve the former, but not the latter problem. Nitrogen oxides have, therefore, become quantitatively more important gaseous pollutants than S02. Monthly averages of NO2 over the average rural areas in Europe range from 2 to 15 ppb, with day maxima reaching 30 ppb (Eliassen et a l . , 1988). The NO, concentrations in urban areas and their surroundings measured as daily or hourly averages reached 10-50 times the rural values, while N O often equalled the NO2 levels. Nitrogen is often considered to be the most important growth-limiting factor in forests, so the increased deposition of anthropogenic nitrogen has probably been beneficial in most situations. But, today, there are indications that nitrogen is no longer growth limiting in certain forest areas, e.g. in the south-west of Sweden (Greenfelt et al., 1983). However, direct physiological

PHOTOSYNTHESIS AND STOMATAL RESPONSES

35

responses to NO, concentrations in the ambient air are disputable, except in combination with other pollutants, primarily SO2 (Section V), or at the high concentrations in commercial greenhouses. In commercial greenhouses it has long been common practice to enrich the atmosphere with C 0 2 to improve plant growth (Law and Mansfield, 1982). The annual plant production in European CO2-enriched greenhouses is worth well above f l billion, and the increase due to COz is 2-5%, or maybe more. Carbon dioxide is added either as pure CO2 from pressure tanks, or is produced in situ by combustion of hydrocarbons (propane, methane and petrol). The latter strategy, however, also produces nitric oxide (NO), which is only slowly oxidized to NO2 in the “clean” greenhouse atmosphere (which contains very little dust and ozone). Nitrogen oxides in greenhouses with special C 0 2 burners typically reach average values of 2-500 ppb with 1ppm COz, while maximum values exceed 1ppm NO, with C02 exceeding 2ppm (Saxe, 1987b,c). When the combustion process was used for primary heating in greenhouses, NO, was found to exceed several parts per million with COz exceeding lOppm, which was more than double the guideline value for human health. The concentration of NO was typically 2-4 times higher than the concentration of NO2. All experiments quoted in Table I1 were carried out in the protected environment of a greenhouse or climate chamber. In general, the applied NO, concentrations were higher than the previously quoted SO2 exposures, since plants were typically less sensitive to NO, than to SOz. B . RESPONSE TO SHORT-TERM NO, EXPOSURE

1. Photosynthesis response to short-term NO, exposure Typical responses. The photosynthesis response to short-term, high level NO and/or NO2 was an instant decline that levelled off within a few hours (Hill and Bennett, 1970; Furukawa et ul., 1984b; Caporn, 1989), though it continued to decline for several hours at the lower exposure concentrations (Srivastava et al., 1975a). The threshold level for photosynthesis response to either gas was about 600ppb with a few hours of exposure (Hill and Bennett, 1970) and 100ppb or less with 20 h of exposure (Capron and Mansfield, 1976). After the fumigations were discontinued, some authors found a quick recovery of photosynthesis (Hill and Bennett, 1970), while others found recovery to be slow (Srivastava et al., 1975a). Hill and Bennett (1970) found a linear dose-response in oats and alfalfa photosynthesis with exposures up to 6-8 pprn for both NO and NO2, while Srivastava et al. (1975a) found the response to NOz to be non-linear in Phuseolus bean above 500-1000ppb. It is to be expected, however, that no dose-response plot to a toxic gas could remain linear with any increase in concentration, since 100% inhibition must be reached far sooner than the

TABLE I1 Changes in net photosynthesis (PS), “stomata1opening” (ST) and respiration (R) in short-term ( I day) nitrogen oxides fumigations” Reference

Species and cultivar

Concentration Duration NO or NO2 (PPb)

PS-response ST-response R-responseb (% control) (% control) (% control)

SHORT TERM Capron and Mansfield (1976)

Lycopersicon esculentum (tomato) cv. “Money Maker”

Sinn et al. (1984) Srivastava et al. (1975a)

Potato, cv. “Kennebec” Phuseolus vulgaris cv. “Pure Gold Wax”

Caporn (1989)

Lactucu sativa L. (lettuce) cv. “Ambassador”

Furukawa et al. (1984b) Hill and Bennett (1970)

Helianthus annuus L. cv. “Russian Mammoth” Medicago sativa (lucerne, alfalfa) cv. “Ranger”

100 NO 100 NO2 100NO+ 100 NO2 500NO 500 NO2 500NO+ 500NO2 120-430 NO2 500 NO2 1000 NO2 3000N02 2000NO+ 500 NO2 2000 NO2 4000 NO2 2000 NO 2000 NO2 2000 NO2 2000 NO +

2h 20 h

89 91

20 h 20 h 20h

82 72 68

20h 5h 2h 2h 2h

59

94 71 53

30 min

90

lh lh 2h 2h

90

2h

is

80 92 87

Avena sativa cv. “Park”

Saxe and Mural1 (1989b)

LONG TERM Sabaratnam et al. (1988)

Lorenc-Plucinska (1988)

Bruggink et al. (1988) Saxe (1986a)

Picea abies (11 cv. ’s), Without pre-exposure With NO2 pre-exposure Without NO pre-exposure With NO pre-exposure Glycine max cv. “Williams”, Immediately after 24 h later Immediately after 24 h later Immediately after 24 h later Pinus sylvestris Tolerant Tolerant Tolerant Susceptible Susceptible Susceptible Lycopersicon lycopersicum (tomato) cv. “Abunda” 8 pot-plant species

2h 2h 2h

93 88 76

ca. 5000 NO ca.5000NO ca. 4000 NO2 ca. 3500 NO2

90 min 90 min 90 min 90 min

88 78 75 65

NS (TR) 87 (TR) 85 (TR) 90 (TR)

100NO2

5 days

200 NO2

5 days

500 NO2

5 days

NS NS 118 123 77 50

NS ( R J NS ( R J NS (Rs) NS (R) NS (Rs) NS (Rs)

500 NO2 1000NO2 2000 NO2 500 NO2 1000NO2 2000 NO2 1000 NO

6 days 6 days 6 days 6 days 6 days 6 days 3 days

NS 52 39 48 39 30 88

79 (Rs)

1000NO 1000NO 1000NO2

5 days 4 days 4h

62 79 NS

NS (Rs) NS (TR) NS (TR)

2000 NO 2000NO2 2000 NO + 2000 NO2

For the meaning of abbreviations and symbols see footnote a to Table I.

-

-

-

-

66 (DR) NS (DR) NS (DR) 144 (DR) NS (DR) NS (DR) 61/156 (DWPR) NSi139 (DRPR) NSRVS (DWPR) 41/125 (DRPR) NSRVS (DRPR) NS/122 (DRPR)

-

NS (DR) 108 (DR)

38

H. SAXE

gas reaches really high concentrations; otherwise, we should not consider the gas to be toxic to the plants. The response to NO + NO2 was usually found to be simple additive (Hill and Bennett, 1970), at least at the lower concentrations (Capron and Mansfield, 1976). Saxe and Murali (1989b) indicated that pre-exposure to high concentrations of NO or NO2 increased the photosynthesis response to the other gas. The response of Picea abies (Saxe and Murali, 1989b) was much less sensitive than the response of pot plants (Saxe, 1986a) to both NO and NO2. Nitrogen oxide was by far the most toxic gas to pot plants, while NO;! was by far the most toxic to trees. The difference in sensitivity was not explained, but could have been related to different levels of nitrate and nitrite reductase in the leaves of the two groups of plants, i.e. the detoxifying mechanism. Response mechanisms. In a recent paper, Caporn (1989) maintained that the response mechanism of photosynthesis to NO (+ NO2) was not yet understood, but that it involves a direct effect on the photosynthetic machinery and was not a result of stomatal closure (although this also sometimes occurred). Influence of the environment. Srivastava et al. (1975b) found NO2 to inhibit photosynthesis in Phaseolus, the greatest inhibition occurring at high irradiance, optimum temperature and high humidities. 2.

Response of diffusive resistance to short-term NO, exposure

Typical responses. All the studies inTable I1 report small or insignificant responses of stomata to short-term NO, exposures. In all cases the stomatal responses were found to be much less sensitive to NO, than the photosynthesis and respiration responses (Furukawa et al., 1984b; Srivastava et al., 1975a). Caporn (1989) found that stomatal opening in lettuce sometimes declined in parallel with photosynthesis when exposed to acute NO, but emphasized that at other times stomatal aperture was unaffected by the pollutant. Stomatal closure did not seem to reduce the concentration of C02 in the intercellular spaces of lettuce leaves. Stomatal uptake of NO, and infuence of the environment. Uptake of NO2 was found generally to follow the stomatal opening and was, therefore, much higher during the day than during the night (Srivastava et al., 1975a; Kaji et al. 1980). Over a wide concentration range (1-7ppm), NO2 uptake by Phaseolus vulgaris increased linearly with concentration, but decreased with time (Srivastava et al., 1975a). Rogers and Campbell (1979) found the specific rate of uptake in corn, soybean, loblolly pine and white oak to be unaffected by exposure concentrations in the tested range of &580 ppb NO2. Sinn et al. (1984), however, found a decrease in specific uptake of NO;! in potato in the tested range of 120-430ppb NO2. Stomatal uptake of NO2

PHOTOSYNTHESIS AND STOMATAL RESPONSES

39

was enhanced by light, caused by a reduction in the total diffusive leaf resistance (Rogers and Campbell, 1979). Murray (1984a), however, found that light affected the deposition of NO2 on the Flacca mutant of tomato (which keep stomata open in darkness) without affecting the rate of transpiration. He suggested, that a light-stimulated ion transport into the photosynthetic active tissue was operating (as mentioned for SO2 by Pfanz et al. (1987a)). Srivastava et al. (1975b) found no influence of photon flux on NO2 uptake in Phaseolus, but the stomatal.uptake of NO2 in this species was enhanced by high temperature, low C 0 2 concentration and high humidity. The relative inhibition of transpiration in Phuseolus by NO2 was increased by increasing temperature, although the absolute transpiration increased (as did NO2 uptake). Kaji et al. (1980) reported that 99% of absorbed NO2 nitrogen after 20 min exposure of sunflower leaves to 6 ppm NO2 had been transformed into reduced, organic nitrogen compounds with only 1 % remaining as nitrate and nitrite in the daytime, while at night 85-89% was reduced to organic forms and the rest was nitrate and nitrite. NO2 was incorporated into the plant via nitrate, nitrite and the glutamine and glutamate synthase system.

3. Respiration response to short-term NO, exposure Srivastava et al. (1975a) found dark respiration to be more inhibited by 1-3 ppm NO2 than photosynthesis. Inhibition increased with temperature (Srivastava et a!., 1975b). Complete recovery was slow. Srivastava et al. (1975b) found no evidence of a photorespiration response to N02. 4. Summary of the responses to short-term NO, exposure Photosynthesis was more sensitive to NO and NO2 than was the stomatal response (Furukawa et al., 1984b; Srivastava et al., 1975a). The stomatal response to short-term NO, exposures was much less important than with SO2 or O3 at ambient pollutant levels. Dark respiration was inhibited, but photorespiration was unaffected. C . RESPONSE TO LONG-TERM NO, EXPOSURE

I. Photosynthesis response to long-term NO, exposure Typical responses. Sabaratnam et al. (1988) found a stimulation of photosynthesis in soybean plants at the lower NO2 concentrations tested for 5 days (100,200 and 300ppb, but only significant with 200 ppb). Concentrations of 3 500 ppb NO2 inhibited photosynthesis. Sabaratnam et al. (1988) found that the effects on photosynthesis were increased rather than reversed 24 h after the exposure was discontinued, while Lorenc-Plucinska (1988) found a general recovery after 24h and 48h, sometimes even with an overshoot. The difference between the two reports may be due to reductions

40

H. SAXE

in chlorophyll content reported by the former workers (Sabaratnam and Gupta, 1988), the beneficial effects of the increased nitrogen content in leaves and the improved leaf area ratio (cm2 g-’) only partly compensating for the reduced chlorophyll. Though Caporn (1989) found an immediate response (within minutes) of lettuce photosynthesis to high levels of NO (+ NO2) in the presence of C 0 2 (enrichment), and Saxe (1988) found a long-term decline in yield, there were no long-term effects of NO, on the photosynthetic capacity in lettuce grown in a C02-enriched NO,-contaminated greenhouse atmosphere. Photosynthesis was only inhibited during the transient periods of NO, (i.e. when intermittent enrichments took place). This is in accordance with the results obtained by Saxe (1986a), who found that the photosynthesis of eight species and cultivars of pot plants was inhibited as much on the first day as on the following three, when exposed to l p p m NO or NO2, but with full recovery on the day that exposure was discontinued. Bruggink et al. (1988) found that photosynthesis did not recover during the night after daytime NO exposures, but they did not test whether a period in light would help recovery of photosynthesis. Saxe (1986a) found only one of eight pot plants to be inhibited by a 4-day exposure to 1pprn NO;?, while seven plants were inhibited by NO.

Response mechanisms. Since stomatal closure does not seem to be responsible for the inhibitory or stimulatory effects of NO and NOz on photosynthesis, there must be more direct effects. Saxe et al. (1989) found photosynthesis in cereals to be inhibited by lower levels of NOz than it took to inhibit thylakoid electron transport in vitro. In vitro activity of RuBPC was found to increase in plants grown in a NO, contaminated C02-enriched atmosphere relative to a “clean” C 0 2 atmosphere (Besford and Hand, 1989).

2. Response of diffusive resistance to long-term NO, exposure Typical responses. As with short-term exposures, NO, effects on stomata were found to be small or insignificant. Sabaratnam et al. (1988) found no effects on stomatal diffusive resistance immediately after 5 days (of 7 h each) exposure to 100-500ppb NO2, or 24h later. Bruggink et al. (1988) found a significant inhibition of stomatal diffusive resistance after 1 , 2 and 3 days of 1OOOppb NO exposure, but not after 4 and 5 days. The nonsignificant trends in both experiments, however, indicate an indirect effect on stomatal closure through effects by NO, on photosynthesis. Okano and Totsuka (1986) found no effects by 2 weeks of 1ppm NO2 on either stomatal or mesophyll resistance in sunflower. Saxe (1986a) found a 4 1 4 % significant decline in three of eight pot plants in response to 4 days of 1ppm NO exposure, and an 8% decline in response to a similar external dose of NO2 in only one of the pot plants. The relative

PHOTOSYNTHESIS AND STOMATAL RESPONSES

41

effects on transpiration, however, were always far smaller than on photosynthesis. On average (of all pot plants), neither gas had any significant effect on transpiration during the 4-day exposure (Table 11). Stomata1 NO, uptake. Saxe (1986b) found NO2 uptake in eight pot plant species or cultivars to correlate with transpiration, while total NO uptake showed no significant dependence on stomata1 opening. Nitrogen oxide was taken up at a constant rate throughout the light period, while the uptake of NO2 decreased towards the end of the day in the same manner as transpiration. Uptake of NO in the dark was as high as in the light, while uptake of NO;! in the dark was reduced by as much as transpiration. Saxe (1986b) suggested that a much smaller proportion of NO than of NOz was taken up by the leaf through stomata, but that it was this “effective” uptake of NO through stomata that affected photosynthesis. Saxe (unpublished data) found neither 1% NO nor 1% NO2 to penetrate isolated cuticles of pot plants. Yoneyama et al. (1980a) found that 2 weeks exposure of several plants increased plant nitrogen by 1-3% with 40 ppb NO2 and &23% with 300 ppb NO2. In the short term, leaf uptake was most important, while in the long term uptake of nitrogen from soil-absorbed gaseous NO2 also became important (Yoneyama et al., 1980b). In contrast to the results obtained with S02, but in accordance with the results of short-term NO, exposures (Srivastava et al., 1975a; Rogers and Campbell, 1979), Okano et al. (1986), Rogers et al. (1979) and Saxe and Murali (1989b) found the specific uptake of NO2 to be unaffected by concentration. This was confirmed for both NO and NO2 (Scots pine, (Skarby et al., 1981)). The specific uptake did not decline with time (as for SO;?),but was constant for 2 weeks, after which it began to rise, probably as a result of uptake via the air-soil-root pathway. Okano and Totsuka (1986) found that a lowered root nitrate supply inhibited the absorption of NO2 in Helianthus leaves exposed to 300ppb NO2. Rowland etal. (1987), on the other hand, found that 300 ppb NO2 only caused significant increases in the nitrogen content of barley leaves when the root nitrate supply was low. In plants that increase their leaf nitrogen when exposed to NO2, the input of nitrate and nitrite ions from the dissociation of NO;! in the extracellular water may initially cause an increase in the nitrate pool in the polluted leaves, which then stimulate the induction of extra nitrate and nitrite reductase activities (Rowland et al., 1987). Eventually the anions of NO2 are turned into reduced nitrogenous compounds, which accounts for the large increases in plant nitrogen content and growth (Rowland, 1986). Only one recent study has dealt with NO, uptake with exposure concentrations relevant to those in rural air remote from pollutant sources (Sweden), i.e. 1-5ppb NO and NO2 (Johansson, 1987). It was found that

42

H. SAXE

the uptake of NO2 became essentially zero at these low concentrations. The primary consequence of this is that more NO, is left in the atmosphere and turned into HN03, which may be more damaging to the plants and ecosystems than NO,, either by dry deposition or through rain-out and acidification.

3. Respiration response to long-term NO, exposure Sabaratnam et al. (1988) found dark respiration in soybean plants to decrease with 5 days of lOOppb NO2, and increase with higher levels (though only significantly at 300ppb). Lorenc-Plucinska (1988) found 6 days of 500 ppb NO2 decreased dark respiration, while higher concentrations had no significant effect. Saxe (1986a) found dark respiration to be inhibited by 4 days of 1ppm NO in only one of eight pot plants, while NO2 stimulated dark respiration in two of the eight pot plants. The average effect on dark respiration of the pot plants was not significant for NO with a small stimulation by NO2 (Table 11) (Saxe, 1986a). Lorenc-Plucinska (1988) found a general stimulation of photorespiration when tolerant or susceptible Scots pine seedlings were exposed to 5002000ppb NO2. One and two days after the exposure was discontinued, however, the stimulation in photorespiration in all treatments reversed to a decline.

4. Summary of the responses to long-term NO, exposure Photosynthesis is generally very responsive to NO, although it takes higher concentrations than with SO2 and 0 3 to elicit comparable effects. Recovery is quicker and more consistent than with the other pollutants and, with few exceptions, there is less visible injury. There is only one example of stimulation of photosynthesis by low NO2 concentrations. It is not yet known how NO and NO2 inhibit photosynthesis in vivo, although the incorporation of NO, in the general metabolic pathways in the plant must diminish the pool of reducing equivalents. Unlike SO2 and 03,NO, has little influence on the diffusive resistance (discussed further in Section V). In long-term fumigation experiments with NO, and COz, the plants always thrive due to the high C02, but growth, photosynthesis and stomata1 opening decline relative to “clean” C 0 2 . The decline is most often without visible symptoms (Saxe and Christensen, 1985). Dark respiration is only slightly affected by NO,. Sometimes it is initially inhibited, but is always eventually stimulated. Photorespiration is initially stimulated, but eventually inhibited.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

43

V. RESPONSE OF PHOTOSYNTHESIS AND DIFFUSIVE RESISTANCE TO SO2 + NO2 A. INTRODUCTION

Sulphur dioxide and NO, normally occur together in polluted air. Responses to the individual gases (see Sections I11 and IV) are, therefore, more of theoretical or mechanistic interest. Mansfield et al. (1987) and Mansfield and McCune (1988) have reviewed the voluminous literature on the yield and growth responses of plants exposed to SO2 and N 0 2 , and a major conclusion was that dry deposition of ambient levels of SO2 + NO2 in rural areas in industrialized countries was likely to produce growth stimulation in some circumstances, and inhibition in others. Initial growth stimulation could turn to long-term deleterious effects. Exposures to the combined gases could, for example, increase sensitivity to frost. B. RESPONSE TO SHORT-TERM SO2 + NO2 EXPOSURE

There are relatively few short- or long-term studies that have analysed the effects of combined SO2 and NO2 on photosynthesis and stomata1 response compared with the number of studies of the physiological effects of the individual gases. Even though responses to gas combinations rather than individual gases are more realistic, all of the quoted studies on SO2 + NO2 were carried out in greenhouses or climate chambers, rather than under field conditions. The combined exposure to the two pollutants may be either simultaneous or sequential. Only results of the former type are discussed here. In the following the term more-than-additive is used to characterize the effects of combined exposures when the combined gases caused an additive response higher than the sum of responses to the single gases at the same concentrations. Similarly, the term less-than-additive is used to characterize effects of combined exposures when the combined gases caused an additive response less than the sum of responses to the single gases at the same concentrations. When responses to the combined gases were less than the response to one or both of the single gases (at the same concentrations), the term antagonistic is used. 1. Photosynthesis response to short-term SO,

+ NO2 exposure

Typical responses. White et al. (1974) found no response of photosynthesis in alfalfa to 1 h of 250 ppb NO2, and only a 6% inhibition by S02, but their combination induced a 24% reduction in photosynthesis (Table 111). Carlson (1983b) found photosynthesis in soybean to be reduced by 2 h exposure to 2400ppb SO2 2400ppb NO2, but not by up to 2 h of 2-600 ppb NO2 alone, and the reduction was significantly greater with the

+

TABLE 111 Changes in net photosynthesis (PS), “stomata1 opening” (ST) and respiration (R) in short-term (< I day) and long-term (>I day) SO1 + NO1 exposures’ Reference

SHORT TERM Natori and Totsuka (1984a)

Species and cultivar

Euonymus japonica

Concentration Duration of pollutant (PPb) 2h 100 so2 2h 100NO2 100 so2 2h 100NO2 Short term 100so2 Short term 100NO2 100so2 + 100NO2 Short term Short term 250 SO2 Short term 250 NO2 250 SO2 + Short term 250 NO2 lh 250 SO2 250 NO2 + lh NO trace 250 SO2 + 250 NO2 lh (+ NO) 105 min 200 so* 105 min 200 NO2 200 so2 + 105 min 200 NO2 2h 800 so2 2h 280 NO2 + double ambient

+

Bull and Mansfield (1974)

White et al. (1974)

Pisum sativum (pea)

Medicago sativa L. (alfalfa) cv. “Ranger”

Carlson (1983b)d

Glycine max L. (soybean)

Hou et al. (1977)

Medicago sativa L. (alfalfa) cv. “Ranger”

+

co2

PS-response ST-response (% control) (% control)

-

-

NS (TR) NS (TR)

R-responseb (% control)

-

-

76 NS 67 48 76 45 98 NS 91 81 NS 75 45 200

-

-

68 (C:) 96 (C:)

NSNS (DRiPR) 97/93 (DRIPR)

60 (C:)

97/93 (DRIPR)

-

-

Saxe (1989)

Picea abies L. (9 half-sibs)

Amundson and Weinstein (1981)

Glycine max (soybean) cv. “Beeson”. sensitive

LONG TERM Ashenden (1979)

Neighbour et al. (1988) Mansfield et al. (1988)

Kumar (1986)

Phaseolus vulgaris cv. “Canadian Wonder”

Befula pendula (birch) water stressed for 5 days after exposure Phleum prateme well watered for 23 days after exposure water stressed for 23 days after exposure Vigna radiata (Mung-bean)

830 SO2 + 3420 NO2 1950SO2 500 NO2

1950SO2 + 500 NO2

100so2 100NO2 100so2 + 100NO2 100 so2 100NO2 100 so2 + 100NO2 40 SO2 40 NO2

+

60 SO2 + 60 NO2 60s02 + 60 NO2 250 SO2 250 NO2 250 SO2 + 250 NO2

6h 2h 2h

2h

1 day 1 day

-

120 (TR) 124 (TR)

1 day 3 days 3 days

-

NS (W 118 (TR) NS (TR)

3 days

-

77 (TR)

30 days

-

202 (TR) 156 (TR)

40 days

NS

-

40 days 40 days 40 days

75 52 59

-

40 days

48

-

For the meaning of the abbreviations and symbols see footnotes a and b to Table I. Diffusive resistance to COz. Responses of PS, TR and R to intermediate-high concentrations are given in Fig. 5.

46

H. SAXE

NO2 concentration (ppm)

o .2 .4 .6 NO2 conccntrition (ppm)

*

NO2 concentration (ppm)

Fig. 5 . The rate of photosynthesis, stomatal conductance, dark respiration and photorespiration in soybean plants during the fourth half-hour of fumigation with SO2 and/or NO2 at concentrations of 6600ppb. The graphs were calculated from the original data by means of least-squares multiple regression analysis. SE, the pooled standard error of the regression surface. (Adapted from Carlson (1983b).)

combined exposure than with SO2 alone (Fig. 5 ) . There was full recovery within 24 h in clean air, and darkness seemed to promote the recovery. The effects of the quoted combined exposures were thus more-than-additive. The degree of synergism between the effects of SO2 and NO2 on photosynthesis decreased as the exposure concentrations increased (Bull and Mansfield, 1974;White et al., 1974; Bennett et al., 1985), and concentrations at or above several hundred parts per billion of both gases did not elicit more-than-additive responses, but could result in less-than-additive or antagonistic responses (Bull and Mansfield, 1974). Hou et al. (1977) found that 645 ppm C02 together with SO2 NO2 pollutants increased rather than decreased photosynthesis, and this C 0 2 concentration was calculated to occur downwind from a coal-fired power plant using western coal when the SO2 concentration was 1ppm. Pollution in the ambient environment may, therefore, not always lead to the declines projected from laboratory experience with COz levels in clean air (350PPm). Response mechanism. Carlson (1983b) did not find carboxylation resistance in soybean to be sensitive to 2 h of either SO2, NO2 or their combination. The stomatal resistance was less than the residual resistance, and the resistance to C02 transport across mesophyll cell walls accounted for nearly all of the substomatal resistance to C02, and was sensitive to the combination of SO2 and NO2, but not to the individually applied pollutants. The apparent quantum yield was reduced by SO2 and NO2 in an additive fashion. It could, however, only be concluded that photosynthesis was inhibited both by the closing of stomata and by the direct effects on mesophyll metabolism. Several effects on this metabolism are mentioned briefly in Section 1II.C.

+

47

PHOTOSYNTHESIS AND STOMATAL RESPONSES

Bennett et al. (1985) emphasised the importance of the stomata, since stornatal closure in snap bean, soybean and cotton leaves accounted for 69-90% of the photosynthesis suppression, depending upon species and the amount of inhibition. Saxe (1989) found the relative response of photosynthesis in Norway spruce to be significantly larger than the relative response of transpiration, and that photosynthesis could continue to decline while transpiration levelled off. Both observations indicate a direct effect of acute SO2 NO2 exposure on photosynthesis.

+

Interaction mechanisms. Wellburn (1982) described the frequently observed more-than-additive effect in biochemical terms. Nitrogen dioxide stimulated nitrite reductuse activity in Lolium perenne, relative to plants in clean air, in SO;?or in SO2 + N02. The presence of SO2 appeared to destroy the ability of the nitrite reductase to respond to NO2, and this inhibition of a potential detoxification mechanism of nitrite is today believed to be one of the main reasons why the SO2 NO2 combination exhibits more-thunadditive effects upon several plants.

+

2.

Response of diffusive resistance to short-term SO2

+ NO2 exposure

Typical responses. Carlson (1983b) found stornatal conductance in soybean to be reduced by 2 h of 2-600 ppb of either SO2or NOz, but more by their combination (Fig. 5). Natori and Totsuka (1984a) found that neither 100 ppb SO2nor 100ppb NO2 alone affected transpiration in 2 h exposures, while the combined exposure to 100ppb of both decreased transpiration significantly (Table 111). Both of these studies demonstrate the typical, more-than-additive effect on diffusive resistance of combined SO2 NO2 exposures. Amundson and Weinstein (1981) demonstrated for three cultivars of soybean (a sensitive, an intermediate and a resistant cultivar) that exposures to high levels of SO2 + NO2 initially increased diffusive resistances significantly above the increase caused by SO2 alone. Nitrogen dioxide alone had no effect. The plants consequently responded with visible injury after SO2 exposure, while there was no injury after SO2 NO2 exposure, due to a smaller pollutant uptake.

+

+

Znfluence of the environment. Bennett et al. (1985) found that SO2 + NO2 elicited the greatest inhibition of photosynthesis under conditions conducive to a maximum photosynthetic rate, 60% RH and high nitrate fertilization.

3. Respiration response to short-term SO2+ NO2 exposure Dark respiration and apparent photorespiration were reduced by NO2 but not by SO2 (Fig. 5 ) , and stimulated by their combined exposures (Saxe, 1989), but there was no interactive effect of SOz and NO2 (Carlson, 1983b).

48

H. SAXE

4. Summary of the response to short-term SO2 + NO2 exposure The lower concentrations of SO2 and NOx typically induce greater effects on photosynthesis and “stomata” than the gases singly. The effects on photosynthesis are both direct and indirect (through effects on stomata); but the relative importance of these effects varies. However, since C 0 2 generally follows ambient SO2 + NO2 pollution, a stimulation in plant productivity due to the beneficial COZcould occur in the ambient environment rather than a reduction due to the toxic SO2 + NO2 mixture. C. RESPONSE TO LONG-TERM SO2 + NO2 EXPOSURE

+

1. Photosynthesis response to long-term SO2 NO2 exposwe

General response. Kumar (1986) found that both 250ppb SO2 and 250 ppb NO2 alone reduced photosynthesis in Vigna radiata more than their combination for the first 30 days of exposure. After 40 days the combined gas exposure reduced photosynthesis the most (Table 111). In other words, an antagonistic effect turned into a less-than-additive effect with time. The unusually strong response of photosynthesis to NO2 as compared with the response to SO2 was probably caused by the NO2 source, which could also have emitted HN03. Mansfield et al. (1988) found SO2 + NO2 to predispose the photosynthetic activity of Phleum pratense to drought. As explained below, this loss in photosynthetic activity is caused by an excessive loss of water due to pollutant-disturbed stomata. 2. Response of diffusive resistance to long-term SO2 + NO2 exposure

General response and drought. Ashenden (1979) found that a 1-day exposure to 100ppb SO2 or 100 ppb NO2 increased transpiration in Phaseolus (Table 111), while the effects of their combined exposure were nonsignificant. From the second or third day of a 5-day exposure, the combined exposure to lOOppb each of SO2 and NO2 inhibited transpiration, while the stimulatory effects of SO;! and NO2 alone gradually disappeared. FreerSmith (1985) found only two significant effects after 40 and 60 days of 40 ppb SO2 or 40ppb NO2 or their combined exposure; one was an occasional, small but significant inhibition of stomatal conductance under low light, the other a positive interaction between SO2 and the photoenvironment, stimulating stomatal conductance. Addition of NO2 seemed to stimulate stomatal conductance further, but Freer-Smith (1985) did not find this or other SO2 NO2 interactions to be significant. Some recent studies indicate that SO2 + NO2 could predispose the plant to climatic stress via their reduction of the structural and functional integrity of the epidermal cells of the leaf (Neighbour et al., 1988; Mansfield et al., 1988). It was found for both broadleaved trees and grasses that the ability of

+

PHOTOSYNTHESIS AND STOMATAL RESPONSES

49

leaves to conserve water, particularly during periods of water stress, was reduced after exposure to small doses of SO2 NO2 at concentrations as low as 10-20ppb. A similar effect occurred with SO2alone, but the magnitude was much greater when SO2 was accompanied by NO2. This is one of the best examples of SO2 NO2 synergism affecting essential physiological processes at ambient pollutant levels, including the previously quoted decline in photosynthesis (Table 111).

+

+

3. Summary of the response to long-term SO2 + NO2 exposure SO2 + NO2 induce larger effects on photosynthesis and diffusive resistance than predicted by the effects of individual gases. The SO2 NO2 concentrations found in the field are lower than the concentrations applied in most of the quoted studies. Taylor and Bell (1988) found that grasses not only adapt to chronic SO2 + NO2 exposures, but as they become tolerant to this pollution they even acquire a demand for low concentrations of NO2. Among agricultural crops new resistant species are automatically selected every year (Gould and Mansfield, 1989). Selection of resistant perennials and trees, by man or by survival of the fittest, takes several years to become effective. Taylor and Bell, (1988) advocate that plants “get used to the pollutants” and that the applied fumigation concentrations are nearly always so high that the results have no indicative value for what will happen in nature. Not all plants in the quoted studies, however, responded positively or were neutral to long-term, low-level (1s.20 ppb) exposures (Mansfield and McCune, 1988; Neighbour etal., 1988), and such low pollutant levels are not the rule in urban environments. In forests, adaptation and new selection may take decades to become effective and, therefore, do not help the present situation.

+

VI. RESPONSE OF PHOTOSYNTHESIS AND DIFFUSIVE RESISTANCE TO 0 3 A. INTRODUCTION

Anthropogenic tropospheric ozone (03) mainly results from reactions between NO, and volatile hydrocarbons and has nearly doubled in the northern hemisphere during the last 100 years. Ozone is the most important photo-oxidant in relation to plant injuries. It has been estimated that a reduction in O3 pollution to the levels of 50 years ago would benefit the annual worldwide agricultural production by up to US $2.4 billion (Adams and Crocker, 1988). Ninety percent of plant injuries in the USA were estimated to be caused by 03.

50

H. SAXE

and 0 3 uptake ( A ) , respond Fig. 6. Rates of net photosynthesis (O), transpiration (0) very differently to short-term, high level O3 exposures (Furukawa ef al., 1984a). (a) Populus euamericana cv. “FS-51”; (b) Populus euamericana cv. “Peace”; (c) Helianthus annuus L. cv. “Russian Mammoth”.

51

PHOTOSYNTHESIS AND STOMATAL RESPONSES

In Europe the average rural background O3 concentration ranges between 25 and 70 ppb, depending on the time of the year and on latitude (Eliassen et al., 1988). One of the most polluted areas, the Los Angeles Basin in California, experiences 60-80 ppb as daily mean concentrations, and maximum hourly mean concentrations of up to 600 ppb, while concentrations in the relatively northerly UK have been recorded to be as high as 250ppb (as quoted by Walmsley et a!. (1980) and Coyne and Bingham (1982)). It is presumably important for some of the experiments quoted in Table IV, and maybe for the conclusions of Adams and Crocker (1988), that emissions from electric discharge ozonators sometimes used in experimental plant fumigations have recently been found to emit N2OSas well as O3(and HN03). N 2 0 5 may by itself elicit effects on the plants. Brown and Roberts (1988) suggested that the problems could be solved by bubbling the ozonator emission through water or by using pure O2 for the O3 production rather than filtered air. B. RESPONSE TO SHORT-TERM

0 3

EXPOSURE

1. Response of photosynthesis and diffusive resistance to short-term exposure

0 3

Typical responses. The typical responses to short-term 0 3 exposures (Table IV) were an immediate inhibition of photosynthesis and diffusive resistance. Matsushima and Yonemori (1985) found that leaf stomata of ‘mandarin’ orange started closing only 3 min after the beginning of a 1.2 ppm fumigation. Recovery was often “sluggish”, though it sometimes began immediately, at least when there was no visible injury, as was often the case with the lower external doses (Hill and Littlefield, 1969; Pel1 and Brennan, 1973). An initially delayed response to O3 was typically followed by a delayed recovery in clean air. Some species were very sensitive to short-term 0 3 exposure (e.g. oats (Myhre et al., 1988)), while others were not very sensitive to short-term exposures even of high O3 concentrations (e.g. grapevines (Roper and Williams, 1988)). There was, however, a variety of response patterns of photosynthesis and diffusive resistance to short-term exposures of 0 3 (Fig. 6). Furukawa et al. (1984a) described an immediate and synchronized decline of photosynthesis and transpiration in response to 2 h of 550ppb 0 3 in two poplar cultivars (“FS-51” and “I-214”), without immediate recovery when the O3 exposure was discontinued (Table IV, Fig. 6). Photosynthesis in another poplar cultivar (“Peace”) responded to 0 3 with a delay, with no change in transpiration, and continued to decline all the way to 0% in the period after the exposure was discontinued, with only a small decline in

TABLE IV Changes in net photosynthesis (PS), “stomatal opening” (ST) and respiration ( R ) in short-term ( I day) fumigations” Reference

SHORT TERM Myhre et al. (1988)

Forberg et al. (1987)

Barnes et al. (1988)

Species and cultivar

Avena sativa cv. “Titus”, 5-day-old plants 11-day-old plants 16-day-old plants 20-day-old plants 52154-day-old plants 2 h later Another 3 h later Avena sativa (oats) cv. “Titus”, 1h later 1h later Pisum sativum (pea) cv. “Feltham First”

cv. “Conquest” Hill and Littlefield (1969) Faensen-Thiebes (1983)

Saxe and Murali (1989~)

Avena sativa (oats) cv. “Park” 12 other species Nicotiana tabacum L. cv. “Be1 W3”, 3 h later Phaseolus vulgaris L. cv. “Saxa”, 17 h later Picea abies L, average of 11 cvs

Concentration Duration of 0 1 (ppb)

90 90 90 90 135-150

lh lh lh lh 2h

70

2h

140

2h

75 75 75 75 400 600 4-700 250

2h 10h 2h 10h 30 min lh 30-120 min lh

150

2h

64

4h

PS response ST response (% control) (% control)

96 92 92 88 75 67 80 94 87 78 67 -

-

67

64 80-22

NS NS 82 85 NS

0 3

R response (% control)

Dijak and Ormrod (1982)

Pisurn ~arivirrnL. cv. “Nugget”, sensitive cv. “Charger“, insensitive

Furukawa et al. (1984a)

Popirlus eiramericana “FS-51”, 1 h later Populirs eiiuiiiericaiia cv. “Peace”, 1 h later 2 h later Helianthirs atinirirs (sunflower) cv. “Russian Mammoth“

150 150 150 150 550

3h 6h 3h 6h Ih

540

Ih

0 3 .

Pel1 and Brennan (1973) Ross and Nash (1983)

Phasaolus vulgaris cv. “Pinto”, 21 h later Resistant and sensitive lichens:

Ramalina menziesii

Roper and Williams (1988) Botkin et al. (1972) LONG T E R M Skarby et al. (1987)

Pseitdoparmelia carperata Low light High light Vitis vinifera (grapevines) cv. “Thompson Seedless”

Pinus sfrobus (white pine) Pinus sylvestris (Scots pine) Subsequent exposure + Subsequent exposure + Subsequent exposure 12 days later

+

720 720 720 720 720 720 720 300

30 min 45 min 60 min 70 min 85 min 95 min 115 min 3h

-

72 5s 88 56 0 57 75 50 70 45 60 42 78 NS

10G780

12h

NS

100 100 200 400 600 5-800

12h 12h 10h 10h 10h 4h

37 49 NS 68 45 93

60 80 125 200

3 days 5 days 4 days 7 days

NS NS NS NS NS

416 ( R , ) 677 (RI) 295 ( R , ) 328 ( R , ) 65 (TR) 44 (TR) NS (TR) NS (TR) 75 (TR) 50 (TR) 68 (TR) 44 (TR) 68 (TR) 40 (TR) 60 (TR) 40 (TR) -

-

-

-

-

-

117 or NS (DR)

-

136 (DR)

79 71 30 28 67

130 (DR) 140 (DR) 160 (DR) 200 (DR) 300 (DR)

(TR) (TR) (TR) (TR)

(TR)

TABLE IV-contd. Reference

Species and cultivar

Concentration Duration of 0 3 (PPb)

PS response ST response (% control) (% control)

R response (% control)

~~~

Reich et al. (1985)

Glycine may, young leaves 130 cv. “Hodgson”, medium age 130 leaves Old leaves 130 Abies fraseri (Fraser fir) 20 Tseng et al. (1988) Amthor and Cumming (1988) Phaseolus vulgaris cv. “Pinto” 90 (Isb) Amundson et al. (1987) Triticum aestivum (winter wheat) 54 (27b) Ross and Nash (1983) Resistant and sensitive lichens: Ramalina menziesii 100 Psuedoparmelia caperata 100 4 days later Amthor (1988) Phaseolus vulgaris 43 (19’) cv. “Pinto” 80 (19‘) Reich and Amundson (1985) Trifolium repens (clover) cv. “Arlington” 45 (19’) Glycine max (soybean) cv. “Hodgson” 90 (50’) Triticum aestivum (wheat) cv. “Vona” 54 (27’) Acer saccharum (sugar maple) 60 (30b) Pinus strobus (eastern white pine) 100 (29b) Populus hybrid 85 (25’) Oshima et al. (1979) Gossypium hirsutum (cotton), cv. “Alcala SJ-2”, young 245 old 245

2-6 days 7-11 days 14-19 days 15 days

-

69 115 (DR‘)

4-9 days

8 days

77

5 days 5 days

NS 51 NS

-

107 (DR‘) 125 (DR‘)

12-17 days 12-17 days 18 days

83

21 days

83

21 days 35 days

76 90

36 days 40 days

91 75

4 days 4 days

85 84

Yang et al. (1983)

Pinus strobus (eastern white pine)

Lehnherr et al. (1988)

Triticurn aestivurn L. cv. “Albis”

Reich (1983)

Populus hybrid deltoides x rrichocarpa Triticum aestivum L. cv. “Albis” Pinus strobus, initially 4 months old (ea. white pine), initially 2 years old Initially 2 years old

Lehnherr et al. (1987) Barnes (1972)

Pinus ellioftii, (slash pine) initially 8 months old Pinus taeda, (loblolly pine) initially 8 months old Pinus elliottii, initially 4 weeks old Pinus taeda, initially 4 weeks old

10 days 20 days 30 days 40 days 50 days 20 days 20 days 50 days 50 days 40 days 40 days 40 days 20 days 60 days ca. 50 days ca. 50 days

NS (DR) 91 (DR) 75 (DR) 81 (DR) 84 (DR) 71 (DR) 75 (DR) 78 (DR) 61 (DR) 108 (DR) 86 (DR) 72 (DR) 263 (DR) 144 (DR)

50

35 days

127 (DR)

50 150 150

36 days 19 days 3 6 7 7 days

NS 90

150

100 100 100 100 100 200 300 200 300 30 (15h) 70 (1.5’) 100 (15’) 85 (25’) 85 (25’) 35 (20’) 100 (20’)

-

-

NS (DR)

-

-

NS

-

175+NS (DR)

36-44 days

NS

-

186-+NS(DR)

150

36434 days

NS

-

141+NS (DR)

50

126 days

91

-

NS (DR)

50

126 days

85

-

NS (DR)

TABLE IV-contd. Reference Arndt and Kaufmann (1985) Greltner and Winner (1988) Reich et d.(1986b) Wallin et a/. (pers.comm.) Temple (1986)

Walmsley et a/. (1980)

Species and cultivar Abies alba (fir), High light Low light Raphanus sativus L. cv. ”Cherrybelle” Glycine mar, cv. “Williams” Glycine mar (soybean) cv. “Hodgson”

Pinus sylvesrris Gossypium hirsutum (cotton), Watered Watered Water stressed Water stressed Raphanus sativus L. (radish) cv. “Cherry Belle” 28 days pre-exposure No pre-exposure As above, but older leaf

Concentration Duration of 0 3 (PPb) 50 50 120 120 120 50 (lob) 90 (19‘) 130 (lo’) 42

42 days 42 days 19 days 25 days 19 days 56 days 56 days 56 days 85 days

44 (12b) 92 (12’) 44 (12’) 92 (12‘) 170 170

99 days 99 days 99 days 99 days 30 days 36 days 2 days 2 days 2 days

170

170 170

PS response S T response (% control) (% control) 61 63 NS 63 NS

90 89 78 60

-

77 NS

R response (% control)

-

65 (Cd 62 (CI) 67 (Cd

-

65 (TR)

-

-

NS NS NS NS

84 (TR) 79 (TR) NS (TR) 84 (TR) NS (R,)

-

NS (4 244 (Rs) 444 (RJ

-

-

-

-

-

-

(DR) (DR) (DR) (DR)

Krause et a / . (1985) Keller and Hasler (1984) Reich and Lassole (1984) Atkinson et a / . (1988a) Keller and Hasler (1987)

Vozzo et a f . (1988)

Acer platanoides

140 140 Picea abies L. (Norway spruce) 225 Picea abies L. (Norway spruce) 150 A bies afba (fir) 150 Populus hybrid, low light 125 (25’) deltoides x trichocarpa , high light 125 (25’) Raphanus sativus L. 200 cv. “Cherry Belle” Picea abies (Norway spruce) 150 Cuttings of 12 year old 150 Grafts of 80 year old 150 150 Glycine m u L. cv. “Young”, Well watered 59 (Isb) 59 (Isb) Water stressed Well watered 85 (Isb) Water stressed 85 (18b)

37 days 58 days 37 days 35 days 35 days 56 days

65 43 69

-

-

-

-

-

116 (CI)

-

84 (Cd

-

-

109 (C,)

-

56 days 37 days

-

37 (CI) NS (TR)

-

56 days 189 days 56 days 189 days

78 44 NS 54

NS (TR) NS (TR)

-

ca. 150 days ca. 150 days ca. 150 days ca. 150 days

74 NS 64 58

For the meaning of the abbreviations and symbols see footnote a to Table I. Reference level as indicated above 0 ppb 0 3 and probably including low levels of SO2 + NO,. Maintenance respiration.

68

-

-

-

-

75 (TR)

-

88 (Rs) NS (4 79 (Rs) NS (R,)

-

58

H . SAXE

transpiration. Faensen-Thiebes (1983), on the other hand, found declines in transpiration with little or no effect on photosynthesis and either a rapid recovery with an overshoot (Nicotiana) or a very slow recovery (Phuseolus). Yet a different response pattern was demonstrated by “Russian Mammoth” sunflower (Furukawa et al., 1984a) exposed to 720 ppb O3 for 2 h: photosynthesis and transpiration declined synchronously, but in an oscillating manner (Table IV, Fig. 6). Two hours of 400ppb 03,however, did not induce oscillations, but an immediate and steady decline in photosynthesis and transpiration, while 200 ppb O3 caused no significant responses (Furukawa et al., 1984a). Short-term exposures to ambient 0 3 concentrations (25-75 ppb) typically elicited a delayed effect on photosynthesis and transpiration declines (Forberg et d . , 1987; Saxe and Murali, 1989c), indicating that the 0 3 injury accumulated gradually, and was associated with a slow detoxification and repair rate.

Photosynthetic response mechanisms. Olszyk and Tingey (1984) confirmed that 0 3 was more reactive and indeed less easily detoxified than SOz. The study on poplar and sunflower by Furukawa et al. (1984a) clearly demonstrates that photosynthesis could decline either with no effects on stomata, or with synchronous responses of the two physiological parameters. Furukawa et al. (1984b) found that a decline in mesophyll conductance rather than in stomatal conductance in 03-exposed sunflower plants explained the observed decline in photosynthesis. Saxe and Murali (1989~) found that photosynthesis declined relatively more than transpiration in 11 cultivars of Picea abies, and interpreted this to mean that the effect of O3on photosynthesis was direct, rather than through an indirect effect by stomatal closure (Fig. 3). Omielan and Pel1 (1988) used soybean mesophyll cell suspensions to study direct effects of O3without indirect effects caused by stomatal closure. They confirmed direct effects of O3 on photosynthesis, as O3 reduced photosynthetic rates to a greater degree than cell viability. The observed effects on photosynthesis were independent of light. Pel1 and Pearson (1983) found up to 80% reduction in the quantity of RuBPC in alfalfa foliage exposed to 250 ppb 0 3 for 2 h, but did not resolve whether this was caused indirectly by an 0 3 effect on membranes or by a direct effect on the sulphydryl groups of the enzyme. The activity of RuBPC was previously shown to be inhibited by 0 3 in both old and young rice plants (Nakamura and Saka, 1978). Olszyk and Tingey (1984) suggested that oxyradicals produced during photosynthetic electron flow add to the toxic effects of O3in an autocatalytic process. An effect on chloroplast membranes was generally among the earliest detected symptoms of 0 3 exposure (Mudd, 1982). Robinson and Wellburn

PHOTOSYNTHESIS AND STOMATAL RESPONSES

59

(1983) found O j impaired the ability of photosynthetic membranes (isolated from oat) to create and maintain effective proton gradients (i.e. the basis for production of A T P ) , and inhibited a secondary dark-dependent partial repair mechanism. The injury was thus enhanced by light. Stomata1 response mechanisms. Faensen-Thiebes (1983) found transpiration in Nicotianum and Phaseolus to be affected by low doses of 03, without a response in photosynthesis, indicating an independent effect on stomata, which may in turn affect the photosynthetic rate. Faensen-Thiebes (1983) speculated that a stornatal response could generally be caused by direct effects on guard cell membranes, inducing loss of turgor, as well as by indirect effects on the mesophyll cells, by increasing abscisic acid (ABA) being transported to stomata and/or by increasing intercellular C 0 2 by inhibiting photosynthesis through effects on the thylakoid membranes. Keitel and Arndt (1983) found a rapid 0 3 induced loss of turgor in Nicotiana, supporting a direct membrane effect as the primary response. In plants experiencing a variety of environmental stresses, surges in the foliar production of ethylene (CzH4) is a well documented phenomenon, and the surges are referred to as “stress ethylene” to distinguish it from the more routine aspects of ethylene metabolism. The physiological significance of “stress ethylene” as a phytohormone is not resolved. Taylor et al. (1988) found a very close correlation between 03-provoked endogenous ethylene production and decline of stornatal conductance (and photosynthesis) (Fig. 7). Aminoethoxyvinylglycine (AVG) blocked ethylene production, with no effects on leaf conductance, but a 17% decline in photosynthesis. Ozone stress with AVG protection reduced ethylene production to 25%, removed a 40% decline in stornatal conductance, and halved the decline in photosynthesis. Taylor et al. (1988) proposed, therefore, that surges in “stress ethylene” are not simply an indicator of stress physiology in plants, but rather are responsible for mediating some of the responses of stomata and photosynthesis in soybean exposed to 1.2 ppm 03.They did not resolve, however, whether this mechanism had any relevance to pulses of ethylene at ambient O3 levels. “Stress ethylene” is discussed further in Section X.3. Stornatal uptake of 0 3 andplant response. In the different photosynthesis responses to 0 3 described by Furukawa et al. (1984a), the uptake of 0 3 always followed the transpiration response, i.e. the “stornatal opening” (Fig. 6). Amiro et al. (1984) found a quantitative relationship between mean 0 3 flux density and the duration of the exposure needed for the occurrence of visual injury in two Phaseolus vulgaris cultivars. Amiro and Gillespie (1985) found that O3 flux density in the same plants decreased leaf conductance to O3in a sensitive cultivar more than in a resistant cultivar, but did not discern whether this was mostly caused by changes in stornatal or in residual resistance. Butler and Tibbits (1979) found that a resistant Phaseolus culti-

60

H . SAXE

160

100 60

100 0

,

l,,l-j

4.

c1)(, producrkn ( n d m4 h-ll Fig. 7. The influence of 0 3 0 nstress ethylene, stornatal conductance and net assimilation in soybean, and the association between ethylene production and stomatal conductance and net assimilation rate. AVG, arninoethoxyvinylglycine-blocks endogenous ethylene production (Taylor er al., 1988).

PHOTOSYNTHESIS AND STOMATAL RESPONSES

61

var had fewer stomata and closed them within 1 h of 1.34 ppm 0 3 exposure compared with a sensitive cultivar. Barnes et ul. (1988) found that frost resistance was affected more by 0 3 in a pea cultivar ("Feltham First", Table IV) that had aslower stomatal response, leading to agreaterflux of 0 3 to the internal tissues. All these studies demonstrated the protective role of stomatal closure. Dijak and Ormrod (1982), on the other hand, found 03-sensitive pea cultivars, as evaluated by visible symptoms, to have a higher increase in diffusive leaf resistance when exposed to O3 than resistant cultivars. Resistance in pea was, therefore, concluded to rely on internal tolerance and repair mechanisms rather than on stomatal closure. Taylor et al. (1982) also found the response of stomatal resistance to be insufficient to explain differences in sensitivity in soybean cultivars, indicating the importance of the residual resistance. Elkiey etal. (1979) found that epidermal characteristics other than diffusive resistance could be important for a lower 0 3 uptake and, thereby, contribute to a lower sensitivity, viz. the number of trichomes, sizes of epidermal cells and the structure of the guard cell cuticle. There may even be a differential, but slow leakage of 0 3 through cuticles (Rich et al., 1970), most likely, however, only where this was broken or weathered. Such leakages, however, were not found by Saxe in four investigated pot plant species (unpublished data). Influence of age and environment. Forberg ef al. (1987) noted that the photosynthetic response of oats to O3 depended on age, with maximum susceptibility to O3 10-20 days after emergence of the panicle. The photosynthesis of a 20-day-old oat leaf was four times as sensitive as a 5-day-old leaf (Myhre ef al., 1988) (Table IV). The physiological response of soybean (Taylor ef al., 1982) and Phaseolus (Amiro and Gillespie, 1985) to 0 3 was likewise found to depend on age (Taylor ef al., 1982). High irradiance did not seem to protect photosynthesis against 0 3 (Omielan and Pell, 1988), as was the case with SO2 (Katainen et a / . , 1987), but rather to increase the damage (Robinson and Wellburn, 1983), as was the case with NO2 (Srivastava e t a / . , 1975b) and SOz + NO2 (Bennett et al., 1985). Lichens may be an exception, since high light intensity (700 pmol m-2 s-' versus 240pmol m-'s-'> partly protected a sensitive lichen against 0 3 inhibition of photosynthesis (Ross and Nash, 1983) (Table IV). Water stress reversibly decreased plant sensitivity to 0 3 by a protective closure of stomata (less O3 uptake), even with small changes in leaf water potential (Tingey et al., 1982). Biochemical or anatomical changes within the leaves were, therefore, not necessary to explain the observed protection during water stress (Tingey and Hogsett, 1985).

2. Respirafiori response to short-term 0 3 exposure It is often difficult to separate the direct effects of O3 o n the processes of

62

H. SAXE

respiration themselves from changes in respiration due to injury or as a result of the action of O3on photosynthesis and thereby on a changed size of metabolic pools. One example of an 0 3 effect separate from effects on photosynthesis has been given by Anderson and Taylor (1973) who found increased C 0 2 evolution in achlorophyllous tobacco callus exposed to 03. Myhre et al. (1988) found dark respiration to be significantly stimulated in oats, but it recovered quickly after exposure was discontinued (Table IV). Pel1 and Brennan (1973) found dark respiration to be only slightly or not inhibited immediately after a 3 h, 300ppb 0 3 exposure of pinto beans, while both photosynthesis and total adenylates (and ATP) were inhibited and stimulated, respectively, without delay. Twenty-one hours after the 3 h O3 exposure had been discontinued, the effects on photosynthesis and adenylates were reduced, but dark respiration had increased. Pel1 and Brennan (1973) concluded that 03-induced changes in respiration are a consequence rather than a cause of cellular injury.

3. Summary of the response to short-term 0 3 exposure Photosynthesis and stomatal opening typically respond to short-term O3 exposures with a decline, but the response pattern varies with cultivar, exposure concentration, environment and age. Recovery is variable in time and degree. Photosynthesis and stomata can both be affected directly, the former by effects on RuBPC activity and quantity or by an effect on the ability to build a proton gradient across thylakoid membranes. Stomata1 closure do not play a consistent role with respect to immunity to visible injury and inhibited photosynthesis. Indeed, the “metabolic capacity” (detoxification and repair) supplemented by certain epidermal characteristics is sometimes much more important than stomatal closure in protection against O3 injuries.

C. RESPONSE TO LONG-TERM

0 3

EXPOSURE

Differences in the responses to long-term O3 exposures among the results quoted in Table IV could, apart from the reasons given at the beginning of Section IIIC, also be caused by the use of different levels of reference. While Oppb was a reasonable choice for SO2 or NOz references, ambient O3 concentrations always had a background level above 10 ppb. Several authors, therefore, applied 10 ppb O3(or higher) as the reference (indicated in Table IV by a superscript 6) for the effects of 0 3 pollution. This abovezero reference often originated from charcoal filtering the forced inlet air in open-top chambers. Ozone effects with a zero reference were somewhat larger, and less realistic. As for the other pollutants, exposure studies on physiological 0 3 effects quite often included investigations in protected environments rather than in

63

PHOTOSYNTHESIS AND STOMATAL RESPONSES

the field, and when investigating the responses of trees, seedlings or cut branches were used more often than mature trees. Among the “positive” exceptions are the field work done by Skarby et al. (1987) and the comparative work in open-top chambers and the laboratory done by Lehnherr et al. (1987, 1988).

1. Response of photosynthesis and diffusive resistance to long-term 0

3

exposure For trees, the growth, even during fumigations, was largely determined by the assimilation the previous year, so an inhibition by O3of photosynthesis should not be expected immediately to inhibit the growth of a tree (Tseng et’ al., 1988). Typical responses. Photosynthesis typically reduced linearly with exposure time and O3 concentration, i.e. linearly with the external O3 dose (Yang et al., 1983; Reich and Amundson, 1985; Krause et al., 1985; Reich, 1987; Skarby et al., 1987). There are examples of photosynthesis being inhibited with no significant effects on stomata (Keller and Hasler, 1987; Lehnherr et a!., 1987; Tseng et al., 1988), after stomata1 adaptation (Walmsley et al., 1980), of stomata being affected with no effect on photosynthesis (Greitner and Winner, 1988; Skarby et al., 1987), and of equivalent and synchronous inhibitions (Keller and Hasler, 1987; Wallin et al., personal communication). Exposure for relatively few hours or days to relatively high O3levels was less inhibiting to photosynthesis than longer exposures to lower levels. The inhibition by low levels of 0 3 was typically “sneaking”, with increasing inhibition in photosynthesis after some delay (Table IV). An inhibition in photosynthesis was observed with 0 3 concentrations (2MOppb) comparable to ambient 0 3 levels (Barnes, 1972; Reich and Amundson, 1985; Arndt and Kaufmann, 1985; Amundson et al., 1987; Skarby et al., 1987;Tseng et al., 1988; Lehnherr et al., 1987,1988; Vozzo et al., 1988; Wallin et al., personal communication). The characteristic opening of stomata and the stimulation of photosynthesis by lower levels of S 0 2 , was not typical for 0 3 (Beckerson and Hofstra, 1979b; Winner et al., 1988), though Barnes (1972) and Krause et al. (1985) observed a “possible stimulation” of photosynthesis in woody species with 50-90ppb 0 3 , and Shertz et al. (1980) reported that stomata of grapevine were opened by 03,and Keller and Hasler (1984) found that O3 increased transpiration in spruce. Tseng et al. (1988) found a decline in photosynthesis with only 15 days of 20 ppb 0 3 , but the effect of 15 days of 50 ppb relative to 20ppb O3 was a stimulation (unknown significance). The ozonator and methods used by Shertz et al. (1980), and maybe others, could also have produced toxic N 2 0 5 as a by-product.

64

H. SAXE

The degree and time of recovery depended on the exposure time and the occurrence of visible injury.

Photosynthetic response mechanisms. Lehnherr et a f . (1987, 1988) studied multiple responses in wheat to three or four levels (different in two experimental years) of long-term O3 exposure: 14C02uptake in situ, C02 response of the net photosynthesis and dark respiration in the laboratory, C 0 2 compensation concentration at 2% and 21% 02,steady-state levels of ribulose-l,5-bisphosphate,3-phosphoglycerate, triose-phosphate, ATP, ADP, AMP, activity of RuBPCO, transpiration, soluble protein and chlorophyll content, and concluded that the response of photosynthesis to elevated O3 (Table IV) was the result of a limitation in the amount of RuBPCO carboxylation activity present in the leaves, and neither to stomatal closure or to diminished RuBPCO synthesis. The results obtained by Lehnherr et a f . (1987, 1988) further suggested that 0 3 induced premature senescence. The gradual decline in photosynthesis during a long-term O3 exposure described by Reich et al. (1986b), correlated with a gradual decline in chlorophyll a and b, which could, however, onlypartfy explain the drop in photosynthesis. Since visible injury did not have to occur for inhibition of photosynthesis to take place (pine (Barnes, 1972)), significant loss of chlorophyll was not essential for a decline. Reich etaf.(1986b) observed a decrease in both chlorophyll and quantum yield, mirroring the effect on the chloroplast function. It was not examined whether either of these declines explained the initial drop in soybean photosynthesis, i.e. was a primary cause of the decline. Since realistic doses of O3 (6 days, 5Oppb) were found to induce ultrastructural injury in chloroplasts (Miyake et a f . , 1989) by peroxidation of unsaturated structural lipids, this is an additional cause for long-term injury to photosynthesis. It is not known, however, if the ultrastructural injury was a primary cause. Most experiments pointed to the direct effects on photosynthesis as the most important, rather than indirect inhibition by 03-induced closure of stomata. Reich and Lassoie (1984) and Reich et a f . (1985), for example, found water use efficiency (WUE, measured as milligrams of C 0 2 fixed per gram H20 lost) of hybrid poplar and soybean to decline significantly with increasing O3 concentration. The effect on WUE depended on the photon fluence rate and the leaf age. Greitner and Winner (1988), on the other hand, found O3 to increase WUE in radish and soybean, suggesting that there are species and circumstances where stomata rather than photosynthesis are the primary target of 03. Response mechanism of stomata. Keller and Hasler (1984, 1987) found that the stomata of O3fumigated spruce and fir needles became “sluggish”in their response to darkness. They interpreted this as a latent injury that

PHOTOSYNTHESIS AND STOMATAL RESPONSES

65

increased the risk of injury by drought, which has often been observed or suspected in areas with air pollution, particularly on days with rapidly changing light conditions (clouds). The sluggishness could originate from impaired membrane function. Temple (1986), however, did not find any “sluggishness” in the stomatal light response in field-grown cotton exposed for a full season to 0 3 . The possible sensitivity of stomatal membranes was, therefore, species dependent. Influence of age and continued exposure. The influence of continuous low exposure levels of 0 3 was to accelerate senescence, as already indicated by Lehnherr el al. (1987, 1988) and as suggested by a plot of specific photosynthesis versus leaf age (Fig. 8) (Reich et al., 1986b). However, Fig. 8 also demonstrates that the inhibition of soybean photosynthesis by 130ppb 0 3 relative to 50ppb 0 3 was largest in relative terms in young and old leaves (the percentage inhibition is indicated above the graphs), while the absolute inhibition was largest in middle-aged leaves. This agrees with results obtained by Reich et aE. (1985). In other continuous exposures, Barnes (1972) found young secondary Pinus strobus needles to be consistently sensitive to 150ppb 0 3 , in contrast to older needles that had been exposed for longer time. Neither primary nor secondary needles of P . strobus responded to 50 ppb 0 3 for 5 weeks. However, exposure of P. elliotti and P. ruedu (Table IV) demonstrated that 126 days with 50ppb O3induced a decline in photosynthesis, while 84 days with 150 ppb 0 3 did not. For these species, therefore, the exposure duration and/or age of the plant material was more important than the exposure concentration.

Fig. 8. Net photosynthesis as affected by leaf age, for soybean exposed to various concentrations of ozone (Reich el al., 1986b).

66

H. SAXE

Walmsley et al. (1980) found that radish in life-long exposures to 170 ppb O3 acquired a tolerance to 03, measured by stomatal resistance and photosynthesis as well as by leaf growth. Stomata in older leaves in clean air were inherently more open, but also more sensitive to 2 days of 170 ppb 0 3 than stomata in younger leaves. Pre-exposed leaves, however, acquired a tolerance to 0 3 , so that they stayed as open in long-term ozonated leaves as when in clean air, while non-pretreated leaves closed their stomata quickly in response to 0 3 (Table IV). With this strategy, stomata did not limit C 0 2 exchange, but neither did they limit 0 3 uptake. Since photosynthesis regained its activity after a prolonged initial decline, even the photochemical and biochemical processes associated with photosynthesis must have required a tolerance to 0 3 . It is not known if either of the physiological aspects of tolerance would occur if the 0 3 concentration had been lower. Coyne and Bingham (1982) agreed that stomatal closure did not diminish photosynthesis in long-term exposed pine trees (described later), but they found the photosynthesis itself to be sensitive. When either young (8 days after maturity) or old (42 days after maturity) cotton plants were fumigated with 245 ppb O3for 4 days over 2 weeks, they reacted in an identical manner, indicating no age dependence in this species (Oshima et al., 1979) (Table IV). Keller and Hasler (1987) compared the response of photosynthesis and transpiration in cuttings of a 12-year-old Norway spruce with responses of grafts of an 80-year-old Norway spruce. Transpiration was only affected in the old spruce, while photosynthesis was more sensitive in the young spruce. It was, however, not possible to conclude whether the differences were due to age or to the methods of cultivation.

Znfluence ofthe environment. Drought stress is a major factor affecting plant yield, and it modifies plant responses to 03.Temple ef al. (1985b, 1988a,b) confirmed for cotton and alfalfa the previously quoted observations for short-term exposures (Tingey and Hogsett, 1985; Tingey et al., 1982) that drought can protect against O3 injury and yield loss. Temple (1986), however, also found drought to inhibit 03-induced stomatal closure in cotton plants, which could diminish the drought protection against 0 3 . Vozzo et al. (1988) found that drought could protect both photosynthesis and the stomatal response in soybean against 59 ppb 0 3 , while higher levels did not protect photosynthesis (Table IV). Reich etal. (1985), on the other hand, found that, even though leaf age, water stress and O3 affected leaf conductance in soybean, there was no interaction among these factors. Tseng et al. (1988) agreed that, while both drought and O3inhibited photosynthesis (Fraser fir seedlings), there was no interaction, i.e. 0 3 and water stress acted independently in affecting physiology. Arndt and Kaufmann (1985) found no significant differences in the response of fir photosynthesis to 0 3 with different photon fluxes (450 and

67

PHOTOSYNTHESIS AND STOMATAL RESPONSES h

J

cfl

0

200

4000

200

4000

200

4000

200

4000

200

*o(

Photon fluence rate (pE m-2 s-1)

Fig. 9. Mean leaf conductance in hybrid poplar, as affected by O3 ( A , 0.125ppm; 0, 0.025 ppm) leaf age and photon fluence rate. Leaf age: (a) 6-9 days; (b) 12-14 days; (c) 19-21 days; (d) 28-35 days; (e) 4&56 days. There was interaction between all three parameters in their effect on conductance (Reich and Lassoie, 1984).

900pmol m-2 s-') (Table IV). Reich and Lassoie (1984), on the other hand, found leaf conductance in hybrid poplar to be increased by O3at low photon fluence rates (PFR = 2-300pmol m-2 s-l), but decreased at high photon fluence rates. The response in leaf conductance, however, also depended upon leaf age, in a complex three-way interaction (O$PFR/leaf-age+ conductance) (Fig. 9). Reich and Lassoie (1984) described the overall effect of O3 to be diminished stomata1 control of water loss. Barnes et al. (1988) found O3 to increase frost injury to pea, and concluded that studies on 03-effects on perennials and over-wintering annuals should always include a full seasonal range of environmental stresses to be meaningful. Stomata1 uptake andplant response. Skarby et al. (1987) found uptake of 0 3 in Scots pine to be up to 50% of the administered quantity during the first hours, but it quickly levelled off as the plant surfaces became saturated, after which there was a linear uptake according to concentration. For an equivalent external dose within a single growing season, agricultural crops were the most sensitive to 0 3 ,with hardwoods intermediately sensitive and conifers the least sensitive. But with equivalent effective doses, all species displayed a similar decline in photosynthesis and growth (Reich, 1987).

2. Respiration response to long-term 0 3 exposure Reich (1983) found a significant increase in dark respiration in hybrid poplar, particularly in young plants, when exposed to 0 3 (Table IV), and Skarby er al. (1987) found increases in dark respiration even after a few days

68

H. SAXE

exposure of 25-year-old Scots pine to low 0 3 levels. So both young and old trees responded to O3 with an increased dark respiration. Amthor (1988) and Amthor and Cumming (1988) found that ambient levels of O3increased maintenance respiration in Phaseolus (Table IV) (i.e. the dark respiration minus the growth respiration, which was not affected by 03).Amthor (1988) supported the idea that the increased maintenance respiration reflected at least a partial repair of the 0 3 damaged tissue. It also, however, represented a diversion of energy and metabolic intermediates from growth processes, and, thereby, decreased growth. The response of dark respiration to O3was often more sensitive than that of photosynthesis (Barnes, 1972; Skarby et al., 1987), suggesting that increased dark respiration was a response to injuries other than those of the photosynthetic apparatus, such as a direct mitochondria1 effect. Skarby et al. (1987) speculated that O3 causes increased respiration at the expense of lipids or proteins or both, and that this has adverse effects on cell membranes, resulting in poor stomata1 regulation. Reich etal. (1986b) and Wallin etal. (personal communication),however, found no response in dark respiration, when soybean was given long-term exposure to 50-130 ppb 0 3 , even though there was a significant inhibition of photosynthesis, and Yang et al. (1983) and Lehnherr et a f . (1988) found a significant decrease in dark respiration, increasing with exposure concentration, but highest in the middle of the exposure period. Lehnherr etal. (1988) expressed this decrease in dark respiration as an increase relative to photosynthesis and concluded that this might help explain increased (ATP + NADPH)/(ADP + orthophosphate + NADP+). As photosynthesis was inhibited relatively more than dark respiration, the absolute dark respiration decreased, and as photorespiration is a consumer of ATP and NADPH, and this was found to be increased, their conclusion regarding the ATP NADPH increase, however, seems inconsistent.

+

3. Summary of the response to long-term 0 3 exposure It is evident that ambient concentrations of O3 can indeed inhibit photosynthesis, as quantified by Adams and Crocker (1988). Photosynthesis, transpiration and dark respiration can each be inhibited separately, without effects on the others, indicating that O3 affects plants at a multiplicity of sites, at least more so than either SO2 or NO2. The effects seem to be due to attacks on general mechanisms such as lipid peroxidation, rather than specific mechanisms, as were typical for SOz, and it is difficult to predict which of the reviewed physiological responses are the most affected. All responses seemed to be very dependent on species, environment, etc. Overall, however, photosynthesis seems to be the most O3affected of the physiological parameters reviewed.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

69

4. Long-term ecological effects of ambient O3 In Section III.C2, SO2 was identified as a modifier of balances in ecosystems. Does O3 also play such a role? Cornelius (1982) studied the influence of O3on the competition between Solidago canadensis and Arternisia vulgaris and found intermittent pulses of 300ppb 0 3 , 4 h each week through two growing seasons to have only short-lived effects on photosynthesis, followed by premature leaf shedding and senescence. Growing the plants in a mixed culture rather than separately protected the plants against natural and 03-induced leaf drop and against O3 effects on chlorophyll, but O3 did not change the competitive balance in mixed cultures. However, O3 did increase leaf drop and, therefore, probably also total production in the mixed culture. Coyne and Bingham (1982) studied the physiology of three injury classes (slight, moderate and severe) in young Pinusponderosa growing for 18 years in the San Bernadino National Forest influenced by oxidants from California’s South Coast Air Basin, USA. The study of the differently affected trees concluded that the loss of photosynthetic capacity was primarily related to the loss of chloroplast function rather than to increased resistance to C 0 2 diffusion through stomata. The ratio of the stomatal COz resistance to the total COz resistance decreased with increasing oxidant injury and needle age. This was in accordance with the previously quoted study of Walmsley et al. (1980), where stomatal resistance in radish in response to long-term 0 3 exposure adapted to being less affected by 0 3 . McLaughlin et al. (1982) studied the physiology of three injury classes in 25-year-old Pinus strobus growing in Tennessee, USA. Growth-ring analysis revealed that sensitive trees experienced a steady decline in average ring width of 70% over 15 years compared with tolerant and intermediate trees. But, in contrast to the results of Coyne and Bingham (1982), McLaughlin etal. (1982) found the photosyntheticcapacity to be identical in all injury classes. The declining annual growth in sensitive trees could be explained by premature needle senescence, 45% shorter needles and 60% increased respiratory activity. The latest results regarding novel forest decline, point to 0 7 as a major factor in this recent large-scale phenomenon. Novel forest decline is discussed further in Section X.D.

VII.

RESPONSE OF PHOTOSYNTHESIS AND DIFFUSIVE RESISTANCE TO 0 3 + SO2 A.

INTRODUCTION

In most investigations of physiological plant responses to gaseous air pollutants, plants were exposed to single gases. However, single pollutants rarely

70

H. SAXE

+

occur alone in the atmosphere. The effects of combined SO2 NO2 exposures have already been described in Section V. Ozone and SO2 (and NO,) also occur together, although the concentration of SO2(and NO,) in Europe are higher during the winter than during the summer, while 0 3 concentrations are highest in the summer. Summertime maximum hourly concentrations for O3 and SO2 were reported to reach 250ppb and 50ppb, respectively, in England (Ormrod et al., 1981), although in the two recent decades, SO2 concentrations have been going steadily down, and 0 3 concentrations have been increasing. Average concentrations of each gas are given in Sections 1II.A and V1.A. Ozone and SO2 are both toxic at ambient levels to physiological processes in plants (Sections I11 and VI). Several investigations have compared their combined effects on photosynthesis and stomata with the effects of the single gases (Table V). B. RESPONSE OF PHOTOSYNTHESIS AND STOMATAL RESISTANCE TO SHORT-TERM 0 3 + SO2 EXPOSURE

Different responses. Ormrod et al. (1981) reported a more-than-additive effect by O3+ SO2 on broad bean photosynthesis with 40 ppb SOz, and 0 3 concentrations up to 90ppb; above 90ppb 03,the combined effect was less-than-additive(Table V). After 2 h recovery was complete for combined exposures with 0 3 up to 70 ppb, and after 20 h recovery was complete for combined exposures with 0 3 up to 90-100 ppb, and above that concentration recovery was incomplete, and visible injury became apparent. The presence of 40 ppb SO2 during the 0 3 exposure did not affect recovery in any way (Black et al., 1982). Natori and Totsuka (1984a) also found a more-thanadditive effect of O3 SO2 (Table V). Shertz et al. (1980) reported on opening of stomata by both 150 ppb and 300 ppb SO2, 200 ppb and 400 ppb 03,and by all combined exposures in two vines (Vitis labruscana, cv. “Ives” or “Delaware”); The effects were always less-than-additive. Chevone and Yang (1985) found that soybean stomata also opened with combined exposures to O3+ SOP,but not with the single gases. This necessity of fumigating with the two gases concurrently to provoke a response was further illustrated by moving the 2 h exposure of either of the two gases 1h ahead (so there was only 1h of overlap); this decreased the photosynthetic response and eliminated the stornatal response (Table V). Other investigations demonstrated stomatal closure in pea in response to 0 3 SOz. Olszyk and Tingey (1986) found an additive response of stomatal conductance in pea exposed to combined 0 3 SO2, and by comparing responses to single-gas, consecutive and concurrent exposures even found evidence for a (metabolic) synergistic effect. Both gases also closed stomata singly, but less so than when combined. Olszyk and Tibbitts (1981a)

+

+

+

TABLE V Changes in net photosynthesis (PS) and stomata1 opening (ST) in short-term (< I day) and long-term (> I day) 0

3

+ SO2 fumigations’

~~

Species and cultivar

Reference

Concentration (ppb) 0 3

SHORT TERM Natori and Totsuka (1984a) Ormrod et al. (1981)

Chevone and Yang (1985)

Euonymoiis japonica Vicia faba L. cv. “Dylan”

Glycine nzax L. cv. “Essex” started 1 h earlier SO2started 1 h earlier Pisum sativiini L. cv. “Alsweet” 0 3

Olszyk and Tingey (1986) Elkley and Ormrod (1979)

Petunia hybrida cv. ‘.White Cascade” 50% RH 90% RH

0 100 100 50 0

so

90 90 180 180 200 0 200 200 200 110 0 110

so2

10~1000 0 100 0 40 40 0 40 0 40 0 700 700 700 700 0 120 120

Duration PS response ( % control) 2-3 h 2-3 h 2-3 h 4h 4h 4h 4h 4h 4h 4h 2h 2h 2h 2h 2h 5 h‘ 5h‘ 5 h‘

-

0 92 87 91 83 74 74 NS NS 33 62 41 -

-

ST response (% control) NS (TR) 72 (TR) 44 (TR)

TABLE V-contd. Reference

Species and cultivar

Concentration (ppb) 0 3

LONG-TERM Sueur-Brymer and Ormrod

Glycine max cv. “McCall”

(1984)

Carlson (1979)

Acer saccharum (sugar maple)

Fraxinus americana (white ash)

Quercus velutina (black oak)

Beckerson and Hofstra (1979a) Phasealus vulgaris cv. “Sanilac”

67 0 67 67 500 0 500 500 0 500 500 0 500 500 0 500 so0 0 500 500 0 500 150 0 150

so-, 0 300

300 300 0 500 500 0 500 500

0 500 500 0 500 500 0 so0 500 0 500 500 0 1.50 150

Duration

1 or 5 days 1 or 5 days 1 day 5 days 1-2 days 1-2 days 1-2 days 4 days 4 days 4 days 1-2 days 1-2 days 1-2 days 4 days 4 days 4 days 1-2 days 1-2 days 1-2 days 4 days 4 days 4 days 5 days 5 days 5 days

PS response (76 control)

ST response (% control)

Beckerson and Hofstra (1979b) Raphanus sativus L. cv. “Champion” Cucumis sativus L. cv. “National Pickling”

150 0 150 150 0

150 150 Glycine max. 0 cv. “Harosoy 63” 150 195 Bytnerowitz and Taylor (1983) Phaseolus vufgaris L. 0 cv. “Bush Blue Lake 274“ 195 514(27~) Amundson er al. (1987) Triticum aestivum 54(27b) cv. “Vona” 50 Jensen (1983) Acer saccharinum 50 (silver maple) 150 Populus deltoides x trichocarpa Jensen (1981) 0 150 Popitlus s p . (yellow poplar) Jensen and Roberts (1986) 150 40% RH 0 150 80% RH 150 0 150

0 150 150 0 150 150 0 150 150

0 195 195 0 39 0 100 0 250 250

0 250 250 0 250 250

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days 4 days 4 days 4 days 22 days 22 days 50 days 50 days 25 days 25 days 25 days

5 days 5 days 5 days 5 days 5 days 5 days

a References are ordered according to increasing external dose of 0 3 . For the meaning of the abbreviations and symbols see footnote a to Table I. Reference level as indicated above 0 ppb 0 3 , and probably including low levels of SO2 + NO,. .. With 2-h pre-exposure in the dark.

74

H. SAXE

previously found 4 h of 140ppb O3+ 400ppb SO2 to close stomata, while SO2 alone closed them, but O3 alone opened stomata in the same pea cultivar. The opening by 0 3 was counteracted at higher concentrations. Ashmore and Onal (1984) found an antagonistic effect by SO2 on 0 3 induced stomatal closure in barley. While 6 h 180ppb O3 closed stomata, and 33-65 ppb opened stomata, and 950 ppb SO2 had no effect on stomata, all O3+ SO2 combinations closed stomata, but less so than with O3 alone. Surprisingly, Ashmore and Onal(l984) found SO2 to protect barley against visible 03-induced injury. That stomatal closure in some cases did protect against visible injury caused by O3 SO2 exposure (100ppb 600 ppb) was indicated by Kobriger et al. (1984), who found that necrosis and loss of chlorophyll in pea was most severe when exposed for 2 h in the middle of the day, rather than in the morning or in the evening when stomatal conductance was 25% less, and uptake presumably lower. Olszyk and Tibbits (1981b) found SO2 exposure to cause less visible leaf injury and to close stomata in pea to a greater extent near the beginning, or the end, than in the middle of a 6 h light period. Evidently, the possible repair mechanisms in light could not compensate for the increased uptake and possible light-dependent production of toxic oxidants from S 0 2 . That the injury was probably uptake dependent more than light dependent was demonstrated by Olszyk and Tingey (1985b), who found no change or less injury in the dark, when pea plants of the same cultivar were exposed during the day and during the night with stomata kept evenly open by fusicoccin. Elkiey and Ormrod (1980) gave a more precise indication. They found that the most sensitive of three Petunia cultivars (cv. “White Cascade”) absorbed most 03,SO2 and 0 3 + SO2 through the stomata (with least surface adsorption), while the least sensitive cultivar (“Capri”) absorbed the least of the single and combined gases through stomata (but with the most surface adsorption). In studies where 0 3 SO2 opened stomata, the RH was somewhat lower (60-70% versus 7 6 8 0 % RH), while the temperature and the photon fluence rates were somewhat higher (2428°C and 360-680pmol m-2 versus 21-25°C and 250-300 pmol m-2 s-l). The apparent contradiction that O3 SO sometimes induced stomatal opening and sometimes closure could, therefore, not be explained by known environmental modification on effects elicited by the single gases, as quoted in Sections 111and VI. Furthermore the relatively few known environmental modifications of the effects on stomata of combined short-term O3 and SO2exposures (quoted below), did not clear up the apparent contradiction. The difference in response was, therefore, likely to have been mainly species dependent.

+

+

+

+

Influence ofthe environment. Elkiey and Ormrod (1979) found that the rapid increase in leaf diffusive resistance in Petunia cultivars induced by O3 or 0 3 + SO2 at 50% RH disappearedjn two of the three studied cultivars at

PHOTOSYNTHESIS AND STOMATAL RESPONSES

75

90% R H . The slight effects of SO2 at 50% R H disappeared at 90% RH. The data reported by Elkiey and Ormrod (1979), quoted in Table V, are for exposures during the early vegetative stage, and indicate a less-than-additive response of 0 3 SO2 compared to the effects of the individual gases. For the prefloral stage, their data indicate an antagonistic effect by SO2 on 03-induced stomatal closure in Petunia, as found previously for barley (Ashmore and Onal, 1984). Elkiey and Ormrod (1980) found stomatal pollutant uptake to correlate with the sensitivity of each Petunia cultivar. The observed influences of the percentage RH could, therefore , be important to the plants, if similar responses were also operative at more realistic exposure concentrations. The previously quoted studies by Olszyk and Tingey (1985b) and Olszyk and Tibbitts (1981b) together indicate that light increases the injury in pea by 0 3 + S 0 2 . Olszyk and Tibbitts (1981b) found soil moisture stress to induce greater stomatal closure with 0 3 + S 0 2 .

+

C . RESPONSE OF PHOTOSYNTHESIS AND DIFFUSIVE RESISTANCE TO

LONG-TERM

0 3

+ SO2 EXPOSURE

Different responses. In long-term exposures of spruce and fir to high levels (2-700 ppb) of either O3or SO2 alone or as concurrent or consecutive exposures, Gross (1987) concluded that net photosynthesis and stomata were affected differently by the two gases. Ozone influenced the photosynthetic apparatus and SO2 influenced stomata. He assumed, therefore, the action of the two gases to be additive, but did not demonstrate in his exposure experiments whether the additive effect was more- or less-thanadditive. Carlson (1979) found the photosynthesis of three trees to respond differently to 500 ppb 03,500 ppb SO2 and their combination, and the response to all exposures differed with species and changed with time as indicated in Table V. The combined exposures changed with time from a more-than-additive to an antagonistic effect by 0 3 on SO2-induced inhibition of photosynthesis in sugar maple, the more-than-additive effect by 0 3 + SO2 fumigation on white ash photosynthesis declined with time, and the lessthan-additive effect by 0 3 SO2fumigation on black oak changed with time to an antagonistic effect by either gas on the effect of the other. Generally speaking, the effects on photosynthesis of single gases increased with time, while the effect of the combined exposures decreased with time. A possible explanation for this is that the combined exposures close stomata more than single gases, whereby the trees are less exposed to further uptake and injury. The exposure concentrations and durations used by Carlson (1979), however, are unlikely to occur in the ambient environment.

+

76

H. SAXE

With a realistic O3concentration, but with a short duration and high SO2 concentration, Sueur-Brymer and Ormrod (1984) found a more-thanadditive effect by the combined exposures on soybean photosynthesis. The more-than-additive effect, however, decreased with time (Table V). With a longer exposure time and somewhat lower concentrations of 0 3 and SO2 than those used by Carlson (1979) and Gross (1987), though still significantly higher than normal ambient levels, Jensen (1981) and Jensen and Noble (1984) found a more-than-additive effect of 0 3 + SO2 in cuttings of a hybrid poplar clone (Table V). The photosynthesis in injured leaves could have been suppressed by a decrease in the photosynthetic leaf area (chlorophyll was not affected), or by an increased dark respiration, indicated by an increased C 0 2 compensation point in fumigated leaves. In another study, Jensen (1983) long-term fumigated silver maple seedlings with even more realistic 0 3 levels and again found O3 SO2 to reduce net assimilation rates. The depression, however, depended on the presence of 100 ppb SO2, which cannot be considered to be a realistic level in rural areas over extended periods. At still more realistic levels of both pollutants, Boyer et al. (1986) found no significant effects in young pine grafts by 50 ppb 03,50 ppb SO2 or their combination during 3-5 days of exposure. The data, however, suggest that O3 depressed photosynthesis while SO2 and O3+ SO2 increased photosynthesis. These trends increased with time, indicating that they could become significant with longer exposure periods. Amundson et al. (1987) fumigated winter wheat with ambient levels of 0 3 , SO2 and their combination from anthesis until harvest. They found no interaction of the two gases in their effects on photosynthesis and stomatal conductance, except with charcoal-filtered versus non-filtered air, where SO2 was a prerequisite for O3 to have an affect on stomatal conductance. With relatively high concentrations of O3 and SO2, Beckerson and Hofstra (1979a) found an impressive more-than-additive effect by 5 days exposure of bean plants. Except for a small initial stomatal closing by O3 and opening by S 0 2 , neither of the single gases ended up having significant effects on the total stomatal resistance in bean, but the effect of the combined exposure was a distinct closure of stomata. This closure prevented visible symptoms, though it did not seem to explain the full protection. Ashmore and Onal (1984) also found protection by combined 0 3 + SO2 exposure (relative exposure to O3 alone), but stomatal closure was not involved. In both these studies, therefore, an antagonistic metabolic interaction between O3 and SO2 is indicated. To study the role of stomata in protecting plants against O3 SO2, Beckerson and Hofstra (1979b) compared visible injury and stomatal response in radish, cucumber and soybean plants fumigated for 5 days with 150 ppb 03,150 ppb SO2 and their combination. The combined exposures caused more-than-additive visible injury in radish and cucumber, while SO2

+

+

PHOTOSYNTHESIS AND STOMATAL RESPONSES

77

acted antagonistic in soybean, protecting against an 03-induced visible injury. The only significant stornatal reactions were an opening caused by SO;! in radish, a closure caused by the combined exposure in cucumber and both these responses in soybean. This indicated a stornatal protection in soybean against visible injury by the gas mixture, but left open the question of why cucumber did not gain similar protection, rather than the observed more-than-additive visible injury. Bytnerowicz and Taylor (1983) found that Phaseofus bean reacted like soybean in that SO;! protected it against 03induced visible injury. As for soybean, this can be explained by a more-thanadditive stomatal closure by 0 3 SOz. From the quoted studies on stomatal closure as a suggestive protectant against gaseous pollutants, it was obvious that such closure was not the only protection strategy, and that closure did not always have the predicted protective effect.

+

Interaction mechanisms. Bennett et a f .(1984) described the antagonistic responses of 0 3 and SO;!in biochemical terms: 0 3 causes part of its injury though a generation of oxyradicals, particularly .OH (which are also generated by fight during normal photosynthesis). Although there was not

L' rImrlmte perwhae (W l y r n 8 i d c

dirmut.lc)

2ds)lydranmhlerodrslve : #I-

cphorph.1e

Fig. 10. Ascorbic acid protects against superoxide and hydrogen peroxide produced in plants during 0 3 and SO2 fumigations. It was proposed that GSH functioned to stabilize enzymes of the Calvin cycle and the cytoplasm,and helped keep ascorbic acid in the reduced form. The indicated pathways are taken from Castillo and Greppin (1988) and Foyer and Halliwell (1976).

78

H. SAXE

much evidence to suggest the involvement of superoxide .O<, superoxide dismutase (SOD) was found to protect against the senescing effect of the oxyradicals. Sulphur dioxide inhibits SOD, which then explains the negative interaction of O3 and SO;?. Additive responses may be understood by the fact that both O3 and SO2 (bisulphite and sulphite) are known to generate free radicals (Mansfield and McCune, 1988). Plants are prepared for detoxifying the common levels of free radicals produced in illuminated chloroplasts through the action of SOD, catalase, and the ascorbateglutathione-NADPH cycle (Fig: lo), and by such compounds as a-tocopherol and carotenes. A small increment in the production of free radicals by pollutants may be scavenged effectively by this normal enzyme system available for this function, but if radical production exceeds the cell’s capacity for scavengingthem, then the damage may appear abruptly, and the more-than-additive response is seen. Another common denominator in O3 and SO2 injury to plants is the production of stress ethylene, where the same mechanism of exceeding a critical value may produce more-than-additive responses. Influence of the environment. Carlson (1979) found both light intensity and relative humidity to affect the photosynthetic response of white ash and sugar maple to 0 3 SO*. At low and high light intensities the combined exposure for 1 day to 500ppb O3 500ppb SO2 inhibited photosynthesis 60-70% more than at medium light intensity, After 4 7 days of combined exposure, this pattern persisted for sugar maple, while white ash was significantly more inhibited only at the low light intensities. The data given by Carlson (1979) (see Table V) were obtained after fumigations at 2045% RH. The response of photosynthesis to O3 S02, however, varied with percentage RH, species and time. After 1 day of combined exposures the photosynthesis of sugar maple declined the most at low percentage RH (2&50% RH), while that of white ash declined equally at low and high percentage RH (55-90% RH). After 5 days of exposure the photosynthetic rate of sugar maple was about equal at low and high humidities, while that of white ash was significantly smaller at the high humidity. In other words, sugar maple was most responsive at low humidities, but the difference recovered with time, while white ash was most responsive at high humidities, although it only showed with time. Jensen and Roberts (1986) monitored the daily variations in leaf diffusive resistance in yellow poplar during 5-day exposures to 150 ppb 03,250 ppb SO2or their combination. As can be seen from Table V, there was a general stimulation by all treatments performed at 40% RH and a decline with all treatments at 80% RH. In both cases the effect was less-than-additive. An additional important effect, not revealed in the table, was stomatal fluctuations in the combined exposure which increased with time. This was an expression of loss of stomatal control.

+

+

+

PHOTOSYNTHESIS AND STOMATAL RESPONSES

79

D. SUMMARY OF THE RESPONSE TO SHORT- AND LONG-TERM 0 3 + SO2 EXPOSURE

Concurrent exposures to O3and SO2 cause more-than-additive or less-thanadditive effects on photosynthesis and diffusive resistance, or the two gases work antagonistically or there may be no interactions. These quite unpredictable responses depend at least on the plant species and cultivar, and on exposure concentration and duration, as well as on the environment.

VIII. RESPONSE OF PHOTOSYNTHESIS AND DIFFUSIVE RESISTANCE TO 0 3 + ACID PRECIPITATION A. INTRODUCTION

In this connection, acid precipitation (AP) will be defined as the oxidation, hydration and dissociation products of SO2 and NO2 when these gases meet atmospheric liquid or vaporous water (i.e. HSO:, S 0 3 2 - , S042-, NOz-, NO3- and H+). If the gases are adsorbed to the leaf surfaces and there, later or immediately, react with water, the situation is roughly comparable to direct exposure to AP. Just as for the gaseous pollutants, only direct effects of AP on plants will be considered here, not effects through the soil, though such cannot always be clearly distinguished or ruled out. Grunwald (1988) gave an example of this ambiguity, when he found the photosynthesis of soybean plants to be stimulated by acid rain (pH4.5) only when the uncovered soil was not nitrogen fertilized. His conclusion, however, was that the stimulation of photosynthesis by AP was due to an increased stornatal opening. The dilute and dissociated strong acids contact the plants as liquid droplets, or bound to particles (wet deposition), or in the case of HN03 in gaseous form (dry deposited, together with the primary gases). The effects of gaseous HN03 are not discussed here. Of wet deposition, only the liquid form has a reasonable chance of penetrating the stornatal barrier, particularly fog or the fine mists of clouds, and has, therefore, been the most studied form of AP interacting with plants. Most studies on the effects of AP on photosynthesis and stornatal conductance have been carried out in combined studies of O3effects and simulated AP. This combination represents a most realistic air pollution environment for trees at high altitudes, where the O3level is higher than at lower altitudes and a major share of the sulphur and nitrogen pollution arrives as AP with the clouds.

TABLE VI Changes in netphotosynthesis (PS) and "stomata1opening" (ST) in response to 0, AP exposures

+

Reference Scott and Hutchinson (1987)

Sigal and Johnston (1986)

Species and cultivar Cladina stellaris 3 days later 3 days later Cladina rangiferina 3 days later Labaria pulmonaria L.

Concentration of 0 3 (PPb) -

3.0

-

2.5

-

3.0

G 177

6 177

Chappelka and Chevone (1988)

Hanson et al. (1988) Prov. "Gales County" Takemoto et al. (1988a)

Liriodendron tulipifera L. (yellow poplar)

Rain pH

50 (20') 100 (20') 2v 20' 100 (20') 100 (20') Pinus tueda (in the field) 167 (14') (loblolly pine) 14' 14' 3.3 (5.2') Capsicum annuum 66 (19') '19" (green pepper) cv. "California Wonder" 19'

Durationb PS response 03,AP (% control)

64 78 59 87 65 118 NS

5.6 or 4.2 2.6 -

4.3 (5.6') 3.0 (5.6') 3.0 (5.6') 3.0 (5.6') -

4.5 (5.2') G, H

-

2.7 17.2') 1.7 (7.2cj

ST response (% control)

I. J J

11

10 NS NS 85 NS 88 76 75 157 NS (C?) 83 NS 72

NS

icii

64 (c;j

The references are ordered according to external O3dose. For the meaning of the abbreviations and symbols see footnote a to Table I and footnote b below. A, no 0 3 ; B, 15mm rain given once over 2 h, and the indicated effects on photosynthesis recorded 3 days later, while recovery levels were recorded 6 days later; C, 5 days of 10h per week for 2 weeks; D, 11mm rain per day for 10 days over 2 weeks; E, 5 days of 4 h for 6 weeks; F, 1h of 7.5 mm rain twice a week for 6 weeks; G, 4 days of 6 h per week for 13 weeks; H, 10mm rain per week for 13 weeks; I, ambient 12-h day average for 11 weeks; J, 2 h of 0.5 mm AP as fog twice a week for 11weeks. concentration as indicated above 0 ppm and probably includes low levels of SOz + NO,, or for AP the reference Reference level (03 pH when different from neutral. Stomata1 conductance to CO2.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

81

B. PHYSIOLOGICAL RESPONSE

Several investigations found no effects on photosynthesis and stomata by AP with or without low levels of 03.Takemoto et a f . (1987) exposed soybean plants for a full season to particle filtered ambient air or charcoal filtered air combined with ambient rain and simulated rain of pH 5.2,4.2 or 3.2. Neither combination of treatments had a significant effect on photosynthesis, stomatal conductance or yield. Reich and Amundson (1985) and Reich et a f . (1986a) found no physiological effects on sugar maple, eastern white pine, northern red oak or hybrid poplar exposed for 7-12 weeks (5.5-7 h per day, 3-7 days per week) to AP (pH 3.0-5.6), and no interactions with the previously quoted effects of ozone O3(Table IV). Elliott et al. (1987) found no statistically significant decline in chlorophyll or growth during 3 years expoeven though the sure of green and white ash to ambient acidicrainfall and 03, 1h mean standard for O3(120 ppb) was exceeded 78 times, and the area (New Jersey) is among the most “acid regions” in the USA (rainfall pH 3.6-4.7). When both AP and O3were found to have physiological effects, they did not always have interactive effects. Takemoto et al. (1988a) exposed green pepper to ambient levels of O3and severe (pH 2.7) or extremely severe (pH 1.7) acid fog. They found ambient O3concentrations to inhibit both photosynthesis and leaf conductance, while with AP only the unrealistic pH 1.7 could inhibit these. There were no interactions between O3 and acid fog, either in the effect on the physiological parameters or in the effects on pigments, dry weight or fruit number. The investigators believe that the decline in photosynthesis was due to stomata1 closure. Temple et al. (1987) found acid fog (pH 2.0 or 3.2) to inhibit both transpiration and photosynthesis, but there was no interaction with 0 3 effects. Sigal and Johnston (1986) (no interaction) could demonstrated that AP (PH 2.6) with or without (03) inhibit photosynthesis directly as lichens do not have stomata. Scott and Hutchinson (1987) demonstrated, that even a realistic AP exposure (pH 3.0; see Table VI) could inhibit photosynthesis in lichens, although this became apparent only some days after treatment. Recovery was obvious 6 days after exposure. It seems realistic that lichen photosynthesis would be inhibited by ambient AP in boreal forests. But some investigators did find interaction between AP and 0 3 in their physiological effects. Hanson et al. (1988) exposed 6-month-old pine seedlings in the field (14 ppb or 167ppb O3 combined with simulated acid rain (pH3.3, 4.5 and 5.2)) and in a laboratory CSTR system (14, 160 and 320ppb O3 with simulated acid rain (pH4.5)) for 13 weeks. The O3 levels applied in the field represented up to 3-4 times ambient concentrations. The acid rain pH represented polluted, ambient and pristine environments, respectively. Ozone inhibited (light saturated) photosynthesis, but only in the field, while AP stimulated photosynthesis. There was an interactive trend (not significant) after only 6 weeks that ozone would abolish photo-

82

H. SAXE

synthesis stimulation by AP, and AP would abolish photosynthesis inhiThe seedlings were more sensitive to O3in the field than in the bitions by 03. laboratory. Dark respiration and stomata were not affected. Chappelka and Chevone (1988) found significant physiological effects in yellow poplar seedlings with realistic O3 episodes (100 ppb) and severe ambient AP (pH 3.0), and an additive effect on photosynthesis by their combination. Ozone alone caused stornatal closure with no significant effects on photosynthesis; AP alone caused small declines in either photosynthesis or leaf conductance, depending on pH; combined ozone and AP caused a linear decline in photosynthesis (Table VI). Ozone sensitized stomata to vapour pressure deficit (VPD), although neither AP nor the combination of O3and AP was found to influence the rfsponse of leaf conductance to VPD. Acid precipitation exposure was, therefore, antagonistic to this response to 0 3 . In contrast, Jensen (1986) found no effects on photosynthesis or stomata of yellow poplar seedlings with external O3 doses exceeding those used by Chappelka and Chevone (1988) in both concentration and duration combined with comparable AP treatments. However, Jensen (1986), found an increased dark respiration. Influence of nutritional status. Selinger et al. (1986) measured the physiological responses of Picea twigs (on whole trees) exposed to O3 and AP. The trees were grown in soils with or without Mg2+ and Ca2+ deficiency. With nutrient deficiency, O3 (“peaks”) reduced net photosynthesis, dark respiration and stornatal conductance. Acid precipitation decreased photosynthesis and dark respiration, but stimulated stornatal conductance. The. combined exposure to O3 and AP generally induced additive effects, with stomatal conductance being significantly reduced. With sufficient nutrient supply the effects on photosynthesis were not-significant , dark respiration was reduced more by single pollutants, while the closing of stomata by 0 3 and their opening by AP ended up as an opening with the combined exposure. The quantitative effect of the individual and combined exposures was thus intensified by nutrient deficiency, and the stornatal response to the combined exposure was reversed. Summary of the responses to 0 3 and A P exposure. Acid precipitation is sometimes found to stimulate, but most often to inhibit, physiological activities. There are sometimes additive or antagonisticinteractions between 0 3 and AP. Ozone is generally found to act at realistic ambient levels, while effects of AP require such high proton concentrations that a negative effect by ambient levels would be dubious in most areas, even in industrialized countries. Temple et al. (1987), however, quote several examples of fog events with pH as low as 2.0 in urban, industrial and agricultural areas of Southern California (where oxidant levels are high).

PHOTOSYNTHESIS AND STOMATAL RESPONSES

83

k A

k

rr 1 0 0 .

.-fa*-

2e

m-

100-

a

Noz+oJ rO?*-q+eJ

SL

Fig. 11. A-C. Effects of SOz, NO2 and 0 3 separately and in combinationsof two and three gases on the net photosynthesis of sunflower leaves relative to the rate prior to fumigation. Vertical bars indicate standard errors. (Adapted from Furukawa and Totsuka (1979).)

84

H. SAXE

IX. RESPONSE OF PHOTOSYNTHESIS AND DIFFUSIVE RESISTANCE TO OTHER AIR POLLUTION COMBINATIONS A. RESPONSE OF PHOTOSYNTHESIS AND TRANSPIRATION TO 0 3 NO2 EXPOSURE

+

The reduction of photosynthesis in sunflower leaves induced by the mixture of O3and NO2 was more-than-additivecompared to the reduction caused by the individual gases, when the concentrations were 200 ppb 0 3 and 2 ppm NO2. With increasing concentrations the combined effect became less-thanadditive or antagonistic: 400 ppb O3inhibited photosynthesis to 65% in 2 h, 4 ppm NO2 inhibited photosynthesis to 10% in 2 h, but their combination only inhibited photosynthesis to 60% of a control (Furukawa et al., 1984b). While neither 200ppb O3 nor 2ppm NO2 affected transpiration in sunflower, their combination reduced transpiration (rnore-than-additive)by 25% (Furukawa et al., 1984b). Effects of 400ppb O3 combined with 4ppm NO2 also inhibited transpiration in a more-than-additive mode. Natori and Totsuka (1984a) found 100ppb 0 3 to reduce transpiration by 26%, 100ppb NO2 by 0%, while the combined exposure reduced transpiration by 24% within 2-3 h. Thus, there was no interacfion in O3+ NO2 exposures of Euonymus japonica as observed with SO2 NO2. Using 13Cassimilation, Ito et al. (1985) found indications of a stimulation of Phaseolus bean photorespiration by high levels of 03,NO2 and their combination. There were no signs of interactions between the two gases in this effect. The rnore-than-additive effect of 0 3 NO2 that was sometimes observed can be explained in biochemical terms, since 0 3 (like S02) is known to inhibit nitrite reductase in corn and soybean (Leffler and Cherry, 1974). This enzyme participates in NO, detoxification.

+

+

B . RESPONSE OF PHOTOSYNTHESIS TO SO2 + NO2 + 0

3

EXPOSURE

The most realistic combination of air pollution fumigations should combine ambient concentrations of all gaseous pollutants and their reaction products, at least AP. Particulates should also be considered. Though there have been several studies on the yield and carbon partitioning effects of several complex gas combinations, the most complex combination yet, with the purpose of photosynthetic studies, included only three gases: SO2 NO2 + O f . Furthermore, the investigators (Furukawa and Totsuka, 1979) restricted the analysis to short-term exposures with high concentrations in their study of combined-pollutant effects on sunflower photosynthesis, so that their results are mainly of academic value (Fig. 11). However, the results of this work do give the impression that the more gases

+

PHOTOSYNTHESIS AND STOMATAL RESPONSES

85

that work together in inhibiting the photosynthetic processes, the lower the concentrations needed to be effective. The conclusion is, therefore, that effects of single gases, or even combinations of two gases or AP, most often underestimate the effects in the ambient environment, where several gases and AP work in unison. Also, the more gases involved, the less predictable the response.

X. DIAGNOSTIC METHODS FOR PREDICTING AIR POLLUTION AND STRESS INJURY TO PLANTS A. GENERAL

Some of the major objectives for investigating plant responses to air pollution are: (1) The need to know the yield loss of commercial crops; (2) to understand the mechanisms of injury; and (3) to devise strategies to predict and minimize losses. There are several biological methods for pxedicting the effects of air pollutants on plants, classified by the use of indicatorplants (visible injury), or by the use of bioindication (invisible injury). This section focuses on quick and sensitive methods for early detection and diagnosis by the use of bioindication. B. INDICATOR PLANTS

Indicator plants are sensitive to specific air pollutants. They are placed at locations with suspected high levels of pollutants. Visible scorching of leaves indicates the integrated load of the pollutant which the indicator plant is sensitive to. Indicator plants thus “measure” long-term air pollution concentrations and integrate their effects, but their response says little about what is going to happen to other species, even under the same environmental conditions. The use of indicator species are limited to air pollution concentrations high enough to cause visible injury. Most plants, however, are affected by air pollution concentrations lower than those that cause visible injury, even to sensitive indicator plants. The use of indicator plants, therefore, does not appear to be a sensitive enough method to help us protect plants efficiently. Another problem is that it takes a long time to obtain results. This subject has been reviewed by Posthumus (1982). Other examples of indicator plants are given by Donagi and Goren (1979), Hirata and Kunishige (1981), Keller (1988), Kress et al. (1982), Laurence et al. (1985) and Nouchi and Aoki (1979).

86

H. SAXE C.

BIOINDICATION

Lower concentrations of air pollution than indicated by indicator plants may be revealed by subtle changes in the biochemical or physiological activities of plants (i.e. bioindication). The advantage of bioindication over the use of the visible signs of injury in indicator plants is that results may be obtained in a matter of hours or days, rather than weeks, months or years. Boindications may, therefore, be used for early detection (prediction) of an approaching or latent injury, before it appears as visible damage and measurable losses in yield. Bioindication may also be used as a diagnostic tool for selecting the most resistant species, cultivars, provenances or clones for a particular purpose, as when replanting forests damaged by air pollution or novel decline symptoms, or in greenhouse production of pot plants or vegetables with C 0 2 enrichment (and NO pollution). Parameters in bioindication should be specific, simple, rapid and unambiguous in use, and yield highly reproducible results. A review of physiological and biochemical tests for the effects of air pollution on plants has been given by Darrall and Jager (1984). Based on the results of more recent references in the literature, the remaining sections of this chapter reassess an expanded range and function of recently used bioindicative methods. 1. An overview of parameters Whenever air pollution or any other environmental stress affects a plant, it will react with a complex response pattern. Not all responses triggered by the broad range of environmental factors are useful for bioindicative purposes with plants growing in the ambient environment, if used alone, since they often do not distinguish particular stress factors. If a single, specific parameter has not been identified for a particular situation, the combined use of several response parameters may be necessary to specify the presence of a specific, harmful pollutant or stress. Sometimes, however, it is not even known which pollutant or stress should be looked for, as we may only know it by its deleterious effects. In the case of novel forest decline, for example, the “stress factors” which induce the well-described symptoms are not known with certainty. Table VII gives an overview of some parameters recently used in bioindication, along with their purpose and value.

Photosynthesis and diflusive resistance. As reviewed in Sections III-IX, photosynthesis and stomata respond to several air pollutants and their combinations, even at low pollutant concentrations that do not induce visible injury. Several of the responses are very rapid, i.e. within minutes or hours. But since photosynthesis and the stornatal response may recover within hours, these parameters can only be used during or immediately after fumigation, and do not necessarily integrate the effects of consecutive episodes over

TABLE VII Methods recently used for the bioindication of air pollutants and environmental stresses and the resulting plant injuries” Reference

Species and cultivar

Photosynthesis andlor diffusive resistance -Dietz er al. (1988) Picea abies L. Fagus sylvatica L. Landhold (1982)

Oleksyn and Bialobok (1986) Reich (1987)

Stressinducer used in test Field conditions

Pinus sylvestris L.

Cause of injury in the field Unknown -

so2 so2

SOz, NO,, HF

0 3

0 3

NO

NO (with COz)

Saxe and Murali (1989a)

Agricultural crops, hardwoods, conifers 8 species or cultivars of pot plants Picia abies L. (11 provenance)

Acute SOz

Unknown

Saxe and Murali (1989b)

Picia abies L. (11 provenance)

Acute NO2

Unknown

Saxe and Murali (1989~)

Picia abies L. (11 provenance)

Acute O3

Unknown

Saxe (1989)

Picea abies L. (9 provenance)

+

Unknown

Wild ef al. (1988)

Picea abies

Leafpigments Mehlhorn ef al. (1988)

Picea abies

Saxe (1986a)

Takemoto ef al. (1988b) Chlorophyllfluorescence Strasser ef al. (1987)

Medicago saliva L. cv. “Moapa” Fagus sylvatica L. (beech) and Populus x euamericana (poplar)

Acute SOz NO2 Field conditions

Unknown

SOz, AP

Unknown

Acid fog

Acid fog

0 3

0 3

0 3

O3

03,

Response parameter

Use and value of bioindication

TET Indicates ‘‘latent’’ forest decline ‘TOz fixation Good correlation between I4COz-f assimilation, S,, and leaf necrosis PS Predicts visible injury in the field (p < 0.05) CS Predicts O3 uptake, PS decline and yield loss PS Selects NO (with COz) resistant, pot plants (p < 0.03) PS Selects NFD resistant spruce (p < 0.01-0.05) PS, TR Selects NFD resistant spruce (p< 0.00.5-0.05) PS, TR Selects NFD resistant spruce (p < 0.005-0.05) PS, TR Selects NFD resistant spruce (p < 0.05) P-700, cyt f, QB Indicates “latent” forest decline VX:AX Early detection of spruce decline (NFD), (p < 0.01) Leaf pigments Over-estimates yield loss Leaf pigments Good estimation of yield loss

Chlorophyll Works well in a chamber fluorescence

TABLE VII-contd.

Reference

Species and cultivar

Endogenous elements and buffer capacity Bytn&owin et al. (1987) Triticum aestivum cv. “Yecora Roio” “spruce” Gasch ef al. (1988)

Huttenen et al. (1985) Legge et al. (1988)

Pinus sylvestris Picea abies Pinus hybrid: contorta x banksiana

Mehlhorn et al. (1988)

Picea abies L.

Endogenous mehbolites Anbazhagen et al. (1988)

Oryza sativa (rice)

Chiment et al. (1986) Gun (1983) Karolewski (1985) Varshney and Varshney (1984)

Endogenous enqme activity Agrawal et al. (1987)

Sedum album Glycine max. cv. “Essex”

Stressinducer used in test

Cause of injury in the field

Field conditions

so2

Field conditions

SO2+ AP

Urban air Urban air Field conditions

SO2 (etc.) SO2 (etc.) SO2 + AP

S02, AP

Unknown

03,

SO2, NOz, NH3 Field conditions 0 3

O3

0 3

O3 SO2

so2

Phaseolur vulgaris cvs Populus cv . “Robusta” Brassica, Phaseolus, Zea (resistantkensitive spp.)

0 3 O3 SO2 Field conditions so2 SO2

Viciafaba L., N. “Local”

so2

Response parameter

S,,

Use and value of bioindication

BC Estimates SO2 stress in the field

SO?S/organicS Early detection of SOz induced spruce decline S,, Best with conifers and winter sampling S, Best with conifers and winter sampling SOiSlorganic S Early detection of decline in PS and yield in SO2 AP exposed forest areas (p < 0.001)) S increase, Early detection of spruce decline Mg decrease (NFD) (ptO.O1)

+

Free proline Too general to be used alone

+

AA DHA Indicates 0 3 stress decrease GSH decline Indicates oxidant stress, e.g. by 0 3 GSH increase Predicts insect growth on stressed plants GSH decline Indicates oxidant stress, e.g. by 0 3 Free proline Too general to be used alone Ascorbic acid High inherent AA and low rate of decline with SO2 indicates SO2 resistance, but only in some species

SO2 SOD inhibition Indicates SO2under well defined conditions

so2

Cicer arietinum L., cv. “T3” Guri (1983)

Phaseolus vulgaris cvs

0 3

Podlecki er al. (1984)

Zea mays (sweet corn)

0 3

SO2 SOD inhibition Indicates SO2 under well defined conditions O3 Inherent GR High GR activity correlates with level low O3sensitivity O3 PO stimulation Screening for 0 9 resistance to visible injury under well defined conditions

Stress ethyleneproducrion

Cape ei al. (1988) Mehlhorn et al. (1988)

Picea abies

Wolfenden er al. (1988) Fuhrer (1985) PeU and Puente (1986) Taylor et al. (1988)

Abies alba Mill. (fir) Avena sativa L. cv. “Ogle” Clycine mar (soybean)

03,

so2, AP

Unknown C2H4 emissions Early detection of novel spruce decline (p <0.01) Unknown C2H4emissions Indicates stages in NFD O3 Stress C2H4 Only C3(not AP) induced stress C2H4 O3 Stress CzH4 Stress C2H4correlates with PS and C, (Fig. 7)

Field conditions 0 3 , AP 03

Anatomical and morphological analysis Benolt ei al. (1982) Pinus sirobus

Field conditions

Mehlhorn et al. (1988)

Picea abies L.

03,SO2, AP

Saxe and Murali (1989a)

Picea abies L. (9 half-siblings)

Tuomisto (1988)

Picea abies L.

Field conditions Field conditions

Roloff (1986)

Fagus sylvatica L.

Field conditions Field conditions

Genetic d y s i s Bergmann and Scholz (1987) Geburek et al. (1987)

Picea abies L. Pinus sylvestris

None

SO2, 03,HF None

General stress ’

Unknown Unknown

General stress Mainly SO2

MRIGlEC Indicates O3 and AP to affect pine growth DW/FWQ,~ Early detection of novel spruce decline (p < 0.01) . H , B, BD, L Selects NFD resistant spruce (p < 0.02-0.0.5) Wax erosion Indicates air pollution load on 1-year-old spruce needles Crown structure Classification and detection of NFD in hardwoods GOT GL and Selects SO2+ 0 3 resistant G-6-PDH GL individuals GDH and Selects SO2 resistant GOT GL individuals

TABLE VII-contd. Reference

Miscellaneous methods Ashenden and Williams

Species and cultivar

Betula pendula Roth.

Stressinducer used in test

Cause of injury in the field

AP

AP

so2

so2

so2

so2

so2703r ws

s02, ws

(1988) EUenson and Amundson (1982) Hartel (1972)

Glycine I M X L. cv. “Beeson” Picea abies L.

Keller (1986) Martin et al. (1988)

Picea abies L. Pinus, Pseudotsuga

Tomiczek (1987)

“spruce”

SOz, N 0 2 , 0 3

SOz

0 3

-

Response parameter

Use and value of bioindication

LR Distinguishes birch canopies which received AP pH 2.5-4.5 (5.5b) (p < 0.05) DLI Early detection of SO2 injury Turbidity Early detection of SO2 damage to

spruce ECD Detects general stress 613Cvalues Mirrors previous exposures of trees ETR Estimates general stress

‘AA, ascorbic acid; AP, acid precipitation; AX, antheraxanthin; B, branching; BC, (cellular) buffer capacity (diminished); BD, branch density; C,, stomatal conductance; Cyt f, cytochrome-f; DHA, dehydroascorbate; DLI, delayed light imaging; DWIEWc,l.2, dry weightlfresh weight ratio differences between current and one- or two-year-old needles; ECD, electrical conductance in leaf diffusate; E 6 , electrical &sue resistance; General stress, water, high temperature, frost- and air pollution stress in the field; GDH, glutamate dehydrogenase; GL, gene loci; GOT, glutamate oxaloacetate transaminase; G-bPDH, glucose-6-phosphate dehydrogenase; GR, GSH-reductase; GSH, glutathione; H, Height; L, total number of Lammas shoots; LR, leaf reflectance; Mg, magnesium; MRIG/EC, mean radial increment growth related to environmental conditions; NFD, novel forest decline; P-700, reaction centre of photosystem I; PS, photosynthesis; Qg, Qg protein as an indicator of the photosystem I1 complex; S,,, total sulphur; TET, thylakoid electron transport; TR, transpiration; Unknown, a stress complex that induces NFD, probably with O3as an important inducing factor; VX, violaxanthin; WS, water stress. Reference pH level.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

91

a longer time period. Rates of photosynthesis and stornatal opening also varied with other parameters, such as light intensity, temperature, leaf and plant age, which complicate the interpretation of data from field exposures with a bioindicative purpose. The physiological parameters, however, were proven to be potentially useful bioindicators in several studies. From the previous sections it has become clear that plants with inherently high photosynthetic rates are generally more sensitive to pollutants than plants with a low photosynthetic rate. A recent example, given by Oleksyn and Bialobok (1986), is quoted in Table VII. Using this rule to select resistant plants, however, would at the same time select less productive plants for a clean environment, which means that the price paid for resistance would often be too high. Other previously quoted studies found an inherently high diffusive resistance or a rapid closure of stomata under stress to protect plants from pollutant uptake and, therefore, from injury. Although this was not always the case, Reich (1987) concluded for a large number of studies and species that an inherited leaf diffusiveconductance does predict both uptake, photosynthesis and yield in agricultural crop plants, hardwoods and conifers. If otherwise appropriate, plants with an inherently high diffusive resistance and/or rapid closure of stomata under stress could, therefore, be selected as being the generally more air pollution resistant cultivars. Saxe (1986a) found a significant correlation between the photosynthesis response of eight species or cultivars of pot plants exposed for 4 days in a controlled environment to NO and the response of pot plants grown for 4 months in a commercial greenhouse with an NO-polluted, CO2-enriched atmosphere. Recent calculations have demonstrated that the photosynthetic NO response over as little as a few hours could also work as a significant bioindicator (Saxe, unpublished data). Measurements of photosynthesis in pot plants exposed short-term to (1 ppm) of NO in a controlled environment may, therefore, help in screening NO resistant pot plants for growth in commercial greenhouses. Saxe and Murali (1989a,b,c) and Saxe (1989) defined a series of tests that each predict the long-term effects of the unknown stress in the field which causes the novel spruce decline. Each test consisted of a 4-5 h acute exposure of spruce seedlings to one of the common air pollutants or their combination in a controlled environment, while recording the photosynthetic and stornatal responses before, during and after pollutant exposure. The investigators did not claim any causal relationship between S02, NOz and novel decline, though they did suggest a causal relationship between 0 3 induced inhibition of net photosynthesis and novel forest decline. Such relationships, however, are irrelevant to the function and usefulness of the given bioindicator methods. Landhold (1982) found a good correlation between visible injury of beech trees fumigated with 75 ppb SO;! for 5 weeks, and 14C02fixation (and total

92

H. SAXE

sulphur content). This bioindication of SO2 injury could be developed into a valid test in the field, and would be easier to implement on a large scale than are physiological measurements in cuvettes. Dietz et al. (1988) and Wild et al. (1988) found thylakoid electron transport, reaction centres I and I1 and cytochrome f to be lower in spruce trees with declined vitality, even if the needles were still green. The investigators considered these bioindications of a latent decline to be among the primary events in novel forest decline. This view seems to be in conflict with previously quoted studies, since several of these found thylakoid electron transport to be merely a secondary event in injury, although air pollution was indeed quoted to be at least partly responsible for the novel forest decline.

Leaf pigments. Numerous studies have used the measurement of chlorophyll and carotenoid contents as a bioindicator for air pollutant levels and effects (Darrall and Jager, 1984). Sulphur dioxide and 0 3 generally decreased the concentration of these pigments, while NO1 was found to increase them, working as a nutrient. Chlorophyll content may be used as a more objective measure of visible injury than visual assessment of injury, but sometimes also to reveal invisible injury (Rabe and Kreeb, 1979). For impartial evaluation of leaf injury (either chlorosis, necrosis or bronzing by ozone) without chemical analysis, Michaels (1988) has suggested the use of digital image analysis. Since the chlorophyll level in plants is sensitive to most abiotic and biotic factors, and varies with nutritional status of the plant, the time of year and leaf age, Darrall and Jager (1984) considered chlorophyll to be toogeneral a bioindicator to be used alone to indicate a particular air pollution stress in the field. Nevertheless, Takemoto et al. (1988b) found changes in chlorophyll and carotenoid contents in long-term O3field fumigated alfalfa to be a precise bioindication of the loss in yield, while losses due to acid fog were over-estimated. Mehlhorn et al. (1988) found the violaxanthin-to-antheraxanthin ratio to be a very powerful bioindicator for early detection of novel decline of spruce. However, neither chlorophylls nor other carotenoids or any other ratios between the investigated leaf pigments correlated significantly with novel forest decline (NFD) in three woody species. Chlorophyll fluorescence. The advantages of using fluorescence characteristics as a bioindicator for a potential injury to plants are that a large number of determinations can be made quickly and with high precision in intact plants (Darrall and Jager, 1984). Schmidt et al. (1988) found the slow induction kinetics to be a more sensitive parameter than either the rapid induction kinetics (Kautsky effect) or decay kinetics, and interpreted their results to mean that the primary injuiy by SO2 in spinach plants was acidificationinterference with the light activated Calvin cycle enzymes. But,

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since the fluorescence characteristics depend on other stress factors and not just on air pollutants, it is too general a bioindicator to be used alone to indicate a particular air pollutant in the field. Strasser et al. (1987) demonstrated, however, that a newly defined light adaptation index which uses measurements of chlorophyll fluorescence kinetics, could function as a sensitive early detection parameter, revealing injury before both the vitality index and the appearance of visible 0 3 symptoms on beech and poplar foliage exposed to 100-300 ppb 0 3 for 5-9 days.

Endogenous elements. With elements such as sulphur from SOz, that adsorb to the surface as well as being taken into the needles of conifers, the time of year at which sampling is done is important, since much sulphur is washed off the needles in the spring (Huttenen et al., 1985). Several investigators, however, have used sulphur successfully as a bioindicator of pollution injury. Bytnerowicz et al. (1987) found total sulphur and buffering capacity to be affected in wheat after 3 weeks exposure to low levels of SO2, and suggested these responses could be used as early indicators of SO2 stress. The responses were more sensitive than changes in chlorophyll or stomata1 diffusion. For bioindication of SOz injury, changes in total sulphur and buffer capacity may be combined with other changes in endogenous elements such as an increase in glutamine and ammonia, as well as stimulation of glutamate dehydrogenase (Jager and Klein, 1977). A 10-yearstudy by Legge etal. (1988) indicated that separation of sulphur into inorganic and organic fractions in pine species exposed to ambient pollution levels in Western Canada, and determining SO2 sulphur/organic sulphur gave a most precise early warning tool for environmental management. Similar results were found by Gasch et al. (1988) for spruce in Germany. Studies by Mehlhorn et al. (1988) indicated that increased sulphur and decreased magnesium content of needles in spruce affected by novel decline may serve as early diagnosis of this NFD (Table VII). The changes of nitrogen and calcium contents were also significant (p < 0.01). Endogenous metabolites. The content of free proline rises in response to water, high temperature and frost stress, but also with high levels of air pollutants (Erickson and Dashek, 1982; Karolewski, 1985; Anbazhagan et al., 1988). Proline constitutes an important osmotic agent in cells, participates in the reconstruction of chlorophyll, activates the Krebs cycle and constitutes an energy source, and is an important part of structural proteins and enzymes. It may participate in important repair mechanisms. Spray application of polyamines have, not surprisingly, been used with some success in reducing symptoms of novel forest decline in small-scale experiments. But, since the level of free proline rises in response to general stress,

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and this reaction depends on the nutritional status of the plants (Anbazhagan et al., 1988), it is too general a bioindicator to be used alone to indicate a particular air pollution stress in the field. Glutathione (GSH) is often found to be the predominant free sulphydryl in water extracts of leaves, and is, therefore, an important element in the plant antioxidant system, probably by preventing the accumulation of H202 and free radicals within subcellular compartments (Chiment et al., 1986). GSH was rapidly decreased in Sedum leaves exposed to 03,while it increased in soybean in response to SO2 exposure (Chiment et al., 1986), and in spruce and fir in response to SO2 and O3(Mehlhorn et al., 1986). The rapid decrease in GSH levels in whole leaves by O3 (Castillo and Grippin, 1988) could be explained by its protection of proteins and fatty acids from oxidants, which directly or indirectly oxidize GSH to GSSH. The GSH increase induced by SO2 and SO2 + 0 3 was not explained, except that GSH reductase (GR) was generally stimulated by SO2 (and 03). The pollutantinduced changes in GSH levels could be used as a general bioindicator for oxidant stress (including certain reaction products of S02, as explained in the following section). GSH reductase (GR) was found to have a higher inherent specific activity in O3 resistant cultivars than in O3 sensitive cultivars of Phaseolus vulgaris (Guri, 1983). The GSH level, therefore, dropped to significantly lower levels in the sensitive bean cultivars. The response in GSH level upon 0 3 exposure, or the inherent GR activity could possibly be used as bioindicators for O3 sensitive plants. Guri (1983) also found ascorbate to decline in response to 0 3 , but the same in both sensitive and resistant bean cultivars. Ascorbic acid (ascorbate, AA) is a powerful reductant. It may detoxify the products of SO2 either by mediating the reduction of SO2 and sulphite to H2S and/or by scavenging free radicals. Varshney and Varshney (1984) found the SO;! resistant Zea mays to have a higher ascorbic acid content in the leaves than the SO2-sensitivePhaseolus radiatus and Brassica n i p , and a slower decline in AA under SO2 exposure. These parameters may be used for bioindication of SO;!resistance with some, but not all, species (Guri, 1983; Senger et al., 1986). Senger et al. (1986) found both GSH and tocopherol to increase after either acid mist or 0 3 exposure of spruce needles, with an antagonistic or a more-than-additive response to the combined exposure. Since the data in the main table in the paper by Senger et al. (1986), however, were contradicted by their conclusion, no deductions as to the possible use of tocopherol or GSH can be drawn from that investigation. While exposure of soybean plants to O3 and SO;!alone, increased the oil content of soybean seeds, combined exposure to both gases had no significant effects (Grunwald and Endress, 1988). Ozone altered the fatty acid content and composition, while SO2 alone or the combined exposures did not significantly change these patterns. This could potentially serve as a

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bioindication for injuries by O3or SO2to soybean, but more work would be needed before this test could be put to practical use. Endogenous enzyme activity. While chlorophyll and protein contents were unaffected or decreased in response to air pollution, enzyme activities were typically found to increase (Rabe and Kreeb 1979; Grundwald and Endress, 1988). Of the numerous reports of changes in enzyme activities associated with pollution fumigation episodes, several authors have described the use of these responses as bioindication of air pollution (Darrall and Jager, 1984). Ozone exposure has often been found to affect peroxidase (PO) and superoxide dismutase (SOD) activities and, as described above, the leaf content of glutathione (GSH) and total ascorbic acid (TAA): ascorbic acid (AA) + dehydroascorbate (DHA) (Castillo and Greppin, 1988; Castillo et a f . , 1988). In a complete analysis of these responses, Castillo and Greppin (1988) found that AA and GSH levels decreased in whole Sedum leaves exposed to 0 3 , with a rapid recovery after exposure, while O3 increased DHA levels and total AA in the apoplast. While neither DHA reductase nor glutathione reductase (GR) was found in the apoplast in Sedum album, Castillo and Greppin (1988) suggested the cycle of reactions depicted in Fig. 10, which is similar to the “AA-GSH-cycle” for chloroplasts suggested by Foyer and Halliwell (1976), also depicted in Fig. 10. In cell extracts O3 exposure activated DHA reductase, while GTH reductase was unaffected. These links in the apoplastkycloplasm and chloroplast regions help explain the functionality of the observed stimulations in PO and SOD activities, changes in DHA reductase and glucose-6-phosphatedehydrogenase, as well as changes in AA, DHA and GSH levels. They are all suggested to be part of a detoxifying response to free radicals like, for example, those produced during O3exposure (Lee and Bennett, 1982;McKersie et al., 1982;Petolino et a f . , 1983; Decleire et al., 1984; Tanaka et a f . , 1985; Jager et al., 1986). In contrast to this conclusion, Patton and Garraway (1986) suggested that PO activity had a predisposing rather than a detoxifying role in visible scorching by O3 exposure in poplar. They found young poplar leaves and leaves of resistant clones to contain little or no PO activity. Patton and Garraway (1986) speculated that phenols from vacuoles and chloroplasts entered the cytoplasm upon O3 destruction of membranes; PO activity would then convert the phenols into quinones which contribute to the formation of cytotoxic polymers. Several investigators have claimed that the peroxidase activity response to O3accurately predicts relative O3sensitivities in screening procedures (e.g. Podleckis et a f . , 1984), while others found a PO response absolutely worthless as a bioindicator (e.g. Endress et al., 1980). PO activity is a complex parameter in bioindication, since it was found to be missing in some plants (Patton and Garraway, 1986), it was found to vary with the time of

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year (Matschke, 1985; Castillo et al., 1988), with leaf age (Patton and Garraway, 1986), with nutritional level (Dohmen, 1986), and since it was also affected by other pollutants (SO2 (Murray, 1984b), or acid rain (Dohmen, 1986)), as well as by biotic stress. The use of PO activity as a quick indicator must, therefore, be restricted to use with certain plants and life cycle stages, under defined conditions and where the pollutant or stress has already been identified (Matschke, 1985). Sulphur dioxide toxicity involves the formation of both sulphite and free radicals. Sandmann and Gonzales (1989) found SO2 to induce radical peroxidative processes quite analogous to the effects of 0 3 . This could make a PO or SOD bioindication possible for S02. An investigation by Katainen et al. (1984), however, concluded that changes in PO activity caused by SO2 fumigations were not a suitable bioindicator for changes in photosynthesis in Scots pine needles. Nor may the inhibited SOD activity (Agrawal et al., 1987), be a very good bioindicator, since effects of leaf age and the usual range of environmental factors affecting enzymes complicate the indication as much as described for the PO response. Several enzymes were more sensitive than PO or SOD activities to low levels of SO2 exposure, e.g. glutamate dehydrogenase activity (as quoted by Darrall and Jager (1984)), and glycosidases (Bucher-Wallin et al., 1979). But, the general conclusion regarding these enzymes as bioindicators was, as for the PO responses, that enzyme responses were dependent on too many factors to be reliable bioindicators for a specific pollutant under the complex ambient conditions. Stress ethylene production. Ethylene is an endogenous metabolite, and volatile phytohormone that moves freely (even out of the plant) to coordinate a wide variety of developmental processes, not least those associated with senescence. Stress ethylene in response to air pollution is formed by the regular pathway from methionine (Meyer et al., 1987), and in some species also by additional pathways (Fig. 12). The production of stress ethylene has been suggested in several studies to be a useful bioindicator of short-term, high level air pollution stress. Stress ethylene was found to correlate with visible injury and reduction in plant growth. It must be recognized, however, that other stresses, e.g. water stress, temperature extremes, insect damage, disease, and mechanical wounding, also stimulate ethylene production. Quoting references preceding 1982, Darrall and Jager (1984) concluded that ethylene evolution should only be used as a bioindicator for air pollutants under short-term, high level pollutant exposures, since ethylene emissions during long-term, low level exposures are variable. Bucher (1981) reported a cyclical pattern of stress ethylene emission from SO2-exposed forest trees, while Kimmerer and Kozlowski (1982) found SO2-exposed birch seedlings to emit ethylene continuously until 80% of the tissues were injured. Chen and Wellburn (1989)

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luWl p t h r y nf ethylene f o r m a t b ~

X-CH2 CH2-Y

kH2CH2-Y

rT,

M m TR

-

C2H4 + Y‘

(Additional scheme

4

Fig. 12. The usual way of ethylene formation from methionine, via (S)-adenosylmethionin (SAM) and 1-aminocyclopropane-1-carboxylicacid (ACC). Synthesis of ACC is the key regulatory step (1, ACC synthase) and 1-malonyl(amino)cyclopropane-1-carboxylic acid (MACC) is formed as a side reaction. Ethylene-forming enzyme (2, EFE) may also play a role in the regulation of stress ethylene. Wellburn (personal communication) found evidence for additional pathways for ethylene formation in Norway spruce (schemes A and B), which depended on the presence of metal ions, but not ACC.

reported a continuous emission of ethylene, even after acid-mist-treated spruce needles had turned completely brown. While the damage developed, ACC (1-aminocyclopropane-1-carboxylicacid) levels and EFE (ethylene forming enzyme) activity gradually rose, but the levels of MACC (1malonyl(amino)cyclopropane-1-carboxylic acid) were unchanged. Completely brown needles lost their capacity to convert ACC to ethylene, but large emissions of ethylene were maintained independently of ACC. When acid-mist treatment was discontinued, ethylene emissions and ACC levels returned slowly to their normal levels within several weeks. Since (AVG), aminoethoxyvinylglycine,a strong inhibitor of ACC synthase, did not inhibit stress ethylene induced in spruce needles by bisulphite or sulphate, an alternative pathway and regulation in ethylene formation by spruce needles was indicated (Wellburn, personal communication) (Fig. 12). Only when rates of ethylene emissions were high and continuous,

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as found in red and Norway spruce exposed to acidic mists, were they suitable for diagnostic purposes in the field. Stan and Schicker (1982) found a given external dose of 0 3 to induce more stress ethylene the shorter the individual exposure period (45, 30 and 15min). Since these short intervals probably differ in the resulting degree of stornatal closure one reason for problems with long-term studies could be the dependence of stress ethylene emission on stornatal opening, as demonstrated by Rodecap and Tingey (1986). If stress ethylene emissions were related to the diffusive resistance of the leaves, it might best be used as a bioindicator with long-term exposures. Light was also found to inhibit O3induced stress ethylene, probably by limiting the conversion of its immediate precursor ACC to ethylene (Rodecap and Tingey, 1983). The environmental conditions and physiological responses and adaptation are of predominant importance when measuring stress ethylene (Meyer et al., 1987). Decoteau et al. (1986) proved the difficulties in using bioindicators in the field, since they found no correlation between stress ethylene production, chlorophyll loss, or even visual injury, and the actual yield reductions in wheat exposed to O3 in the field. Other recent studies, however, have successfully used ethylene as a bioindicator. Pel1 and Puente (1986) found both 294ppb O3 and acid rain (pH 2.8-3.8) to induce visible injury in oats. Only 03,however, induced stress ethylene. Fuhrer (1985) reported a correlation between novel forest decline (NFD) and ethylene responses. The chronic stress that induced NFD had two different ethylene responses: a low ethylene production (3 times the control) and accumulation of ACC (as conjugated l-aminocyclopropane-lcarboxylic acid, MACC) associated with reduced needle growth, and a high ethylene production (10-20 times the control) associated with chlorophyll breakdown, followed by premature abscission of the needles in Abies alba. Mehlhorn and Wellburn (1987) found that stress ethylene indicated the sensitivityof pea seedlings to O3exposure. Seedlings exposed for 3 weeks to 150ppb 03,however, had half the ethylene emissions as controls and showed no visible injury, while those exposed for only 1day after 3 weeks in clean air emitted twice the ethylene emission of the controls and were visibly injured by 03.Plants sprayed with AVG inhibited visible injury in pea (Mehlhorn and Wellburn, 1987) and, as previously mentioned, also stomatal closure and declining photosynthesis in soybean (Fig. 7) (Taylor et al., 1988). Combined exposure with O3and other pollutants (NO and/or NOz) increased ethylene emissions and visible injury in pea. NO, exposure alone also increased ethylene emissions and predisposed pea plants to subsequent visible O3 injury. As previously mentioned, since stress ethylene could be provoked by several environmental stresses, Mehlhorn and Wellburn (1987) suggested that O3 and ethylene are major factors in NFD, reasoning that there is more O3and general environmental stress, and more severe NFD in

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the mountains. Mehlhorn et al. (1988) consequently reported ethylene emissions to be a highly significant bioindication of novel spruce decline. These aspects are discussed further in Sections X.D and X.E. Anatomical and morphological analysis. Changes in anatomy and morphology are not per se bioindications of air pollutants or any other specific environmental factor, but the result of complex environmental interactions with the genetic capability of the plants. Using radial growth in trees as an indicator in relation to known environmental and genetic parameters has, however, found some use. Benoit et al. (1982) found general reductions in the radial growth of native eastern white pine over a 446 km long forest expanse in the eastern USA during the period 1955-1978. Precipitation was positively correlated with radial growth until 1964, but negatively after that year. The mean annual radial increment growth was significantly smaller in O3sensitive than in O3resistant trees. A similar study by Puckett (1982) on the comparative radial growth of white pine, pitch pine, eastern hemlock and chestnut oak, with temperature and precipitation in three time periods (1901-1920, 1926-1945 and 19541973) suggested that the reduced growth in the last time period was non-climatic. It correlated with suspected increase in acid rain and air pollution in the south-eastern part of New York state in the early 1950s. The pollution stress may have had a direct effect and/or it may have caused the prevailing climatic conditions to be more limiting to tree growth. Saxe and Murali (1989a) found several morphological characteristics of young Picea abies half-siblings to correlate with novel decline symptoms of their clonal parents in a Danish seed orchard: height, branch density, lammas shoots. Roloff (1986) has developed a classification of the branching structure in hardwoods to determine and predict forest decline. A use of existing anatomical and morphological parameters for bioindication has been demonstrated by other investigators (Table VII). Tuomisto (1988) found the erosion of basic wax structures on 1-year-old Norway spruce needles to be a specific, reliable and reproducible bioindicator of air pollution stress. The erosion could easily be distinguished from mechanical injury and damage by insects, but included indirect effects of air pollution through the soil ecosystem. Mehlhorn et al. (1988) found the ratio of dry weight and fresh weight in current-year needles relative to 1-to 2-year-old needles of Picea abies to indicate novel decline in Western Europe. Genetic analysis. Air pollution was demonstrated to exert a distinct selection pressure on isozyme-gene systems in Norway spruce (Scholz and Bergmann, 1984). Geburek et al. (1987) identified 46 relatively sensitive and 45 relatively tolerant individuals of Pinus sylvestris stressed with air pollution in a field trial. They found that allele and/or genotype frequencies at gene loci of glutamate dehydrogenase (GDH) and glutamate oxaloacetate

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transaminase (GOT) differed significantly between sensitive and tolerant plants. Bergmann and Scholz (1987) performed a similar analysis for Picea abies, and found that glucose-6-phosphate dehydrogenase (G-6-PDH) and GOT differed in SO2 + O3fumigated and non-fumigated samples, and that fumigated samples were consistent with field-grown samples in Hartzen with a similar ambient pollutant stress. By electrophoretic detection of these loci, it should, therefore, be possible to select individuals tolerant to general or more specific air pollution stress.

Miscellaneous methods. Ellenson and Amundson (1982) found that a 2ppm SO2 exposure of soybean for 3.5 h induced a delayed light emission (DLE) after only 10min of fumigation, and visible injury after 5 days. The DLE phenomenon is universal in green plants and results when lightgenerated photosynthetic intermediates recombine in the dark to produce an electronically excited state of chlorophyll that gives rise to fluorescence. The response was so sensitive that lower pollutant concentrations should have been tried, to evaluate the use of this method as an early detection bioindication with ambient pollutant stress. Swedish investigations in this respect are underway (Bjorn, Lund University, Institute of Plant Physiology, personal communication). The electrical conductivity in needle diffusate (ECD) gave a rough estimate of the “state” of stressed spruce plants. Water stress and SO2 exposure were found to increase the conductivity, while O3lowered it (Keller, 1986). This bioindication was so sensitive to SO2 that 6 months exposure to just 25 ppb SO2 gave a highly significant response, and without visible symptoms of injury. Water stress worked antagonistically with SO2 reducing the SO2 induced increase in ECD. There was little response with 03.The method detected changes due to air pollution, but it did not distinguish between ions leached from the apoplast and those from the cell interior, and thus did not provide proof of changes in membrane permeability. Another method reported to involve an electrical measurement included the assessment of the impedance in the tissues under the bark layer of Norway spruce (Salema et al., 1987; Tomiczek, 1987). Spruce trees fumigated 6 hours daily for one or two weeks with 250ppb O3 showed an increase of 38% and 52%, respectively in electrical resistance in cambium, phloem and xylem tissues of the inner bark. Tomiczek (1987) speculated that the increase was caused by a decline in photosynthesis andlor diffusive conductance, both of which would lead to a reduction of water and ion transport in the inner bark tissues. The sensitivity of the method could make it a valid bioindication for air pollution and general stress, but its low specificity would make results difficult to interpret. Hartel (1972) found that the turbidity of aqueous extracts of spruce needles increased in plants exposed to SO2 at concentrations that did not induce visible injury. The increase in turbidity was probably the result of

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higher solubility of substances such as polysaccharides, proteins, polyphenols, pectins and tannins. The turbidity correlated positively with sulphite content in needles and annual radial increment growth. This bioindication is quick to perform but, as it has not been evaluated with other pollutants and general stress, its specificity is unknown, although it has been used several times since the early 1950s (as quoted by Darrall and Jager (1984)). Cape et a f .(1988) found spruce trees in less polluted sites in Britain to have a higher Hartel-turbidity than more polluted sites in the Netherlands. The Hartel bioindication was, therefore, dependent on factors other than air pollution. Greitner and Winner (1988) found significant increases of negative SL3C values in radish and soybean plants exposed for 19 days to 120ppb 01,in parallel with reduced growth and stomata1 closure. Martin et al. (1988) found similar increases of negative S13C values in a range of woody and herbaceous plants exposed for 25-28 days to combinations of 47-165 ppb SO2, 50-161 ppb 0 3 and 109-190ppb NOz. But the correlation between visible leaf injury at the last day of exposure and changes in S13C values was low. There were eight cases of changed S13C values with no visible injury, eight cases of visible injury without changes in SI3C values, and six cases where both changes in 6I3Cvalues and visible injury occurred with the same species and exposure regime. This method, therefore, may at the best be used as a non-specific bioindicator of pollution stress, and only for selected species. Martin et al. (1988), however, found a good correlation between air pollution levels in 1972-1976 and 1982-1986 and corresponding 613C values in wood, with less negative values and smaller radial increment growth in the more polluted air (from a local source) during the early period. This could be used to evaluate previous levels of air pollution, though the specificity of the method would still be doubtful. Ashenden and Williams (1988) distinguished birch canopies exposed to simulated acid rain (pH 2.5,3.5 and 4.5) for 34 and 75 weeks by different leaf reflectance in the visible and near-infra-red wavelength regions, and found significance after 34 weeks. The investigators believe the method has potential as a remote sensing bioindication (satellite photography), but emphasized that plants in the field vary more than their experimental material with respect to within-species variation. age, fungal and insect infestation, foliage and crown structure, mixed species canopies, understorey vegetation and soil background. D. BIOINDICATIONS FOR EARLY DETECTION OF NOVEL FOREST DECLINE

Forest decline induced by natural causes (biotic or abiotic) has been recognized and recorded for centuries, while anthropogenic air pollution induced decline came with the industrial revolution. The novel (recent) decline, caused by a complex of stress factors, has only been recognized for a few decades.

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As stated in SectionX.C, analyses of tree-ringgrowth patterns indicated that novel forest decline, currently affecting forests throughout Europe and North America, started between 20 and 30 years ago in the USA (LeBlancet al., 1987). The scientific discussion about novel forest decline in Western Europe was initiated by reports on fir dieback in southern Germany. Because research activities have so far failed to isolate pathogens that may be responsible for this disease, most scientists are now inclined to view that a non-biological cause is responsible for the problem. At first, the novel decline was thought to be caused by the impact of SO2 and its reaction products leading to soil acidification processes. Today, we recognize that direct effects of air pollution and climate play a more important role. Suggested causations in forest decline have been widely published in recent years (e.g. Ashmore et al., 1985; Blank, 1985; Schiitt and Cowling, 1985; Kozlowski, 1986a,b; Krause et al., 1986; Hinrichsen, 1986, 1987; Innes, 1987; Franklin et al., 1987; Matzner and Ulrich, 1987; Prim et al., 1987; Prinz, 1987, 1988; Cape et al., 1988; Gross et al., 1988; Klein, 1988; Mehlhorn et al., 1988; Prinz and Krause, 1988; Seufert et al., 1988), and the multiple symptoms have been illustrated by Hartmann et al. (1988). Since 1980 and 1982in southern and other parts of Germany, respectively, damage to Norway spruce and other forest trees has significantly increased. The most important symptom observed is the so-called mountainous needle yellowing, associated with the deficiency of magnesium. Although spruce and fir decline culminated in the mid 1980s, the decline of deciduous trees has increased steadily since 1982. However, the relative roles of climate, soil and air pollutants in causing the various symptoms are not yet clear. Final scientificproof for the causation of novel forest decline may never materialize. But, at any one time, we will have a favourite or mostprobable explanation. Recent research has especially answered questions about the symptom of needle yellowing. The long-term temporal development in needle yellowing correlates with tropospheric O3(Prinz 1987,1988; Prinz et al., 1987; Prinz and Krause, 1988), which together with NO, are the major pollutants to increase during the last decades (Pfeffer and Buck 1985), and with a depletion of nutrients (except nitrogen) in the soil due to progressing soil acidification. The vertical profile of O3 as well as fog events seem to account for the increase in decline with altitude, on top of which geogenic soil factors induce the spatial differentiation. The short-term temporal development, on the other hand, seems to be incited by climaticfactors. The development of damage in all affected tree species would not have been apparent without climate as a predisposing factor. However, climate alone could not have caused forest decline to such an extent without appropriate unfavourable soil and/or pollution conditions (Prinz and Krause, 1988). The elucidation of the interaction of these factors is a major task in future research, since major gaps still exist, despite all the scientific progress made so far.

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A recent search for bioindicators of novel forest decline has also brought possible mechanisms in a new perspective. Since SOz partly inhibits photosynthesis by inhibition of RuBPC in experimental fumigations, Weidner and Kraus (1987) looked for activities of this enzyme in four needle generations, but concluded that RuBPC was not involved in spruce decline. Cape et al. (1988) tested 30 different variables (reviewed by Wolfenden et al. (1988)), among which most of the bioindications are described above (Section X.C), on three tree species (Picea abies (Norway spruce), Pinus sylvestris (Scots pine) and Fagus sylvatica (beech)) collected on 14 different sites from northern Scotland to southern Germany. The 30- to 40-year-old trees were also scored with the EEC system (Bauer, 1984) for symptoms of novel decline, and records of soil, climate and air pollution were taken from each site. They found significant (p < 0.05) differences between the sites for 14 of the tested variables or variable ratios for Norway spruce. However, genotype, soil type, climate, etc., played a major role. The 14 variables were: needle contents of calcium, sulphur and nitrogen, tissue pH, contact angle (of a drop on the leaf surface), magnesium, dust as percent dry weight, violaxanthin/antheraxanthin,chlorophylls/xanthophylls,chlorophyll a and b , potassium, violaxanthin/lutein, a-tocopherol, dry weight as percent fresh weight. Regarding photosynthesis, Cape et al. (1988) found no significant changes until leaves showed yellowing. This parameter was, therefore, not a valid early detection parameter. It is generally accepted that the operation of the xanthophyll cycle of violaxanthidantheraxanthin protects photosynthetic membranes against internally generated photo-oxidative reactions (Krinsky, 1966; Goodwin and Mercer, 1983; Elstner and Osswald, 1984), but the site-dependent change was particularly interesting since it suggested a carotenoid mitigation or absorption of harmful oxidations from outside the plant. Mehlhorn et al. (1988) further analysed the data reported by Cape et al. (1988) by logistic regression in search for site-independent relationships between possible diagnostic or explanatory variables and recorded visible damage. The investigators found three such variables for spruce which could make it possible to assess tree damage related to forest decline in the future without an over-reliance on subjective visual observations and classifications. These variables were mentioned in Section X.C (ethylene emission from 2-year-old needles, the violaxanthidantheraxanthin ratio, and dryweight/freshweight ratio differences between current and 1- or 2-year-old needles). The best fit was obtained when the three major explanatory variables identified by Mehlhorn et al. (1988) were combined. To test the explanatory value of these three parameters for spruce, their response to individual and combined 0 3 (up to 185ppb), SO2 (up to 28 ppb) and acid rain (pH 4.G5.0) exposures were tested in a 4-year exposure experiment with 4- to 8-year-old spruce trees in open-top chambers (Seufert et al., 1988). Ozone had the strongest single effect, which was further

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enhanced by additional SO2 and, possibly, also acid rain. There was a significant interaction between O3 and SO2 in causing increased rates of (stress) ethylene formation (p < 0.01). The claim by Prinz and colleagues that O3 is the major cause of novel forest decline was thus supported by Mehlhorn et al. (1988). This was also suggested by recent data of Saxe and Murali (1989c), and by very recent investigations where scientists at Lancaster University have found that 0 3 fumigated Norway spruce exhibited the same symptoms as those found by Prinz and colleagues, which again were identical to those found in forest areas affected by novel forest decline. The “problem” with inducing these symptoms were that they did not appear the first year, but only during the second year with low level O3 exposures (Wellburn, personal communication). E. BIOINDICATION AS A DIAGNOSTIC TOOL FOR SELECTING PLANTS RESISTANT TO NOVEL FOREST DECLINE AND SPECIFIC AIR POLLUTANTS

It has been conjectured that we have no realistic chance of coping with novel forest decline in Europe by breeding more resistant plant species, or by improving forest management (Anon., 1987). I do not agree with this view. As we are not yet certain of the causes of novel decline, we cannot fight the recent forest symptoms efficiently. The work of Cape etal. (1988), Mehler et al. (1988) and Seufert et al. (1988) all suggesting a sensitive means of early diagnosis of novel decline in Norway spruce caused by O3 + SO2 + AP, will only help protect our forest if air pollution is diminished in the identified regions. Furthermore a quick reduction in the present levels of certain air pollutants, however good and necessary a remedy it may turn out to be, may not solve our problem. Factors other than 0 3 , SO2 and AP could at a later time prove sufficient to induce the novel decline by themselves. Whether or not it is accepted that photochemical oxidants in combination with SO2, acid precipitation and other new and old stress factors are responsible for novel forest decline in Europe and/or North America, we need to select resistant tree provenances and individuals for cloning and propagation and replanting of forest areas affected by the recent decline. Saxe and Murali (1989a,b,c) and Saxe (1989) have suggested a series of quick methods for this selection, as was mentioned in Section X.C. When comparing the relative severity of novel decline symptoms on nine clones of 20- to 24-yearold Norway spruce growing in a Danish seed orchard, and the relative responses of photosynthesis and transpiration under acute laboratory exposures of 4- to 5-year-old half-sibling progeny, to 0 3 , S02, NO2 and the combination of the latter two gases, they found significant correlations. They concluded that acute air pollution exposures of young clones in a controlled environment would deliver a precise diagnosis (p < 0.0025-0.05) for selecting the trees most resistant to novel decline. It should be tested if

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even younger trees can be used in this diagnosis, and the tests should be extended to relevant species and provenances. Another example of the use of short-term physiological responses to select plants resistant to a specific air pollutant has also been quoted above, i.e. that regarding pot plants resistant to NO occurring in commercial greenhouses. Finally, it must be stressed that responses during short-term, high level exposure to air pollutants do not automatically agree with those during long-term, low level exposures. This is clear from the work reviewed in Sections 11-VII, while a specific example with the species described in the present section was given by Garsed and Rutter (1982) for three Picea and five Pinus populations. F. CONCLUSION

We now know of so many bioindicative methods that, even though their individual specificity is often broad, their combined use could significantly participate in protecting plants, crops and ecosystems against air pollution injury in the future. The practical use of several new bioindicative methods is ready to move from “the academic desk” to large-scale field trials, as already exemplified by a few successful studies. Comparative research of plant responses to air pollution affected by the complex and unstable biotic and abiotic environment in the open field, with the plant responses in the controlled environment must inevitably proceed to define the proper use of bioindicative methods for specific situations.

ACKNOWLEDGEMENTS I am grateful to The National Forest and Nature Agency, and The Danish Agricultural and Veterinary Research Council for their support. I am also grateful to the libraries at Rise and The Royal Veterinary & Agricultural university for their helpful assistance, and to R. Rajagopal and B. Saxe for proof-reading.

REFERENCES Adams, R. M. and Crocker, T. D. (1988). Model requirements for economic evaluations of pollution impacts upon agriculture. In “Assessment of Crop Loss from Air Pollutants” (W. W. Heck, 0.C. Taylor and D. T. Tingey, eds), Chap. 19. Elsevier Applied Science, London. Adams, W. W., 111, Winter, K. and Lanzl, A. (1989). Light and the maintenance of photosynthetic competence in leaves of Populus balsamifera L. during shortterm exposures to high concentrations of sulfur dioxide. Planta 177, 91-97.

106

H. SAXE

Agrawal, M., Nandi, P. K., Agrawal, S. B. and Rao, D. N. (1987). Superoxide dismutase: an indicator of SOz-tolerance in plants. Current Science 56,35-36. Alscher, R. (1984). Effects of SOz on light-modulated enzyme reactions. In “Gaseous Air Pollutants and Plant Metabolism” (M. J. Koziol and F. R. Whatley, eds), Chap. 14. Butterworths, London. Alscher, R., Bower, J. L. and Zipfel, W. (1987). The bases for different sensitivities of photosynthesis to SOz in two cultivars of pea. Journal of Experimental Botany 38, 99-108. Amiro, B. D. and Gillespie, T. J. (1985). Leaf conductance response of Phaseolus vulgaris to ozone flux density. Atmospheric Environment 19,807-810. Amiro, B. D., Gillespie, T. J. and Thurtell, G. W. (1984). Injury response of Phaseolus vulgaris to ozone flux density. Atmospheric Environment 18,12071215. Amthor, J. S. (1988). Growth and maintenance respiration in leaves of bean (Phaseolus vulgaris L.) exposed to ozone in open-top chambers in the field. New Phytologist 110, 319-325. Amthor, J. S. and Cumming, J. R. (1988). Low levels of ozone increase bean leaf maintenance respiration. Canadian Journal of Botany 66,724-726. Amundson, R. G. and Weinstein, L. H. (1981). Joint action of sulfur dioxide and nitrogen dioxide on foliar injury and stomata1 behaviour in soybean. Journal of Environmental Quality 10,204-206. Amundson, R. G., Kohut, R. J., Schoettle, A. W., Raba, R. M. and Reich, P. B. (1987). Correlative reductions in whole plant photosynthesis and yield of winter wheat caused by ozone. Phytopathology 77,75-79. Anbazhagan, M., Krishnamurthy, R. and Bhagwat, K. A. (1988). Proline: an enigmatic indicator or air pollution tolerance in rice cultivars. Plant Physiology 133,122-123. Anderson, W. C. and Taylor, 0. C. (1973). Ozone induced carbon dioxide evolution in tobacco callus cultures. Physiologia Plantarum 28, 419-423. Anderson, L. E., Muschinek, G. and Marques, I. (1988). Effects of SO2 and sulfite on stromal metabolism. In “Air Pollution and Plant Metabolism” (S. SchulteHolstede, N. M. Darrall, Blank, L. W. and A. R. Wellburn, eds), pp. 134-147. Elsevier Applied Science, London. Anon. (1987). “Air Quality Guidelines for Europe”. WHO Regional Publications, European Series No. 23, p. 400. Regional Office for Europe, Copenhagen. Arndt, U. von and Kaufmann, M. (1985). Wirkungen von Ozone auf die apparente Photosynthese von Tanne und Buche. Allgemeine Forstzeitschrift 40,19-20. Ashenden, T. W. (1979). Effects of SOz and NOz pollution on transpiration in Phaseolus vulgaris L. Environmental Pollution 18,45-50. Ashenden, T. W. and Williams,J. H. (1988). Differencesin thespectralcharacteristicsof birch canopies exposed to simulated acid rain. New Phytologist 109, 79-84. Ashenden, T. W., Tabner, P. W., Williams, P., Whitmore, M. E. and Mansfield, T. A. (1982). A large scale system for fumigating plants with SO2 and NOz. Environmental Pollution (Ser. B) 3, 21-26. Ashmore, M. R. and Onal, M. (1984). Modification by sulphur dioxide of the responses of Hordeum vulgare to ozone. Environmental Pollution (Ser. A ) 36, 31-43. Ashmore, M., Bell, N. and Rutter, J. (1985). The role of ozone in forest damage in West Germany. Ambio 14, 81-87. Assrnann, S. M. (1988). Stomata1 and non-stomata1 limitations to carbon assimilation: an evaluation of the path-dependent method. Plant, Cell and Environment 11,577-582.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

107

Atkinson, C. J. and Winner, W. E. (1987). Gas exchange characteristics of Heteromeles arbutjolia during fumigation with sulphur dioxide. New Phytologist 106,423-436. Atkinson, C. J., Robe, S. V. and Winner, W. E. (1988a). The relationship between changes in photosynthesis and growth for radish plants fumigated with SO2 and 03.New Phytologist 110,173-184. Atkinson, C. J., Winner, W. E. and Mooney, H. A. (1988b). Gas exchange and SO2 fumigation studies with irrigated and unirrigated field grown Diplacus aurantiacus and Heteromeles arbutifolia. Oecologia 75,386-393. Ayazloo, M., Garsed, S. G. and Bell, J. N. B. (1982). Studies on the tolerance to sulphur dioxide of grass populations in polluted areas. 11. Morphological and physiological investigations. New Phytologist 90, 109-126. Barnes, R. L. (1972). Effects of chronic exposure to ozone on photosynthesis and respiration of pines. Environmental Pollution 3, 133-138. Barnes, J. D., Reiling, K. Davison, A. W. and Renner, C. J. (1988). Interaction between ozone and winter stress. Environmental Pollution 53, 235-254. Barton, J. R., McLaughlin, S . B. and McConathy, R. K. (1980). The effects of SO2 on components of leaf resistance to gas exchange. Environmental Pollution (Ser. A ) 21,255-265. Bauer, F. (1985). “Diagnosis and Classification of New Types of Damage Affecting Forests”. Special edition Commission of the European Communities, DG IV, F3 Forests and Silviculture, Rue de la Loi 200, B-1049 Brussels, Belgium. Beckerson, D.W. and Hofstra, G. (1979a). Stornatal responses of white bean to O3 and SO2 singly or in combination. Atmospheric Environment 13,533-535. Beckerson, D. W. and Hofstra, G. (1979b). Response of leaf diffusive resistance of radish, cucumber and soybean to O3 and SO2 singly or in combination. Atmospheric Environment 13, 1263-1268. Beckerson, D. W. and Ormrod, D. P. (1986). Polyamines as antiozonants for tomato. HortScience 21, 1070-1071. Bell, J. N. B., Rutter, A. J. and Relton, J. (1979). Studies on the effect of low levels of sulphur dioxide on the growth of Loliumperenne L. New Phytologist 83,627-643. Bennett, J. H. and Hill, A. C. (1973). Inhibition of apparent photosynthesis by air pollutants. Journal of Environmental Quality 2, 526-530. Bennett, J. H., Lee, E. H. and Heggestad, H. E. (1984). Biochemical aspects of plant tolerance to ozone and oxyradicals: superoxide dismutase. In “Gaseous Air Pollutants and Plant Metabolism” (M. J. Koziol and F. R. Whatley, eds), Chap. 27. Butterworths, London. Bennett, J. H., Lee, E. H. and Heggestad, H. E. (1985). Physiologicaleffectsof SO2 and NO2: dose-response modeling. Plant Physiology 77 (Suppl. 4), 161 (Abstr. 883). Benoit, L. F., Skelly, J. M., More, L. D. and Dochinger, L. S. (1982). Radial growth reductions of Pinus strobus L. correlated with foliar ozone sensitivity as an indicator of ozone-induced losses in eastern forests. Canadian Journal of Forest Research 12, 673-678. Bergmann, F. and Scholz, F. (1987). The impact of air pollution on the genetic structure of Norway spruce. Silvae Genetica 36, 80-83. Besford, R. T. and Hand, D. W. (1989). The Effects of COz enrichment and nitrogen oxides on some Calvin cycle enzymes and nitrite reductase in glasshouse lettuce. Journal of Experimental Botany 40,329-336. Biggs, A. R. and Davis, D. D. (1980). Stornatal response of three birch species exposed to varying acute doses of SOz. Journal of the American Society for Horticultural Science 105, 514-516.

108

H. SAXE

Biggs. A. R. and Davis, D. D. (1982).Effects of sulfur dioxide on water relations of hybrid poplar foliage and bark. Canadian Journal of Forest Research 12, 612-6 16. Biscoe, P. V . , Unsworth, M. H. and Pinckney, H. R. (1973). The effects of low concentrations of sulphur dioxide on stomatal behaviour in Vicia faba. New Phytologist 72, 1299-1306. Black, V .J. (1984).The effect of air pollutants on apparent respiration. h “Gaseous Air Pollutants and Plant Metabolism” (M. J . Koziol and F. R. Whatley, eds), Chap. 17. Butterworths, London. Black, C. R. and Black, V. J. (1979). The effects of low concentrations of sulphur dioxide on stomatal conductance and epidermal cell survival in field bean (Viciafaba L.). Journal of Experimental Botany 30, 291-298. Black, V . J. and Unsworth, M. H. (1979). Effects of low concentrations of sulphur dioxide on net photosynthesis and dark respiration of Vicia faba. Journal of Experimental Botany 30,473-483. Black, V. J. and Unsworth, M. H. (1980).Stomata1 responses to sulphur dioxide and vapour pressure deficit. Journal of Experimental Botany 31,667-677. Black, V . J., Ormrod, D. P. and Unsworth, M. H. (1982).Effects of low concentrations of ozone. singly, and in combination with sulphur dioxide on net photosynthesis rates of Viciafubu L. Journalof Experimental Botany 33,1302-1311. Blank, L. W. (1985).A new type of forest decline in Germany. Nature314,311-314. Bonte, J. and Cormis, L. de (1977). Inhibition, en anaerobiose, de la reaction de fermature des stomates du Pelargonium en presence de dioxyde de soufre. Environmental Pollution 12, 125-133. Bonte, J. and Louguet, P. (1975). Interrelations entre la pollution par le dixoyde de soufre et le mouvement des stomates chez le Pelargonium x hortorum: effects de I’humidte relative et de la teneur en gaz carbonique de l’air. Physiologie VkgCtale 13, 527-537. Bonte, J., Bonte, C., Cormis, L. de, and Louguet, P. (1977).Contribution a 1’6tude des caract6res de resistance de Pelargonium a un pollutant atmosphkrique, le dioxyde de soufre. Physiologie Vkgktale 15, 15-27. Botkin, D. B., Smith, W. H. andCarlson, R. W. (1972).Ozone suppressionofwhite pine net photosynthesis. Journal of the Air Pollution Control Association 21, 778-80. Boyer, J. N . , Houston, D. B. and Jensen, K. F. (1986). Impacts of chronic S02,03, and S 0 2 + 0 3 exposures on photosynthesis of Pinus strobw clones. European Journal of Forest Pathology 16, 293-299. Brenninger, C. and Tranquillini, W. (1983). Photosynthese, Transpiration und Spaltoffnungsverhalten verschiedener Holzarten nach Begasung mit S 0 2 . European Journal of Forest Pathology 13,228-238. Brown, K. A. and Roberts, T. M. (1988). Effects of ozone on foliar leaching in Norway spruce (Picea abies L. Karst): Confounding factors due to NO, production during ozone generation. Environmental Pollution 55,55-73. Bruggink, G. T., Wolting, H. G., Dassen, J. H. A. and Bus, V. G. M. (1988). The effect of nitric oxide fumigation at two C02 concentrations on net photosynthesis and stornatal resistance of tomato (Lycopersiconlycopersicum L. cv. Abunda). New Phytologist 110, 185-191. Bucher, J. B. (1981). SO2-induced ethylene evolution of forest tree foliage and its potential use as an indicator. European Journal of Forest Pathology 11,369-373. Bucher-Wallin, I . K. von, Bernhard, L. and Bucher, J. B. (1979). Einfluss niedriger SO2-Konzentrationenauf die Aktivitat einiger Glykosidasen der Assimilationsorgane verklonter Waldbaume. European Journal of Forest Pathology 9,6-15.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

109

Bull, J. N. and Mansfield, T. A. (1974). Photosynthesis in leaves exposed to SO2 and NOz. Nature 250, 443-444. Butler, L. K. and Tibbitts, T. W. (1979). Stomata1 mechanisms determining genetic resistance to ozone in Phaseolus vulgaris L. Journal of the American Horticultural Society 104, 213-216. Bytnerowicz, A. and Taylor, 0. C. (1983). Influence of ozone, sulfur dioxide, and salinity on leaf injury, stornatal resistance, growth, and chemical composition of bean plants. Journal of Environmental Quality 12,397-405. Bytnerowicz, A., Olszyk,D. M.,Kats, G.,Dawson,P. J., Wolff, J.andThompson,C. R. (1987). Effects of SO2on physiology, elemental content and injury development of winter wheat. Agriculture Ecosystems and Environment 20, 37-47. Caemmerer, S. von & Farquhar, G. D. (1981). Some relationships between the biochemistry and gas exchange of leaves. Planta 153,376-387. Cape, J. N., Paterson, L. S., Wellburn, A. R., Wolfenden, J., Mehlhorn, H., Freer-Smith, P. H. and Fink, S. (1988). “The Early Diagnosis of Forest Decline: A Report”. NERC Institute for Terrestial Ecology, Merlewood Research Station, Grange-over-Sands, Cumbria LA11 6JU, UK. Caporn, S. J. M. (1989). The effects of oxides of nitrogen and carbon dioxide enrichment on photosynthesis and growth of lettuce (Lactuca sativa L.), New Phyrologist 111,473-481. Capron, T. M. and Mansfield, T. A. (1976). Inhibition of net photosynthesis in tomato in air polluted with NO and N02. Journal of Experimental Botany 27, 1181-1186. Caput, C. and Belot, Y. (1978). Absorption of sulphur dioxide by pine needles leading to acute injury. Environmental Pollution 16,3-15. Carlson, R. W. (1979). Reduction in the photosynthetic rate of Acer, Quercus and Fraxinus species caused by sulphur dioxide and ozone. Environmental Pollution 18, 159-170. Carlson, R. W. (1983a). The effects of SO2 on photosynthesis and leaf resistance at varying concentrations of C 0 2 . Environmental Pollution (Ser. A ) 30,309-321. Carlson, R. W. (1983b). Interaction between SO2 and NO2 and their effects on photosynthesis properties of soybean Glycine m a . Environmental Pollution (Ser. A ) 32, 11-38. Carlson, R. W. and Bazzaz, F. A. (1982). Photosynthetic and growth response to fumigation with SO2 at elevated C 0 2 for C3 and C4 plants. Planta 54, 50-54. Castillo, F. J. and Greppin, H. (1988). Extracellular ascorbic acid and enzyme activities related to ascorbic acid metabolism in Sedum album L. leaves after ozone exposure. Environmental and Experimental Botany 28,231-238. Castillo, F. J., Ogier, G., Miller, P. R. and Greppin, H. (1988). Variations saisonnikres d’indicateurs biochimiques chez I’epicka (Picea abies L. [Karst] de la for& Genevoise: corrklation avec le taux en polluants atmosphkriques (dioxyde de soufre et ozone). Archives des Sciences (Geneve) 41, 345-363. Cecil, R. and Wake, R. G. (1962). The reactions of inter- and intra-chain disulfide bonds in proteins with sulphite. Biochemical Journal 82,401-406. Cerovic, G. Z., Kalezic, R. and Plesnicar, M. (1982). The role of photophosphorylation in SO2 and S03* inhibition of photosynthesis in isolated chloroplasts. Planta 156, 249-254. Chappelka, A. H. and Chevone, B. I. (1988). Growth and physiological responses of yellow-poplar seedlings exposed to ozone and simulated acidic rain. Environmental Pollution 49, 1-18. Chen, Y.-M. and Wellburn, A. R. (1989). Enhanced ethylene emissions from red and Norway spruce exposed to acidic mists. Plant Physiology 91, 357-361.

110

H.SAXE

Chevone, B. I. and Yang, Y. S. (1985). COz exchange rates and stomata1 diffusive resistance in soybean exposed to 0 3 and SO*. Canadian Journal of Plant Science 65,267-274. Chiment, J. J., Alscher, R. and Hughes, P. R. (1986). Glutathione as an indicator of SOz-induced stress in soybean. Environmental and Experimental Botany 26, 147-152. Cooley, D. R. and Manning, W. J. (1987). The impact of ozone on assimilate partitioning in plants: a review. Environmental Pollution 47,95-113. Cornelius, R., von (1982). Der Einfluss von Ozone auf die Konkurrenz von Solidago canadensis L. und Artemisia vulgaris L. Angewandte Botanik 56,243-251. Comic, G. (1987). Interaction between sublethal pollution by sulphur dioxide and drought stress. The effect on photosynthetic capacity. Physiologia Plantarum 71, 115-119. Cowling, D. W. and Koziol, M. J. (1978). Growth of ryegrass (Loliumperenne L.) exposed to SOz. I. Effects on photosynthesis and respiration. Journal of Experimenal Botany 29,1029-1036. Coyne, P. I. and Bingham, G. E. (1982). Variation in photosynthesis and stomata1 conductance in an ozone-stressed Ponderosa pine stand: light response. Forest Science 28,257-273. Darrall, N. M. (1986). The sensitivity of net photosynthesis in several plant species to short-term fumigations with sulphur dioxide. Journal of Experimental Botany 37,1313-1322. Darrall, N. M. (1989). The effects of air pollutants on physiological processes in plants. Plant, Cell and Environment 12, 1-30. Darrall, N. M. and Jager, H. J. (1984). Biochemical diagnostic tests for the effect of air pollution on plants. In “Gaseous Air Pollutants and Plant Metabolism”, Chap. 22. Butterworths, London. Davis, J. M. and Rogers, H. H. (1980). Wind tunnel testing-of open-top field chambers for plant effects assessment. Journal of the Air Pollution Control Association 30, 905-908. Davison, A. W., Barnes, J. D. and Renner, C. J. (1988). Interactions between air pollutants and cold stress. In “Air Pollution and Plant Metabolism”. (S. Schulte-Hostede, N. M. Darrall, L. W. Blank and A. R. Wellburn, eds), pp. 272-287. Elsevier Applied Science, London. Decleire, M., Cat, W. de, Temmerman, L. de and Baeten, H. (1984). Modifications de lkctivitie des peroxydase, catalase et superoxyde dismutase dans des feuilles d’tpinard traite h 16zone. Journal of Plant Physiology 116,147-152. Decoteau, D. R., Grant, L. and Craker, L. E. (1986). Failure of ozone susceptibility tests to predict yield reductions in wheat. Field Crops Research 13, 185-191. Dietz, B., Moors, I., Flammersfeld, U., Riihle, W. and Wild, A. (1988). Investigation on the photosynthetic membranes of spruce needles in relation to the occurrence of novel forest decline I. The photosynthetic electron transport. Zeitschriffur Naturforschung Teil c 43,581-588. Dijak, M. and Ormrod, D. P. (1982). Some physiological and anatomical characteristics associated with differential ozone sensitivity among pea cultivars. Environmental and Experimental Botany 22,395402. Dohmen, G . P. von (1986). Einfluss von Mineralstoffernahrung, Ozone und Saurem Nebel auf Peroxidase-Aktivitaten in Fichtennadeln, Picea abies (L.) Karst. Forshvissenschaftliches Centralblatt 105, 252-254. Donagi, A. E. and Goren, A. I. (1979). Use of indicator plants to evaluate atmospheric levels of nitrogen dioxide in the vicinity of a chemical plant. Environmental Science and Technology 13, 986-989.

.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

111

Downs, R. J. (1980). Phytotrons. The Botanical Review. 46, 447-489. Eliassen, A., Hov. 0., Iversen, T., Saltbones, J. and Simpson, D. (1988). “Estimates of Airborne Transboundary Transport of Sulphur and Nitrogen over Europe”, EMEPIMSC-W Report 1/88, August 1988. The Norwegian Meteorological Institute, Meteorological Synthesizing Centre, West (MSC-W) of EMEP. Elkiey, T. and Ormrod, D . P. (1979). Leaf diffusion resistance responses of three Petunia cultivars to ozone andlor sulfur dioxide. Journal of the Air Pollution Control Association 29, 622-625. Elkiey, T. and Ormrod, D. P. (1980). Sorption of ozone and sulfur dioxide by Petunia leaves. Journal of Environmental Quality 9, 93-95. Elkiey, T., Ormrod, D. P. and Pelletier, R. L. (1979). Stomata1 and leaf surface features as related to the ozone sensitivity of Petunia cultivars. Journal ofthe American Society for Horticultural Science 104, 510-514. Ellenson, J. L. and Amundson, R. G. (1982) Delayed light imaging for the early detection of plant stress. Science 215, 1104-1106. Elliott, C. L., Eberhardt, J. C. and Brennan, E. G. (1987). The effect of ambient ozone pollution and chlorophyll content of green and white ash. Environmental Pollution 44,61-70. Elstner, E. F. and Osswald, W. (1984). Fichtensterben in “Reinluftgebieten”: Strukturresistenzverlust. Naturwissenschafltliche Rundschau. 3 7 , 5 2 4 1 . Endress, A. G . , Suarez, S. J. and Taylor, 0.C. (1980). Peroxydase activity in plant leaves exposed to gaseous HC1 or ozone. Environmental Pollution (Ser. A ) 22, 47-58. Erickson, S.S. and Dashek, W. V. (1982). Accumulation of foliar soluble proline in sulphur dioxide-stressed Glycine m u . cv. “Essex” and Hordeum vulgare cvs “Proctor” and “Excelsior” seedlings. Environmental Pollution (Scr. A ) 28, 89-108. Evans, L. S. and Thompson, K. H. (1984). Comparison of experimental designs used to detect changes in yields of crops exposed to acidic precipitation. Agronomy Journal 76, 81-84. Faensen-Thiebes, A. (1983). Veranderungen im Gaswechsel Chlorophyllgehalt und Zuwachs von Nicotiana tabacum L. und Phaseolus vulgaris L. durch Ozone und deren Beziehung zur Ausbildung von Blattnekrosen. Angewandte Botanik 57, 181-191. Farrar, J. F., Relton, J. and Rutter, A. J. (1977). Sulphur dioxide and the growth of Pinus sylvestris. Journal of Applied Ecology 14, 861-875. Feiler, S.(1985). Einfliisse von Schwefeldioxid auf die Membranpermeabilitat und Folgen fur die Frostempfindlickeit der Fichte (Picea abies (L.) Karst). Flora 177,217-226. Forberg, E., Aarnes, H. and Nilsen, S. (1987). Effects of ozone on net photosynthese in oat (Avena sativa) and duckweed (Lemna gibba). Environmental Pollution 47, 285-291. Fowler, D. and Cape, J. N. (1982). Air pollutants in agriculture and horticulture. In “Effects of Gaseous Air Pollution in Agriculture and Horticulture” (M. H. Unsworth and D. P. Ormrod, eds), Chap. 1. Butterworth Scientific, London. Foyer, C. H. and Halliwell, B. (1976). The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133,21-25. Franklin, J. F., Shugart, H. H. and Harmon, M. E. (1987). Tree death as an ecological process. Bioscience 37,550-556. Freer-Smith, P. H. (1985). The influence of SO2 and NO2 on the growth, development and gas exchange of Betulapkndula Roth. New Phytologist 99,417-430.

112

H. SAXE

Fuhrer, J. (1985). Ethylene production and premature senescence of needles from fir trees (Abies alba). European Journal of Forest Pathology 15, 227-236. Furukawa, A. and Totsuka, T. (1979). Effects of N02, SO2 and O3 alone and in combinations on net photosynthesis in sunflower. Environmental Control in Biology 17, 161-166. Furukawa, A., Isoda, O., Iwaki, H. and Totsuka, T. (1979a). Interspecific differences in respohses of transpiration to SOz. Environmental Control in Biology 17, 153-159. Furukawa, A., Koike, A., Hozumi, K. and Totsuka, T. (1979b). The effects of SOz on photosynthesis in poplar leaves at various C02 concentrations. Journal of the Japanese Forestry Society 61, 351-356. Furukawa, A., Natori, T. and Totsuka, T. (1980). The effects of SO:! on net photosynthesis in sunflower leaf. Research Report,of the National Institute for Environmental Studies, Japan 11, 1-8. Furukawa, A., Katase, M., Ushijima, T. and Totsuka, T. (1984a) Inhibition of photosynthesis in poplar species and sunflower by 0 3 . Research Report from the National Institute for Environmental Studies, Japan 65, 77-87. Furukawa, A., Yokoyama, M., Ushijima, T. and Totsuka, T. (1984b). The effects of NO2 and/or O3on photosynthesis of sunflower leaves. Research Report from the National Institute for Environmental Studies, Japan 65,89-98. Garsed, S . G. and Rutter, A. J. (1982). Relative performance of conifer populations in various tests for sensitivity to SO2 and the implications for selecting trees for planting in polluted areas. New Phytologist 92, 349-367. Garsed, S. G., Farrar, J. F. and Rutter, A. J. (1979). The effects of low concentrations of sulphur dioxide on the growth of four broadleaved tree species. Journal of Applied Ecology 16,217-226. Gasch, G., Griinhage, L., Jager, H.-J. and Wentzel, K.-F. (1988). Das Verhaltnis der Schwefelfraktionen in Fichtennadeln als Indikator fiir Immissionsbelastungen durch Schwefeldioxid. Angewandte Botanik 62, 73-84. Geburek, Th., Scholz, F., Knabe, W. and Vornweg, A. (1987). Genetic studies by isoenzyme gene loci on tolerance and sensitivity in an air polluted Pinus sylvestris field trial. Silvae Genetica 36, 49-53. Gezelius, K. and Hallgren, J.-E. (1980). Effects of SO3 on the activity of ribulose bisphosphate carboxylase from seedlings of Pinus silvestris. Physiologia Plantarum 49,354-358. Gmur, N. F., Evans, L. S. and Lewin, K. F. (1983). Effects of ammonium sulfate aerosols on vegetation. I. Chamber design for long-duration exposures. Atmospheric Environment 17,707-714. Goodwin, T. W. and Mercer, E. I. (1983). “Introduction to Plant Biochemistry”, 2nd edn. Pergamon Press, Oxford. Gould, R. P. and Mansfield, T. A. (1989). The sensitivity of early 20th century cultivars of wheat to air pollution. Environmental Pollution 56,31-37. Greitner, C . S. and Winner, W. E. (1988). Increases in 613C values of radish and soybean plants caused by ozone. New Phytologist 108,489494. Grennfelt, P, Bengtson, C. and Skarby, L. (1983). Deposition and uptake of atmospheric nitrogen oxides in a forest ecosystem. Aquilo Series Botanica 19, 208-221. Gross, von K. (1987). Gaswechselmessungen an jungen Fichten und Tannen wahrend Begasung mit Ozone und Schwefeldioxid (allein und in Kombination) im Kleinphytotron. Allgemeine Forst- und Jagdzeitung 158, 31-36. Gross, von K., Vollbrecht, P., Franzen, J., Dietz, J. and Wagner, E. (1988). Untersuchungen zum Mechanismus der neuartigen Waldschaden: Hemmung

PHOTOSYNTHESIS AND STOMATAL RESPONSES

113

der Photosynthese und Vergilbung von Fichtenkeimlingen und jungplflanzen durch Begasung mit beta-Pinen. Allgemeine Forst- und Jagdzeitung 159, 42-48. Grunwald, C. (1988). Impact of acid precipitation on nitrogen and COz fixation, and biomass accumulation by soybean. Environmental Pollution 53, 430-431. Grunwald, C. and Endress, A. G. (1988). Oil, fatty acid, and protein content of seeds harvested from soybeans exposed to 0 3 and/or SO2.Botanical Gazette 149,283-288. Guri, A. (1983). Variation in glutathione and ascorbic acid content among selected cultivars of Phaseolus vulgaris prior to and after exposure to ozone. Canadian Journal of Plant Science 63,733-737. Hanson, P. J.. McLaughlin, S. B. and Edwards, N. T. (1988). Net COz exchange of Pinus taeda shoots exposed to variable ozone levels and rain chemistries in field and laboratory settings. Physiologia Plantarum 74, 635-642. Hartmann, G., Nienhaus, F. and Butin, H. (1988). “Farbatlas Waldschaden: Diagnose von Baumkrankheiten”. Eugen Ulmer, Stuttgart. Heagle, A. S., Body, D. E. and Heck, W. W. (1973). An open-top field chamber to assess the impact of air pollution on plants. Journal of Environmental Quality 2, 365-368. Heath, R. L. (1988). Biochemical mechanisms of pollutant stress. In “Assessment of Crop Loss from Air Pollutants” (W. W. Heck, 0.C. Taylor and D . T. Tingey, eds), Chap. 1 1 . Elsevier Applied Science, London. Heck. W. W., Philbeck, R. B. and Dunning, J. A. (1978). A Continuous Stirred Tank Reactor (CSTR) System for Exposing Plants to Gaseous Air Contaminants. Principles, Specifications, Construction, and Operation”. Agricultural Research Service, US Department of Agriculture, ARS-S-181. Science and Education Administration, P.O. Box 53326, New Orleans, Louisiana 70153, USA. Heck, W. W., Krupa, S.V. and Linzon, S. N. (eds) (1979). “Methodology for the Assessment of Air Pollution Effects on Vegetation. Proceedings APCA Specialty Conference”, pp. 1 .l-18.3. Air Pollution Control Association, Pittsburgh, PA. Hill, A. C. and Bennett, J. H. (1970). Inhibition of apparent photosynthesis by nitrogen oxides. Atmospheric Environment 4,341-348. Hill, A. C. and Littlefield, N. (1969). Ozone. Effects on apparent photosynthesis, rate of transpiration, and stomata1 closure in plants. Environmental Science and Technology 3 , 52-56. Hinrichsen, D. (1986). Multiple pollutants and forest decline. Ambio 15,258-265. Hinrichsen, D. (1987). The forest decline enigma. What underlies extensive dieback on two continents? BioScience 37,542-546. Hirata, Y and Kunishige, M. (1981). Studies on the survey and application of new indicate plants to sulfur dioxide 11. The responses of Hydrangea “Enziandom” (Hydrangea hortensis SM.) as an indicator plant at an air polluted area. Bulletin of the Vegetable Ornamental Crops Research Station 5C, 51-62. Hofstra, G., Wukasch, R. T. and Drexler, D. M. (1983). Ozone injury on potato foliage as influenced by the antioxidant EDU and sulphur dioxide. Canadian Journal of Plant Pathology 5, 115-119. Hou, L.-Y., Hill, A. C. and Soleimani, A. (1977). Influence of COz on the effects of SO2 and NO2 on alfalfa. Environmental Pollution 12, 7-16. Houpis, J. L. J. and Helms, J. A. (1985). Patterns of photosynthesis of Pinus ponderosa seedlings fumigated for two years with S02. Plant Physiology 77, 60,(abstr. 322).

114

H. SAXE

Hunt, G. A. and Black, V. J. (1988). Environmental stress: amplifier of physiological responses to SO2 in plants. Environmental Pollution 53,433435. Huttunen, S . and Soikkeli, S. (1984). Effects of various gaseous pollutants on plant cell ultrastructure. In “Gaseous Air Pollutants and Plant Metabolism”, (M. J. Koziol and F. R. Whatley, eds), Chap. 9. Butterworths, London. Huttunen, S., Laine, K. and Torvele, H. (1985). Seasonal sulphur contents of pine needles as indices of air pollution. Annals Botanica Fennici 22, 343-359. Hallgren, J.-E. and Gezelius, K. (1982). Effects of SO2 on photosynthesis and ribulose bisphosphate carboxylase in pine tree seedlings. Physiologia Plantarum 54, 153-161. Hallgren, J.-E. Linder, S., Richter, A., Troeng, E. and Granat, L. (1982). Uptake of SO2 in shoots of Scots pine: field measurements of net flux of sulphur in relation to stomatal conductance. Plant, Cell and Environment 5, 75-83. Hartel, 0. (1972). Langjahrige Messreihen mit dem Triibungstest an abgasgeschadigten-Fichten. Oecologia 9, 103-111. Innes, J. L. (1987). Air pollution and forestry. Forestry Commission Bulletin 70, 5-39. Ito, O., Mitsumori, F. and Totsuka, T. (1985). Effects of NO2 and O3 alone or in combination on kidney bean plants (Phaseolus vulgaris L.): products of 13C02 assimilation detected by I3C nuclear magnetic resonance. Journal of Experimental Botany 36,281-289. Jensen, K. F. (1981). Growth analysis of hybrid poplar cuttings fumigated with ozone and sulphur dioxide. Environmental Pollution (Series A ) 26, 243-250. Jensen, K. F. (1983). Growth relationships in silver maple seedlings fumigated with O3 and SOz. Canadian Journal of Forest Research 13,298-302. Jensen, K. F. (1986). Photosynthetic response of Liriodendron tulipifera L. seedlings to acid rain treatment and ozone fumigation. American Journal of Botany 73, 720 (Abstr. 323). Jensen, K. F. and Noble, R. D. (1984). Impact of ozone and sulfur dioxide on net photosynthesis of hybrid poplar cuttings. Canadian Journal of Forest Research 14,385-388. Jensen, K. F. and Roberts, B. R. (1986). Changes in yellow poplar stomatal resistance with SO2 and 0 3 fumigation. Environmental Pollution (Series A ) 41, 235-245. Johansson, C. (1987). Pine forest: a negligible sink for atmospheric NO, in rural Sweden. Tellus 39B, 426-438. Jones, T. and Mansfield, T. A. (1982). The effect of SO2on growth and development of seedlings of Phleum pratense under different light and temperature environments. Environmental Pollution (Series A ) 27, 57-71. Jager, H. J. and Kiein, H. (1977). Biochemical and physiological detection of sulfur dioxide injury to pea plants (Pisum sativum). Air Pollution Control Association 27, 464-466. Jager, H.-J. von, Wiegel, H.-J. and Griinhage, L. (1986). Physiologische und biochemische Aspekte der Wirkung von Immissionen auf Waldbaume. European Journal of Forest Pathology 16,98-109. Kaji, M., Yoneyama, T., Totsuka, T. and Iwaki, H. (1980). Absorption of atmospheric NO2 by plants and soils. VI. Transformation of NO2 absorbed in the leaves and transfer of the nitrogen through the plants. Research Report of the National Institute for Environmental Studies, Japan 11,51-58. Karolowski, P. (1985). The role of free proline i the sensitivity of poplar (Populus “Robusta”)plants to the action of S02. European Journal of Forest Pathology 15, 199-206.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

115

Katainen, H.-S., Karjalainen, R., Makinen, E., Okinen J. and Kellomaki, S . (1984). European Journal of Forest Pathology 14, 33-42. Katainen, H.-S., Makinen, E., Jokinen, J., Karjalainen, R. and Kellomaki, S. (1987). Effects of SO2 on the photosynthetic and respiration rates in Scots pine seedlings. Environmental Pollution 46, 241-25 1 . Keitel, A . and Arndt, U. (1983). Ozoninduzierte Turgeszenzverluste kei Tabak (Nicotiana tabacum var. Be1 W3) - ein Hinweis auf schnelle Permeabilitatsveranderungen der Zellmembranen. Angewandte Botanik 57, 193-204. Keller, T. (1978). Einfluss niedriger SO2-Konzentrationen auf die C02-Aufnahme von Fichte und Tanne. Photosynthetica 12, 316-322. Keller, T. (1986). The electrical conductivity of Norway spruce needle diffusate as affected by certain air pollutants Tree Physiology 1, 85-94. Keller, T. (1988). Growth and premature leaf fall in American aspen as bioindications for ozone. Environmental Pollution 52, 183-192. Keller, T. and Hasler, R. (1984). The influence of a fall fumigation with ozone on the stomatal behaviour of spruce and fir. Oecologia 64, 284-286. Keller, T. and Hasler, R. (1986). The influence of a prolonged SO2 fumigation on the stomatal reaction of spruce. European Journal of Forest Pathology 16, 110-1 15. Keller, T. and Hasler, R. (1987). Some effects of long-term ozone fumigations on Norway spruce. I. Gas exchange and stomatal response. Trees 1, 129-133. Kimmerer, T. W. and Kozlowski, T. T. (1981). Stomata1 conductance and sulfur uptake of five clones of Populus tremuloides exposed to sulfur dioxide. Plant Physiology 67,990-995. Kimmerer, T. W. and Kozlowski, T. T. (1982). Ethylene, ethane, acetaldehyde, and ethanol production by plants under stress. Plant Physiology 69, 840-848. Klein, H., Jager, H.-J. Domes, W. and Wong, C. H. (1978). Mechanisms contributing to differential sensitivities of plants to SO2. Oecologia 33, 203-208. Klein, R. M. (1988). Causation in forest decline. Environmental Science and Technology 22, 148-149. Kobriger, J. M., Tibbitts, T. W. and Brenner, M. L. (1984). Injury, stomatal conductance, and abscisic acid levels of pea plants following ozone plus sulfur dioxide exposures at different times of the day. Plant Physiology 76, 823-826. Koziol, M. J. (1980). Monitoring gas concentrations in pollutant exposure systems: defining exposure concentrations. Journal of Experimental Botany 31, 14131423. Koziol, M. J. and Whatley, F. R. (1984). “Gaseous Air Pollutants and Plant Metabolism”. Butterworths, London. Koziol, M. J., Shelvey, J. D., Lockyer, D. R. and Whatley, F. R. (1986). Response of SO2-sensitive and resistant genotypes of ryegrass (Lolium perenne L.) to prolonged exposure to S02. New Phytologist 102,345-357. Kozlowski, T. T. (1986a). Responses of woody plants to environmental pollution. I. Sources and types of pollutants and plant responses. Forestry Abstracts 47, 5-51. Kozlowski, T. T. (1986b). Environmental pollution and tree growth. 11. Factors affecting responses to pollution and alleviation of pollution effects. Forestry Abstracts 47, 106-132. Krause, G. H. M., Jung, K.-D. and Prinz, B. (1985). Experimentelle Untersuchungen zur Aufklarung der neuartigen Waldschaden in der Bundesrepublik Deutschland. VDZ Berichte 560, 627-655.

116

H. SAXE

Krause, G. H. M., Arndt, U., Brandt, C. J., Bucher, J., Kenk, G. and Matzner, E. (1986). Forest decline in Europe: Development and possible causes. Water, Air, and Soil Pollution 31, 647-668. Kress, L. W., Skelly, J. M. and Hinkelmann, K. H. (1982). Relative sensitivity of 18 full-sib families of Pinus taeda to 0 3 . Canadian Journal of Forest Research 12, 203-209. Krinsky, N. I. (1966). The role of carotenoid pigments as protective agents against photosensitized oxidations in chloroplasts In “Biochemistry of Chloroplasts” (T. W. Goodwin, ed), Vol. 1, pp. 423-430. Academic Press, London. Krizek, D. T., Wergin, W. P. and Semeniuk, P. (1985). Morphological and physiological properties of Poinsettia leaves and bracts in relation to sulfur dioxide sensitivity. Environmental and Experimental Botany 25, 165-173. Kropff, M. J. (1987). Physiological effects of sulphur dioxide. 1 . The effect of SO2 on photosynthesis and stomata1 regulation of Vicia faba L. Plant, Cell and Environment 10,753-760. Kumar, N. (1986). Response of Vigna radiata to SO2 and NO2 pollution. Acta Botanica Indica 14, 139-144. Laisk, A., Pflanz, H . , Schramm, M. J. and Heber, U. (1988a). Sulfur-dioxide fluxes into different cellular compartments of leaves photosynthesizing in a polluted atmosphere. I. Computer analysis. Planta 173, 23CL240. Laisk, A., Pfanz, H. and Heber, U. (1988b). Sulfur dioxide fluxes into different cellular compartments of leaves photosynthesizing in a polluted atmosphere. 11. Consequences of SO2 uptake as revealed by computer analysis. Planta 173, 241-252. Landhold, W., von (1982). Der Einfluss einer praxisnahen SO2 Begasung auf das 14C0~-Fixierungsmiinster von Buchen (Fagus sylvatica L.). European Journal of Forest Pathology 12, 331-339. Laurence, J. A., Maclean, D. C., Mandl, R. H., Schneider, R. E. and Hansen, K. S. (1982). Field tests of a linear gradient system for exposure of row crops to SO2 and HF. Water, Air, and Soil Pollution 17, 399-407. Laurence, J. A., Reynolds, K. L. and Greitner, C. S. (1985). Bioindicators of SO2: response of three plant species to variation in dosage kinetics of SO2. Environmental Pollution (Series A ) 37, 43-52. Law, R. M. and Mansfield, T. A. (1982). Oxides of nitrogen and the greenhouse atmosphere. In “Effects of Gaseous Air Pollution in Agriculture and Horticulture” (M. H. Unsworth and D. P. Ormrod, eds), Chap. 5. Butterworths, London. LeBlanc, D. C., Raynal, D. J. and White, E. H. (1987). Acidic deposition and tree growth. 11. Assessing the role of climate in recent forest declines. Journal of Environmental Quality 16,334-340. Lee, E. H. and Bennett, H. (1982). Superoxide dismutase: a possible protective enzyme against ozone injury in snap beans (Phaseolus vulgaris L.). Plant Physiology 69, 1444-1449. Leffler, H. R. andcherry,J. H. (1974). Destructionofenzymaticactivitiesofcorn and soybean leaves exposed to ozone. Canadian Journal of Botany 52,1233-1238. Legge, A. H., Jaques, D. R., Harvey, G. W., Krouse, H. R., Brown, H. M., Rhodes, E. C., Nosal, M., Schellhase, H. U., Mayo, J., Hartgerink, A. P., Lester, P. F., Amundson, R. G. and Walker, R. B. (1981). Sulphur gas emissions in the boreal forest: the West Whitecourt case study. Water, Air, and Soil Pollution 15,77-85. Legge, A. H., Bogner, J. C. and Krupa, S. V. (1988). Foliar sulphur species in pine: a new indicator of a forest ecosystem under air pollution stress. Environmental Pollution 55, 15-27.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

117

Lehnherr, B., Grandjean, A + ,Machler, F. andFuhrer, J. (1987). Theeffect of ozone in ambient air on ribulose bisphosphate carboxylaseloxygenase activity decreases photosynthesis and grain yield in wheat. Journal of Plant Physiology 130,189-200. Lehnherr, B., Machler, F., Grandjean, A. and Fuhrer, J. (1988). The regulation of photosynthesis in leaves of field-grown spring wheat (Triticum aestivum L., cv. Albis) at different levels of ozone in ambient air. Plant Physiology 88, 11151119. L‘Hirondelle, S. J. and Addison, P. A. (1985). Effects of S0260n leaf conductance, xylem tension, fructose and sulphur levels of jack pine seedlings. Environmental Pollution (Series A ) 39, 373-386. L’Hirondelle, S. J., Addison, P. A. and Huebert, D. B. (1986). Growth and physiological responses of aspen and jack pine to intermittent SO2 fumigation episodes. Canadian Journal of Botany 64,2421-2427. Lockyer, D. R., Cowling, D. W. and Jones, L. H. P. (1976). A system for exposing plants to atmospheres containing low concentrations of sulphur dioxide. Journal of Experimental Botany 27, 397-409. Lorenc-Plucinska, G. (1982). Effect of sulphur dioxide on SOz-tolerant and S02susceptible Scots pine seedlings. Photosynthetica 16, 140-144. Lorenc-Plucinska, G. (1988). Effects of nitrogen dioxide on C02 exchange in Scots pine seedlings. Photosynthetica 22, 108-1 11. Lucas, P. W., Cottam, D. A. and Mansfield, T. A. (1987). A large-scale fumigation system for investigating interactions between air pollution and cold stress on plants. Environmental Pollution 43, 15-28. Luttge, U., Osmond, C. B., Ball, E., Brinckmann, E. and Kinze, G. (1972). Bisulfite compounds as metabolic inhibitors: nonspecific effects on membranes. Plant and Cell Physiology 13,505-514. Luxmore, R. J. (1988). Assessing the mechanisms of crop loss from air pollutants with process models. I n “Assessment of Crop Loss from Air Pollutants” (W. W. Heck, 0. C. Taylor and D. T. Tingey, eds), Chap. 17. Elsevier, Applied Science, London. Maas, F. M., Kok, L. J. de, Hoffmann, I and Kuiper, J. C. (1987). Plant responses to H2S and SO2fumigation. I. Effects on growth, transpiration and sulfur content of spinach. Physiologia Plantarum 7 0 , 713-721. Mackay, C. E., Senaratna, T., McKersie, B. D. and Fletcher, R. A. (1987). Ozone induced injury to cellular membranes in Triticum aestivum L. and protection by triazole $3307. Plant Cell Physiology 28, 1271-1278. Majernik, 0. and Mansfield, T. A. (1971). Effects of SO2 pollution on stomata1 movements in Vicia faba. Phytopatologische Zeitschrift 7 1 , 123-128. Majernik, 0.and Mansfield, T. A. (1972). Stomata1 responses to raised atmospheric C 0 2 concentrations during exposure of plants to SO2 pollution. Environmental Pollution 3, 1-7. Mandl, R. H., Kohut, R. J. and Laurence, J. A. (1988). An integrated system for evaluating the effects of ozone and acidic precipitation on the nutrition, growth and physiology of red spruce and sugar maple. Environmental Pollution 53, 444-447. Mansfield, T. A. and McCune, D. C. (1988). Problems of crop loss assessment when there is exposure to two or more gaseous pollutants. In “Assessment of Crop Loss from Air Pollutants” (W. W. Heck, 0.C. Taylor and D. T. Tingey, eds), Chap. 13. Elsevier Applied Science, London. Mansfield, T. A., Whitmore, M. E., Pande, P. C. and Freer-Smith, P. H. (1987). Responses of herbaceous and woody plants to the dry deposition of SO2 and

118

H. SAXE

NOz. In “Effects of Atmospheric Pollutants on Forests, Wetlands and Agricultural Ecosystems” (T. C. Hutchinson and K. M. Meema, eds), NATO AS1 Series, Vol. G16, pp. 131-144. Springer-Verlag, Berlin. Mansfield, T. A., Wright, E. A., Lucas, P. W. and Cottam, D. A. (1988). Interaction between air pollution and water stress. In “Air Pollution and Plant Metabolism” (S. Schulte-Holstede, N. M. Darrall, L. W. Blank and A. R. Wellburn, eds), pp. 288-306. Elsevier Applied Science, London. Martin, B., Bytnerowicz, A. and Thorstenson, Y. R. (1988). Effects of air pollutants on the composition of stable carbon isotopes tiL3C,of leaves and wood, and on leaf injury. Plant Physiology 88, 218-223. Matschke, J. (1985). Peroxydase - ein Marker in der Forstpflanzenziichtung. Beitrage fur die Forstwirtschaft 19, 166-173. Matsuoka, Y. (1978). Experimental studies of sulphur dioxide injury on rice plants and its mechanism. Special Bulletin of the Chiba-Ken Agricultural Experimental Station 7 , 251-265. Matsushima, J. and Yonemori, K. (1985). Sensitivity of Satsuma mandarin to ozone as related to stomata1 function indicated by transpiration rate, change of stem diameter and leaf temperature. Journal of the American Society for Horticultural Science 110, 106-108. Matzner, E. and Ulrich, B. (1987). Results of studies on forest decline in northwest Germany. NATO AS1 Series, Vol. G16. In “Effects of Atmospheric Pollutants on Forests, Wetlands and Agricultural Ecosystems” (T. C. Hutchinson and K. M. Meema, eds), NATO AS1 Series, Vol. G16, pp. 25-42. SpringerVeriag, Berlin. McFarlane, J. C. and Pfleeger, T. (1987). Plant exposure chambers for study of toxic chemical-plant interactions. Journal of Environmental Qualify 16, 361-371. McKersie, B. D., Beversdorf, W. D. and Hucl, P. (1982) The relationship between ozone insensitivity, lipid-soluble antoxidants and superoxide dismutase in Phaseolus vulgaris. Canadian Journal of Botany 60,2686-2691. McLaughlin, S. B., Shriner, D. S., McConathy, R. K. and Mann, L. K. (1979). The effects of SO2 dosage kinetics and exposure frequency on photosynthesis and transpiration of kidney beans (Phaseolus vulgaris L.). Environmental and Experimental Botany 19, 179-191. McLaughlin, S. B., McConathy, R. K., Duvick, D. and Mann, L. K. (1982). Effects of chronic air pollution stress on photosynthesis, carbon allocation, and growth of white pine trees. Forest Science 28, 60-70. McLeod, A. R. and Baker, C. K. (1988) The use of open field systems to assess yield response to gaseous pollutants. In “Assessment of Crop Loss from Air Pollutants” (W. W. Heck, 0. C. Taylor and D. T. Tingey, eds), Chap. 8. Elsevier Applied Science, London. McLeod, A. R., Roberts, T. M., Alexander, K and Cribb, D. M. (1988). Effects of open-air fumigation with sulphur dioxide on the growth and yield of winter barley. New Phytologist 109,67-78. Mehlhorn, H. and Wellburn, A. R. (1987). Stress ethylene formation determines plant sensitivity to ozone. Nature 327, 417-418. Mehlhorn, H., Seufert, G . , Schmidt, A. and Kunert, K. J. (1986). Effect of SO2 and 0 3 on production of antioxidants in conifers. Plant Physiology 82, 336-338. Mehlhorn, H., Francis, B. J. and Wellburn, A. R. (1988). Prediction of the probability of forest decline damage to Norway spruce using three simple site independent diagnostic parameters. New Phytologist 110, 525-534. Meyer, A., Muller, P. and Sembdner, G. (1987). Air pollution and plant hormones. Biochemistry and Physiology Pflanzen 182, 1-21.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

119

Michaels, T. E. (1988). A digital image analysis method for selecting ozoneinsensitive white beans. Canadian Journal of Plant Sciences 68, 627-632. Miszalski, Z. and Lorenc-Plucinska, G. (1988). Effects of SOz on K+ efflux from Vicia faba epidermal strips. Photosynthetica 22, 467-469. Miyake. H.. Matsumura, H., Fujinuma, Y . and Totsuka. T. (1989). Effects of low concentrations of ozone on the fine structure of radish leaves. New Phytologist 111. 187-195. Mooney, H. A.. Kuppers, M., Koch, G., Gorham, J., Chu, C. and Winner, W. E. (1988). Compensating effects to growth of carbon partitioning changes in response to SOz-induced photosynthetic reduction in radish. Oecologia 75, 502-506. Mortensen. L. M. (1982a). Growth responses of some greenhouse plants to environment. I. Experimental techniques. Scientia Horticulturae 16, 39-46. Mortensen, L. M. (1982b). Growth responses of some greenhouse plants to environment. 111. Design and function of a growth chamber-prototype. Scientia Horticulturae 16, 57-63. Mudd. J. B. (1982). Effects of oxidants on metabolic function. I n “Gaseous Air Pollutants and Plant Metabolism” (M. J. Koziol and F. R. Whatley, eds), Chap. 9. Butterworths, London. Mueller, P. W. and Garsed, S. G . (1984). Microprocessor-controlled system for exposing plants to fluctuating concentrations of sulphur dioxide. New Phytologist 97, 165-173. Muller, R. N., Miller, J. E. and Sprugel, D. G. (1979). Photosynthetic response of field-grown soybeans to fumigations with sulphur dioxide. Journal of Applied Ecology 16, 567-576. Murray, A. J. S. (1Y84a). Light affects the deposition of NO2 to the Flacca mutant of tomato without affecting the rate of transpiration. New Phytologist 98, 447-450. Murray, F. (1984b). Responses of subterranean clover and ryegrass to sulphur dioxide under field conditions. Environmental Pollution (Series A ) 36, 239-249. Murray, F. (1985) Changes in growth and quality characteristics of Lucerne (Medicago sativa L.) in response to sulphur dioxide exposure under field conditions. Journal of Experimental Botany 36. 44W57. Musselman, R. C. (1985). Protecting grapevines from ozone injury with ethylenediurea and benomyl. American Journal of Enology and Viticulture 36.38-42. Musselman. R. C., McCool, P. M., Oshima, R. J. and Teso, R. R. (1986). Fieldchambers for assessing crop loss from air pollutants. Journal of Environmental Quality 15, 152-157. Myhre. A , , Forberg, E., Aarnes. H. and Nilsen, S. (1988). Reduction of net photosynthesis in oats after treatment with low concentrations of ozone. Environmental Pollution 53, 265-271. Nakamura, H. and Saka, H. (1978). Photochemical oxidants injury in rice plants. 111. Effect of ozone on physiologica1 activities in rice plants. Japanese Journal of Crop Science 47, 704-714. Natori. T. and Totsuka, T. (1984a). Effects of mixed gas on transpiration rate of several woody plants. 2. Synergistic effects of mixed gas on transpiration rate of Euonymus japonica. Research Report from the National Institute of Environmental Studies, Japan 65,55-62. Natori, T. and Totsuka. T. (1984b). An evaluation of high resistance in Polygonum cuspidaturn to sulfur dioxide (SOz).Research Report of the National Institute of Environmental Studies, Japan 69, 99-107.

120

H. SAXE

Neighbour, E. A., Cottam, D. A. and Mansfield, T. A. (1988). Effects of sulphur dioxide and nitrogen dioxide on the control of water loss by birch (Betula spp.). New Phytologist 108, 149-157. Norby, R. J. and Kozlowski, T. T. (1982). The role of stomata in sensitivity of Betula papyrifera seedlings to SO2 at different humidities. Oecologia 53, 34-39. Nouchi, I. and Aoki, K. (1979). Morning glory as a photochemical oxidant indicator. Environmental Pollution 18, 289-303. Noyes, R. D. (1980). The comparative effects of sulfur dioxide on photosynthesis and translocation in bean. Physiological Plant Pathology 16, 73-79. Nystrom, S. D., Hendrickson, R. C., Pratt, G. C. and Krupa, S. V. (1982). A computerized open-top field chamber system for exposing plants to air pollutants. Agriculture and Environment 7,213-221. Okano, K. and Totsuka, T. (1986). Absorption of nitrogen dioxide by sunflower plants grown at various levels of nitrate. New Phytologist 102, 551-562. Okano, K., Fukuzawa, T., Tazaki, T. and Totsuka, T. (1986). I5N dilution method for estimating the absorption of atmospheric NO; by plants. New Phytologist 102.73-84. Oleksyn, J. and Bialobok, S. (1986). Net photosynthesis, dark respiration and susceptibility to air pollution of 20 European provenances of Scots pine Pinus sylvestris L. Environmental Pollution (Series A ) 40,287-302. Olszyk, D. M. and Tibbitts, T. W. (1981a). Stomatal response and leaf injury of Pisum sativum L. with SO2 and 0 3 exposures. I. Influence of pollutant level and leaf maturity. Plant Physiology 67, 539-544. Olszyk, D. M. and Tibbitts, T. W. (1981b). Stomatal response and leaf injury of Pisum sativum L. with SO2 and 0 3 exposures. 11. Influence of moisture stress and time of exposure. Plant Physiology 67,545-549. Olszyk, D. M. and Tingey, D. T. (1984). Phytotoxicity of air pollutants: evidence for the photo-detoxification of SO2 but not 0 3 . Plant Physiology 74, 999-1005. Olszyk, D. M. and Tingey, D. T. (1985a). Interspecific variation in SO2 flux. Leaf surface versus internal flux, and components of leaf conductance. Plant Physiology 79,949-956. Olszyk, D. M. and Tingey, D. T. (1985b). Metabolic basis for injury to plants from combinations of 0 3 and SO*.Studies with modifiers of pollutant toxicity. Plant Physiology 77,935-939. Olszyk, D. M. and Tingey, D. T. (1986). Joint action of 0 3 and SO2 in modifying plant gas exchange. Plant Physiology 82, 401-405. Olszyk, D. M., Kats, G. Dawson, P. J., Bytnerowicz, A., Wolf, J. and Thompson, C. R. (1986). Characteristics of air exclusion systems vs. chambers for field air pollution studies. Journal of Environmental Quality 15, 326-334. Olszyk, D. M., Bytnerowicz, A., Fox, C. A., Kats, G., Dawson, P. J. and Wolf, J. (1987). Injury and physiological responses of Larrea tridentata (DC) Coville exposed in situ to sulphur dioxide. Environmental Pollution 48, 197-211. Omasa, K., Hashimoto, Y., Kramer, P. J., Strain, B. R., Aiga, I. and Kondo, J. (1985). Direct observation of reversible and irreversible stomata1 responses of attached sunflower leaves to SOz. Plant Physiology 79, 153-158. Omielan, J. A. and Pell, E. J. (1988). The role of photosynthetic activity in the response of isolated Glycine max mesophyll cells to ozone. Canadian Journal of Botany 66, 745-749. Ormrod, D. P., Black, V. J., and Unsworth, M. H. (1981). Depression of net photosynthesis in Viciafaba L. exposed to sulphur dioxide and ozone. Nature 291,585-586. Ormrod, D. P., Marie, B. A. and Allen, 0. B. (1988). Research approaches to

PHOTOSYNTHESIS AND STOMATAL RESPONSES

121

pollutant crop loss functions. I n “Assessment of Crop Loss from Air Pollutants” (W. W. Heck, 0. C. Taylor and D. T. Tingey, eds). Chap. 2. Elsevier Applied Science, London. Oshima, Y., Ushijama, T. and Tazaki, T. (1973). Effects of atmospheric SO2 on photosynthesis and transpiration rate of Helianthus annuus L. Environmental Control in Biology 11, 103-108. Oshima, R. J., Braegelmann, P. K., Flagler, R. B. and Teso, R. R. (1979). The effects of ozone on the growth, yield, and partitioning of dry matter in cotton. Journal of Environmental Pollution 8, 474-479. Pande, P. C. (1985). An examination of the sensitivity of five barley cultivars to SO2 pollution. Environmental Pollution (Series A ) 37, 27-41. Patton, R. L. and Garraway, M. 0. (1986). Ozone-induced necrosis and increased peroxidase activity in hybrid poplar (Populus s p . ) leaves. Environmental and Experimental Botany 26, 137-141. Pell, E. J. and Brennan, E. (1973). Changes in respiration, photosynthesis, adenosine 5’-triphosphate, and total adenylate content of ozonated pinto bean foliage as they relate to symptom expression. Plant Physiology 51, 378-381. Pell, E. J. and Pearson, N. S. (1983). Ozone-induced reduction in quantity of ribulose 1.5-bisphosphate carboxylase in alfalfa foliage. Plant Physiology 73, 185-187. Pell, E. J. and Puente, M. (1986). Emission of ethylene by oat plants treated with ozone and simulated acid rain. New Phytologist 103, 709-715. Petolino, J. F., Mulchi, C. L. and Aycock, M. K. Jr. (1983). Leaf injury and peroxydase activity in ozone-stressed tobacco cultivars and hybrids. Crop Science 23, 1102-1106. Pfanz, H., Martinoia, E., Lange, 0.-L. and Heber, U. (1987a). Mesophyll resistance to SO2 fluxes into leaves. Plant Physiology 85,922-927. Pfanz, H., Martinoia, E., Lange, 0.-L. and Heber, U. (1987b). Fluxof SO2 into leaf cells and cellular acidification by S02. Plant Physiology 85, 928-933. Pfeffer, H. U. and Buck, M. (1985). Messtechnik und Ergebnisse von Immissionsmessungen in Waldgebieten. VDI Berichte 560, 127-155. Pierre, M. and Queiroz, Q. (1988). Air pollution by SO2 amplifies the effect of water stress on enzymes and total soluble proteins of spruce needles. Physiologia Plantarum 73.412417. Podleckis, E. V., Curtis, C. R. and Heggestad, H. E. (1984). Peroxidase enzyme markers for ozone sensitivity in sweet corn. Phytopathology 74, 572-577. Posthumus, A. C. (1982). Biological indicators of air pollution. In “Effects of Gaseous Air Pollution in Agriculture and Horticulture” (M. H. Unsworth and D. P. Ormrod, eds), Chap. 2. Butterworths, London. Preston, K. P. (1988). Effects of sulphur dioxide pollution on a Californian coastal sage scrub community. Environmental Pollution 51, 179-195. Prinz, B. (1987). Causes of forest damage in Europe. Major hypothesis and factors. Environment 29(9), 10-37. Prinz, B. (1988). Ozone effects on vegetation. I n “Tropospheric Ozone” (I. S. A. Isaksen, ed). D. Reidel, New York. Prinz, B. and Krause, G. H. M. (1988). State of scientific discussion about the causes of the novel forest decline in the Federal Republic of Germany and surrounding countries. Paper presented at the 15th International Meeting for Specialists in Air Pollution Effects on Forest Ecosystem, IUFRO. Air Pollution and Forest Decline, Interlaken, Switzerland, 2-8 October 1988. Prinz, B., Krause, G. H. M. and Jung, K.-D. (1987). Development and causes of novel forest decline in Germany. I n “Effects of Atmospheric Pollutants on

122

H. SAXE

Forests, Wetlands and Agricultural Ecosystems” (T.C. Hutchinson, and K. M. Meema, eds), NATO AS1 Series, Vol. (316, pp. 1-24. Springer Verlag, Berlin. Puckett. L. J . (1982). Acid rain, air pollution, and tree growth in Southeastern New York. Journal of Environmental Quality 11, 376-381. Rabe, R. and Kreeb. K. H. (1979). Enzyme activities and chlorophyll and protein content in plants as indicators of air pollution. Environmental Pollution 19, 119-137. Rao. I. M., Amundson, R. G., Alscher-Herman, R. and Anderson, L. E. (1983). Effects of SOz on stomata1 metabolism in Pisum sativum L. Plant Physiology 72,573-577. Reich. P. B. (1983). Effects of low concentrations of O3on net photosynthesis, dark respiration, and chlorophyll contents in aging hybrid poplar leaves. Plant Physiology 73,291-296. Reich, P. B. (1987). Quantifying response to ozone: a unifying theory. Tree Physiology 3, 63-91. Reich. P. B. and Lassoie, J . P. (1984). Effects of low level 0 3 exposure on leaf diffusive conductance and water use efficiency in hybrid poplar. Plant, Cell and Environment 7 , 661-4568. Reich. P. B. and Amundson, R. G. (1985). Ambient levels of ozone reduce net photosynthesis in tree and crop species. Science 230, 566-570. Reich, P. B., Schoettle, A. W. and Amundson, R. G. (1985). Effects of low concentrations of 0 3 . leaf age and water stress on leaf diffusive conductance and water use efficiency in soybean. Physiologia Plantarum 63, 58-64. Reich, P. B., Schoettle, A. W. and Amundson, R. G . (1986a). Effects of 0 3 and acidic rain on photosynthesis and growth in sugar maple and northern red oak seedlings. Environmental Pollution (Series A ) 40, 1-15. Reich, P. B., Schoettle, A. W., Raba, R. M. and Amundson, R. G. (1986b). Response of soybean to low concentrations of ozone: I. Reductions in leaf and whole plant net photosynthesis and leaf chlorophyll content. Journal of Environmental Quality 15, 31-36. Rich, S., Waggoner, P. E. and Tomlinson, H. (1970). Ozone uptake by bean leaves. Science 169, 79-80. Rist, D. L. and Davis. D. D. (1979). The influence of exposure temperature and relative humidity on the response of pinto bean foliage to sulfur dioxide. The American Phytopathological Society 69, 23 1-235. Robinson, D. and Wellburn, A. R. (1983). Light-induced changes in the quenching of 9-amino-acridine flourescence by photosynthetic membranes due to atmospheric pollutants and their products. Environmental Pollution (Series A ) 32, 109-120. Rodecap, K. D. and Tingey, D. T. (1983). The influence of light on ozone-induced 1-aminocyclopropane- 1-carboxylic acid and ethylene production from intact plants. Zeitschrift Pflanzenphysiologie 110, 419-427. Rodecap, K. D. and Tingey, D. T. (1986). Ozone-induced ethylene release from leaf surfaces. Plant Science 44. 73-76. Rogers, H. H. and Campbell, J. C. (1979). Nitrogen-15 dioxide uptake and incorporation by Phaseolus vulgaris (L.). Science 206,333-335. Rogers, H. H., Jeffries, H. E. and Witherspoon, A. M. (1979). Measuring air pollutant uptake by plants: nitrogen dioxide. Journal of Environmental Quality 8, 551-557. Roloff, A. (1986). “Morphologie der Kronenentwicklung von Fagus sylvatica L. (Rotbuche) unter besonderer Beriicksichtigung moglicherweise neuartiger

PHOTOSYNTHESIS AND STOMATAL RESPONSES

123

Veranderungen”. Berichte des Forschungszentrum Waldokosystemel Waldsterben, Bd. 18. Universitat Gottingen. Roper, T. R. and Williams, L. E. (1988). Response of grape leaf photosynthesis to chronic and acute ozone exposure. HortScience 23,724 (Abstr. 034). Ross, L. J. and Nash, T. H. I11 (1983). Effect of ozone on gross photosynthesis of lichens. Environmental and Experimental Botany 23,71-77. Rowland, A. J. (1986). Nitrogen uptake, assimilation and transport in barley in the presence of atmospheric nitrogen dioxide. Plant and Soil 91,353-356. Rowland, A. J., Drew, M. C. and Wellburn, A. R. (1987). Foliar entry and incorporation of atmospheric nitrogen dioxide into barley plants of different nitrogen status. New Phytologist 107, 357-371. Sabaratnam, S. and Gupta, G. (1988). Effects of nitrogen dioxide on biochemical and physiological characteristics of soybean. Environmental Pollution 55, 149-158. Sabaratnam, S . , Gupta, G. and Mulchi, C. (1988). Nitrogen dioxide effects on photosynthesis in soybean. Journal of Environmental Quality 17, 143-146. Salema, M., Jukola-Sulonen, E.-L. and Lappalainen, T. (1987). Vaihtovirtavastuksen Kayttokelpoisuudesta kuusen (Picea abies) vitaliteettitunnuksena. Aquilo Series Botanica 25, 161 (Abstr. in English). Sandmann, G. and Gonzales, H. G. (1989). Peroxidative processes induced in bean leaves by fumigation with sulphur dioxide. Environmental Pollution 56, 145-154. Sato, S., Umezawa, T. and Ishikawa, H. (1979). Effects of Continuous Exposure of Low Concentration Sulphur Dioxide on the Growth of Radish (Raphanus sativus L.). Bulletin of the Bioenvironment Laboratory, CRIEPI, 478014, Japan. Saxe, H. (1979). A structural and functional study of the coordinated reactions of individual Commelina communis L. stomata (Commelinaceae). American Journal of Botany 66, 1044-1054. Saxe, H. (1983). Long-term effects of low levels of SO2 on bean plants (Phaseolus vulgaris). I. Immission-response pattern of net photosynthesis and transpiration during life-long, continuous measurements. Physiologia Plantarum 57, 101-107. Saxe, H. (1986a). Effects of NO, NO2 and C02 on net photosynthesis, dark respiration and transpiration of pot plants. New Phytologist 103, 185-197. Saxe, H. (1986b). Stomatal-dependent and stomatal-independent uptake of NO,. New Phytologist 103, 199-205. Saxe, H. (1987a). Stomate-dependent and stomate-independentuptake of NO,, and effects on photosynthesis respiration and transpiration of potted plants. In “Effects of Atmospheric Pollutants on Forests, Wetlands and Agricultural Ecosystems” (T. C . Hutchinson and K. M. Meema, eds), pp. 463-479 NATO AS1 Series, Vol. G16 R). Springer Verlag, Berlin. Saxe, H. (1987b). Kvaelstofilter i C02-berigede vaeksthuse. Gartner Tidende 42, 11941199. Saxe, H. (1987~).COz fordeling i vaeksthuse. Cartner Tidende 42, 1202-1205. Saxe, H. (1988). Kuldioxid ti1 vzksthusgreintsager, med og uden kvelstofilte. Ugeskriftf o r Jordbrug 18,666467. Saxe, H. (1989). Diagnostic parameters for selecting against novel spruce (Picea abies) decline: IV. Response of photosynthesis and transpiration to SO,+NO2 exposures. Physiologia Plantarum 76,362-367. Saxe, H. and Christensen, 0. V. (1985). Effects of carbon dioxide with and without nitric oxide pollution on growth, morphogenesis and production time of pot plants. Environmental Pollution (Series A ) 38, 159-169.

124

H . SAXE

Saxe, H. and Murali, N. S. (1989a). Diagnostic parameters for selecting against novel spruce (Picea abies) decline: I. Tree morphology and photosynthesis response to acute SO2 exposures. Physiologia Plantarum 76,340-348. Saxe, H. and Murali, N. S. (1989b). Diagnostic parameters for selecting against novel spruce (Picea abies) decline: 11. Response of photosynthesis and transpiration to acute NO2-exposures. Physiologia Plantarum 76: 349-355. Saxe, H. and Murali, N. S. (1989~).Diagnostic parameters for selecting against novel spruce (Picea abies) decline: 111. Response of photosynthesis and transpiration to 0 3 exposures. Physiologia Plantarum 76: 362-367. Schmidt, W., Schreiber. U. and Urbach, W. (1988). SO2 injury in intact leaves, as detected by chlorophyll fluorescence. Zeitschrift Naturforschung Teil c 43, 269-274. Scholz, F. and Bergmann, F. (1984). Selection pressure by air pollution as studied by isozyme-gene-systems in Norway spruce exposed to sulphur dioxide. Silvae Genetica 33, 238-241. Schulte-Hostede, S., Darrall, N. M., Blank, L. W. and Wellburn, A. R. (1988). “Air Pollution and Plant Metabolism”. Elsevier Applied Science, London. Schutt, P. and Cowling, E. B. (1985). Waldsterben, a General decline of forests in Central Europe: symptoms, development and possible causes. Plant Disease 69,548-558. Scott, M. G. and Hutchinson, T. C. (1987). Effects of a simulated acid rain episode on photosynthesis and recovery in the Caribouforage lichens, Cladina stellaris (Opiz.) Brodo and Cladina Rangiferina (L.). New Phytologist 107, 567-575. Selinger, von H., Knoppik, D. and Ziegler-Jons, A. (1986). Einfluss von Mineralstoffernahrung, Ozone und saurem Nebel auf Photosynthese-Parameter und stomatare Leitfahigkeit von Picea abies (L.) Karst. Forstwissenschaftliches Centralblatt 105,239-242. Senger, H., Osswald, W., Senser, M., Greim, H. and Elstner, E. F. (1986). Gehalte an chlorophyll und den Antioxidantien Ascorbat, Glutathion und Tocopherol in Fichtennadeln (Picea abies [L.] Karst.) in Abhangigkeit von Mineralstoffernahrung. Ozone und saurem. Forstwissenschaftliches Centralblatt 105, 264-267. Seufert, G., Arndt, U., Jager, H. J., Bender, J. and Schweizer, B. (1988). Longterm effects of air pollutants on spruce (Picea abies) and fir (Abies alba) in open-top chambers. In “Effects of Atmospheric Pollutants on the Spruce-fir forests of the Eastern United States and the Federal Republic of Germany”. U.S. Forest Service Technical Report, October, 1987. Shertz, R. D., Kender, W. J. and Musselman, R. C. (1980). Effects of ozone and sulfur dioxide on grapevine. Scientia Horticultura 13, 37-46. Shimazaki, K.-I. (1988). Thylakoid membrane reactions to air pollutants. In “Air Pollution and Plant Metabolism”. (S. Schulte-Hostede, N. M. Darrall, L. W. Blank and A. R. Wellburn, eds), pp. 116-133. Elsevier Applied Science, London. Shimizu, H., Furukawa, A. and Totsuka, T. (1980). “Effects of Low Concentrations of SO2 on the Growth of Sunflower”. Research Report from the National Institute for Environmental Studies 11, 9-17. Sigal, L. L. and Johnston, J. W., Jr. (1986). Effects of acidic rain and ozone on nitrogen fixation and photosynthesis in the lichen Laboria pulmonaria (L.). Environmental and Experimental Botany 26, 59-64. Sigal, L. L. and Suter, G. W. 11. (1987). Evaluation of methods for determining adverse impacts of air pollution on terrestrial ecosystems. Environmental Management 11, 675-694.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

125

Sij, J. W. and Swanson. C. A. (1974). Short-term kinetic studies on the inhibition of photosynthesis by sulfurdioxide. Journalof Environmental Quality 3: 103-107. Silvius. J. E.. Ingle. M. and Baer, C. H . (1975). Sulfur dioxide inhibition of photosynthesis in isolated spinach chloroplasts. Plant Physiology 56,434-437. Sinn, J. P.. Pell, E. J. and Kabel, R. L. (1984). Uptake rate of nitrogen dioxide by potato plants. Air Pollution Control Association 34, 668-669. Sisson. W. B.. Booth, J. A. and Throneberry, G . 0. (1981). Absorption of SO2 by pecan (Carya illinoensis (Wang) K. Koch) and alfalfa (Medicagosativa L.) and its effect on net photosynthesis. Journal of Experimental Botany 32,523-532. Skarby, L.. Bengtson, C.. Bostrom. C.-A., Greenfelt, P. and Troeng, E. (1981). Uptake of NO, in Scots pine. Silva Fennica 15, 496-498. Skarby, L. Troeng, E. and Bostrom. C.-A. (1987). Ozone uptake and effects on transpiration, net photosynthesis, and dark respiration in Scots pine. Forest Science 33, 801-808. Smith. G . ,Greenhalgh, B., Brennan, E. and Justin, J. (1987). Soybean yieldinNew Jersey relative to ozone pollution and antioxidant application. Plant Disease 71, 121-125. Srivastava, H. S., Joliffe. P. A. and Runecles, V. C. (1975a). Inhibition of gas exchange in bean leaves by NOz. Canadian Journal of Botany 53,466-474. Srivastava. H. S., Joliffe, P. A. and Runecles, V. C. (1975b). The effects of environmental conditions on the inhibition of leaf gas exchange by N02. Canadian Journal of Botany 53,475-482. Stan. H.-J. and Schicker. S. (1982). Effect of repetitive ozone treatment on bean plants-stress ethylene production and leaf necrosis. Atmospheric Environment 16.2267-2270. Strasser, R. J., Schwarz, B. and Bucher, J. B. (1987). Simultane Messung der Chlorophyll Fluoreszenz-Kinetik bei verschiedenen Wellenlangen als rasches Verfahren zur Fruhdiagnose von Immissionsbelastungen an Waldbaumen: Ozoneinwirkungen auf Buchen und Pappeln. European Journal of Forest Pathology 17, 149-157. Sueur-Brymer, N. M. and Ormrod, D. P. (1984). Carbon dioxide exchange rates of fruiting soybean plants exposed to ozone and sulphur dioxide singly or in combination. Canadian Journal of Plant Science 64,69-75. Sugahara, K. (1984). Effects of air pollutants on light reactions in chloroplasts. In “Gaseous Air Pollutants and Plant Metabolism” (M. J. Koziol and F. R. Whatley, eds), Chap. 13. Butterworths, London. Takemoto, K. and Noble, R. D. (1982). The effect of short-term SO2 fumigation on photosynthesis and respiration in soybean Glycine max. Environmental Pollution (Series A ) 28, 67-74. Takemoto, B. K., Shriner, D. S. and Johnston, J. W. Jr. (1987). Physiological responses of soybean (Glycine m u L. Merr) to simulated acid rain and ambient ozone in the field. Water, Air, and Soil Pollution 33, 373-384. Takemoto, B. K., Bytnerowicz, A. and Olszyk, D. M. (1988a). Depression of photosynthesis, growth, and yield in field-grown green pepper (Capsicum anuum L.) exposed to acidic fog and ambient ozone. Plant Physiology 88, 477-482. Takemoto, B. K., Hutton, W. J. and Olszyk, D. M. (1988b). Responses of fieldgrown Medicago sativa L. to acidic fog and ambient ozone. Environmental Pollution 54, 97-107. Tanaka, K., Suda, Y., Kondo, N. and Sugahara, K. (1985). 0 3 tolerance and the ascorbate-dependent H 2 0 z decomposing system in chloroplasts. Plant Cell Physiology 26, 1425-1431.

126

H. SAXE

Taylor, G. E., Jr. and Tingey, D. T. (1983). Sulfur dioxide flux into leaves of Geranium carolinianum L. Evidence for a non-stomata1 or residual resistance. Plant Physiology 12,237-244. Taylor, G. E., Jr., Tingey, D. T. and Ratsch, H. C. (1982). Ozone flux in Glycine max (L.) Merr.: sites of regulation and relationship to leaf injury. Oecologia 53, 179-186. Taylor, G. E., Jr., Tingey, D. T. and Gunderson, C. A. (1986). Photosynthesis, carbon allocation, and growth of sulfur dioxide eco-types of Geranium carolinianum L. Oecologia 68, 350-357. Taylor, G. E., Jr., Ross-Todd, B. M. and Gunderson, C. A. (1988). Action of ozone on foliar gas exchange in Glycine max L. Merr. a potential role for endogenous stress ethylene. New Phytologist 110, 301-307. Taylor, H. J. and Bell, J. N. B. (1988). Studies on the tolerance to SO2 of grass populations in polluted areas. V. Investigations into the development of tolerance to SO2 and NO2 in combination and NO2 alone. New Phytologist 110,327-338. Temple, P. J. (1986). Stomatal conductance and transpirational responses of fieldgrown cotton to ozone. Plant, Cell and Environment 9,315-321. Temple, P. J., Fa, C. H. and Taylor, 0. C. (1985a). Effects of SOz on stomatal conductance and growth of Phaseolus vulgaris. Environmental Pollution (Series A ) 31,267-279. Temple, P. J., Taylor, 0. C. and Benoit, L. F. (1985b). Cotton yield responses to ozone as mediated by soil moisture and evapo-transpiration. Journal of Environmental Quality 14,5540. Temple, P. J., Lennox, R. W., Bytnerowicz, A. andTaylor, 0. C. (1987). Interactive effects of simulated acidic fog and ozone on fieldgrown alfalfa. Environmental and Experimental Botany 21,409417. Temple, P. J., Benoit, L. F., Lennox, R. W., Reagan, C. A. and Taylor, 0. C. (1988a). Combined effects of ozone and water stress on alfalfa growth and yield. Environmental Quality 11, 108-113. Temple, P. J., Kupper, R. S., Lennox, R. L. and Rohr, K. (1988b). Physiological and growth responses of differentially irrigated cotton to ozone. Environmental Pollution 53, 255-263. Tingey, D. T. and Hogsett, W. E. (1985). Water stress reduces ozone injury via a stomatal mechanism. Plant Physiology 77, 944-947. Tingey, D. T., Thutt, G. L., Gumpertz, M. L. and Hogsett, W. E. (1982). Plant water status influences ozone sensitivity of bean plants. Agriculture and Environment 7,243-254. Tomiczek, C. (1987). Stressuntersuchungen an “Ozone-begasten Fichten” mittels Digital-Impullstromgerat (Conditiometer AS-1). Centralblatt fur die Gesamte Forstwesen 104,219-224. Tschanz, A., Landolt, W., Bleuler, P. andBrunold, C. (1986). Effectsof SOzon the activity of adenosine 5’-phosphosulfate sulfo-transferase from spruce trees (Picea abies) in fumigation chambers and under field conditions. Physiologia Plantarum 61,235-241. Tseng, E. C., Seiler, J. R. and Chevone, B. I. (1988). Effects of ozone and water stress on greenhouse grown Fraser fir seedling growth and physiology. Environmental and Experimental Botany 28,3741. Tuomisto, H. (1988). Use of Picea abies needles as indicators of air pollution: epicuticular wax morphology. Annales Botanici Fennici 25, 35 1-364. Unsworth, M. H., Biscoe, P. V. and Pinckney, H. R. (1972). Stomatal responses to sulphur dioxide. Nature 239,458459.

PHOTOSYNTHESIS AND STOMATAL RESPONSES

127

Ushijima, T. and Tazaki, T. (1977). The influence of sulphur dioxide on the photosynthetic and transpiration rate in several higher plants. Proceedings of the Fourth International Clean Air Congress, pp. 84-87. International Union of Air Pollution Prevention Association, Brighton, UK. Varshney, S. R. K. and Varshney, C. K. (1984). Effects of SO2 on ascorbic acid in crop plants. Environmental Pollution (Series A ) 35, 285-290. Vozzo, S. F., Miller, J. E., Heagle, A. S. and Pursley, W. A. (1988). Effects of ozone and water stress on net photosynthetic rate of field grown soybean leaves. Environmental Pollution 53, 471-473. Walmsley, L., Ashmore, M. R. and Bell, J. N. B. (1980). Adaptation of radish Raphanus sativus L. in response to continuous exposure to ozone. Environmental Pollution (Series A ) 23, 165-177. Weidner, M. and Kraus, M. (1987). Ribulose 1,5-bisphosphate carboxylase activity and influence of air pollution in spruce. Physiologia Plantarum 70, 664-672. Wellburn, A. R. (1982). Effects of SO2 and NO2 on metabolic function. I n “Effects of Gaseous Air Pollution in Agriculture and Horticulture” (M. H. Unsworth and D. P. Ormrod, eds), Chap. 8. Butterworths, London. Wellburn, A. (1988). “Air Pollution and Acid Rain. The Biological Impact”. Longman Scientific & Technical, London. Wentzel, K. F. (1985). Smogbelastung im Wald am hochsten. Hessischer Waldbesitzerverband 33(5),33-35. White, K. L., Hill, A. C. and Bennett, J. H. (1974). Synergistic inhibition of apparent photosynthesis rate of alfalfa by combinations of sulfur dioxide and nitrogen dioxide. Environmental Science and Technology 8,574-576. Wild, A., Flammersfeld, W.,Moors, I . , Dietz, B. and Riihle, W. (1988). Investigation on the photosynthetic membranes of spruce needles in relation to the occurrence of novel forest decline 11. The content of Q,-protein, cytochrome f, and P-700. Zeitschrift fur Naturforschung, Teil c, 43,589-595. Winner, W. E. and Mooney, H. A. (1980a). Ecology of SO2 resistance: I. Effects of fumigations on gas exchange of deciduous and evergreen shrubs. Oecologia 44,290-295. Winner, W. E. and Mooney, H. A. (1980b). Ecology of SO2 resistance: 11. Photosynthetic changes of shrubs in relation to SO2 absorption and stomata1 behaviour. Oecologia 44, 296-302. Winner, W. E. and Mooney, H. A. (1980~).Ecology of S02resistance: 111. Metabolic changes of C3 and C4 Atriplex species due to SO2 fumigations. Oecologia 46,49-54. Winner, W. E. and Mooney, H. A. (1985). Ecology of SO2 resistance. V. Effects of volcanic SO2 on native Hawaiian plants. Oecologia 66, 387-393. Winner, W. E., Gellespie, C., Shen, W . 4 . and Mooney, H. A. (1988). Stomata1 responses to SO2 and 03.I n “Air Pollution and Plant Metabolism” (S. Schulte-Holstede. N. M. Darrall, L. W. Blank and A. R. Wellburn, eds), pp. 255-271. Elsevier Applied Science, London. Wolfenden, J . , Robinson, D. C . , Cape, J . N., Paterson, I. S., Francis, B. J., Mehlhorn, H. and Wellburn, A. R. (1988). Use of carotenoid ratios, ethylene emissions and buffer capacities for the early diagnosis of forest decline. New Phytologist 109, 85-95. Yang, Y.-S., Skelly, J. M., Chevone, B. I . and Birch, J. B. (1983). Effects of long-term ozone exposure on photosynthesis and dark respiration of eastern white pine. Environmental Science and Technology 17,371-373. Yoneyama, T., Totsuka, T., Hayakawa, N. and Yazaki, J. (1980a). Absorption of atmospheric NO2 by plants and soils. V. Day and night NO2-fumigation effect

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on the plant growth and estimation of the amount of NO*-nitrogen absorbed by plants. Research Report of the National Institute for Environmental Studies, Japan 11, 31-50. Yoneyama, T., Yasuda, T . , Yazaki, J. and Totsuka, T. (1980b). Absorption of atmospheric NO1 by plants and soil. VII. NO2 absorption by plants: reevaluation of the air-soil-root route. Research Report of the National Institute for Environmental Studies, Japan 11, 59-67. Ziegler. I. (1972). The effects of SO3 on the activity of ribulose-l,5-diphosphate carboxylase in isolated spinach chloroplasts. Planta 103, 155-163.

Transport and Metabolism of Carbon and Nitrogen in Legume Nodules

JOHN . G . STREETER Department of Agronomy. Ohio State University and Ohio Agricultural Research and Development Center. .Wooster. Ohio 44691. USA

I . Introduction

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

I1. Nodule Anatomy and Terminology . . . . . A . Tissues and Cell Types . . . . . . . . . . . . B . Organization in Infected Cells C . Bacteroids . . . . . . . . . . . . . .

. . . . . . . . .

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

130 131 131 134 139

111. Carbon Processing . . . . . . . . . . . . . . . . . . . . . A . HostFunctions . . . . . . . . . . . . . . . . . . . . B . Bacteroid Functions . . . . . . . . . . . . . . . . . .

141 141 146

IV . Nitrogen Processing . . . . . . . . . . . . . . . . . . . . A . Bacteroid Functions . . . . . . . . . . . . . . . . . . B . Host Functions . . . . . . . . . . . . . . . . . . . .

153 153 154

V . RestrictionsImposedbyMicroaerobicConditions . . . . . . . A . The Oxygen Regulation System . . . . . . . . . . . . . B . Impact of Low 0 2 on Metabolism . . . . . . . . . . . .

161 161 161

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163

VI . Summary and Suggestions for Future Work

Acknowledgements

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165

References . . . . . . . . . . . . . . . . . . . . . . . . 165 Copyright 01991Academic Press Limited Advances in Botanical Research Vol . 18 ISBN 0-12-00591%5

All rights of reproduction in any form reserved

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I.

INTRODUCTION

The subject area chosen for this review provides extraordinary challenges and opportunities. The opportunities relate to the fact that in the legume nodule, we find two diverse organisms living side by side. Thus, as we might expect, the metabolism of the plant and the metabolism of the bacteria are interconnected and even show signs of interdependence. Although metabolism in nodules may be complex, there may be unique opportunities to broaden our knowledge about the regulation and control of plant metabolism in general. The challenges afforded by the subject area relate to the breadth and diversity of the literature. The subject material is scattered across the microbiology, biochemistry, plant physiology, and applied plant science literature. There is also more literature than can be realistically reviewed as a single collection. For some subjects (e.g. nitrogenase biochemistry) it has been necessary to cite only sample references, but for some sections an attempt has been made to review essentially all of the recent literature. A few reviews specific to particular subject areas of this chapter have been published, and these will be cited in the pertinent sections. A recent major review of carbon metabolism in rhizobia has been published, but that review emphasized cultured bacteria (Stowers, 1985); as we will see, metabolic capabilities of cultured bacteria and bacteroids may be quite different. Another previous review is that by Schubert (1986) on metabolism and transport of fixed nitrogen in legume nodules. Finally, Rawsthorne et al., (1980) reviewed several aspects of the present subject, but that review is now over 10 years old. The emphasis in this chapter is on what may be called the “organization of metabolism”. To repeat, legume nodules include two different sets of genes and two different sets of metabolic capabilities. Superimposed on this metabolic complexity is a system of specialized tissues and compartments within cells, and superimposed on most of the system is a very low free oxygen concentration. All this leads to some uncommon biochemistry and to some opportunities for regulation of transport and metabolism which we do not find in more typical plant tissues. It is on these unique qualities of nodules that this chapter will focus, in the hope that there are some useful lessons to be learned both by the general plant physiologist and by those struggling to understand the chemical interactions of the plant and bacterium. A good place to start is with the realization that the overall function of legume nodules requires very large amounts of reducing equivalents (electrons, or, more to the point here, reduced carbon). Various estimates have been published, but there seems to be a consensus for a requirement of about 12.2g of carbohydrate for each gram of nitrogen fixed for soybean (Glycine max (L.) Merr.) nodules (Rainbird et al., 1984; Heytler and Hardy,

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1984). This includes a “cost” of 7.3 g for the operation of nitrogenase, 2.7g for nodule maintenance, 1.9g for assimilation and transport of fixed nitrogen, and 0.26g for growth (Rainbird et al., 1984). Thus, to support a typical seasonal amount of nitrogen fixed by a crop of soybeans-namely, 75 kg of nitrogen per hectare-about 1.5 tonnes of sucrose would be required. Not all authors would agree with this estimate and an example of another careful analysis has produced a slightly higher value (17 g carbohydrate per gram of fixed nitrogen) based on an analysis of clover (Trifoliurn repens, T. prarense), alfalfa (Medicugo sativa), sainfoin (Onobrychis viciifolia) and pea (Pisurn sativurn) nodules (Witty et al., 1983).

11. NODULE ANATOMY AND TERMINOLOGY A. TISSUES AND CELL TYPES

Various aspects of the subject of nodule structure have been reviewed by Newcomb (1981) and by Sprent (1980); also, Schubert (1986) has recently provided a useful summary of export products of a wide variety of nodules. Legume nodules can be classified conveniently into two groups. “Determinate” nodules have no meristem, are usually spherical in shape, have infected cells lacking vacuoles and generally export ureides. Arachis and Lotus nodules are exceptions to this rule in that they form determinate nodules which do not export ureides (Peoples et al., 1986; J. I. Sprent, personal communication). “Indeterminate” nodules have a meristem and, because of their continuing growth, are generally cylindrical in shape. In addition, indeterminate nodules have vacuolated infected cells and export amides (principally asparagine). Some other features of the two types of nodules have been summarized by Sprent (1980). A selection of publications containing high-quality micrographs which illustrate the anatomy of the two nodule types is given in Table I. In spite of the simple classification shown in Table I, it should be emphasized that there is considerable diversity among legume nodules (Sprent, 1980, 1989) and most legume nodules have not been studied (deFaria et al., 1989). Although various nodules differ in some structural details, the general organization of tissue regions is similar in most nodules and will be described based on the most thoroughly studied nodules, namely soybean. Beginning from the outside, the outer cortex is a loosely organized region wherein it is often difficult to locate a true epidermis (Fig. 1). This loose organization is consistent with the importance of gas exchange to the operation of the nodule. Also consistent with the need for gas transport is the presence of prominent lenticels on the surface of some nodules (Pankhurst and Sprent, 1975). Curiously, not all nodules have lenticels, and this is even true for some large, spherical nodules (Sen et al., 1986). A layer of sclerenchyma

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cells is sometimes present in the outer cortex (Fig. l), but many nodules lack this tissue. Its function is undoubtedly to strengthen the nodule; there are gaps in the sclerenchyma layer indicating that it is not a significant barrier to gas exchange (Selker and Newcomb, 1985). Further into the cortex, four to seven cell layers from the infected tissue, are found the vascular bundles, each of which is surrounded by an endoderma1 layer (Pate et al., 1969; Newcomb et al., 1989). Transfer cells found within the vascular bundles of indeterminate nodules probably facilitate transport of nitrogenous products out of the nodule (Pate et al., 1969). Between the vascular bundles and the infected cells lies a specialized layer of the cortex, referred to simply as the inner cortex. Plastids and abundant starch grains (especially in young nodules) are found in these cells along with peroxisomes, which are responsible for a terminal step of nitrogen processing in determinate nodules (Newcomb et al., 1989). Thus, the inner cortex appears to be important in the processing of both carbon and nitrogen. Also, it is within this layer that a barrier to gas diffusion is localized (Tjepkema and

TABLE I Summary of selected reports on legume nodule structure Legume

Typea

Reference

Medicago sativa L.

Ind

Glycine max (L.) Merr

Det

Pisum sativum L.

Ind

Phaseolus vulgaris L.

Det

Lotus pedunculatus Cav. Arachis hypogaea L. Vigna unguiculata L. Trifolium repens L. Pisum arvense L. Vigna radiata L. Sesbania rostrata L.' Oxytropis maydelliana Trautv. Oxytropis arctobia Bunged Astragalus alpinus L.d

Det Det Det Ind Ind Det Ind? Ind Ind Ind

Vance et al. (1980) Pate1 and Yang (1981) Truchet et al. (1989) Goodchild and Bergersen (1966) Pankhurst and Sprent ( 1975)h Werner and Morschel (1978) Newcomb et al. (1979) Newcomb (1976) Newcomb et al. (1979) VandenBosch et al. (1985) Lafontaine et al. (1990) Pankhurst et al. (1979) Sen et al. (1986) Sen et al. (1986) Pate et al. (1969) Pate et al. (1969) Newcomb and McIntyre (1981) Tsien et al. (1983) Newcomb and Wood (1986) Newcomb and Wood (1986) Newcomb and Wood (1986)

~~

"Det, determinate; Ind, indeterminate (see text). 'Surface features; emphasis on lenticels. %tern nodules. dLegumes from the high Arctic tundra.

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Yocum, 1974); this barrier and its implications for nodule function will be discussed in a later section. A detailed analysis of the vascular anatomy of soybean nodules has recently led to the important realization that the system is not continuous, i.e. each strand ends in a loop surrounded by a closed endodermal sac (Walsh et af.,1989a). This finding is consistent with previous results showing that nitrogen fixing activity of nodules is relatively insensitive to fluctuations in transpiration (Minchin and Pate, 1974; d e v i s e r and Poorter, 1984). This finding also has major ramifications for transport of metabolites into and out

Fig. 1. Cross-sectional view of a portion of a soybean nodule; magnification X 400. VB, vascular bundle; SC, sclerenchyma; OC, outer cortex; IC, inner cortex; IR, infected region. Smaller, lighter cells in the infected region are uninfected. Inclusions in these uninfected cells and in the inner cortex are starch grains.

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of nodules because it means that the supply of water to support xylem export is dependent on phloem import, not on transpirational movement of water through the root (Walsh et al., 1989b). However, Raven et al. (1989) have questioned this proposition on theoretical grounds and have suggested that some water from the root is required to support xylem export. They suggest diffusion of water through the parenchyma connecting the root and nodule, but the matter remains unresolved. The infected zone generally forms a well-defined region in the centre of the nodule (Fig. 1). It is important to note that, within this region, uninfected cells are also present. In soybean nodules, uninfected cells have been estimated to occupy 21% of the volume, and every infected cell is in contact with at least one uninfected cell (Selker and Newcomb, 1985). This intimate contact of infected cells and uninfected cells is a critical feature because it is in the uninfected cells that the key step of ureide synthesis (discussed later) is carried out (Newcomb et al., 1985). In most determinate nodules, some of the uninfected cells are organized into rays, and the three-dimensional organization of these rays in the soybean nodule has recently been beautifully portrayed (Selker, 1988). Uninfected cells are also present in the infected zone of indeterminate nodules, but organization into rays seems to be absent, and whether or not these cells play any unique role in nitrogen processing has not been elucidated (Vance et al., 1980; Patel and Yang, 1981; Newcomb and Wood, 1986). Aruchis nodules again seem to be an exception in that they appear to lack uninfected cells in the infected region (Sen et al., 1986); the implication of this arrangement for nodule function is discussed later. Because of differences in the metabolic role of various cell types, there have been several attempts to purify them for biochemical studies. The infected zone and cortex can be separated by mechanical means (Shelp etal., 1983) or with the use of commercially available hydrolytic enzymes (Streeter, 1982); the latter method is generally used. With soybean nodules, cellulase plus pectinase treatment results in disintegration of the infected zone whereas the cortex remains intact (Streeter, 1982). The infected and uninfected cells from the infected region can then be separated using sucrose gradient centrifugation (Shelp et al., 1983; Hanks et al., 1983) or Percoll gradient centrifugation (Kouchi et al., 1988, 1989). The differences in the biochemical function of these cell types are discussed later.

B. ORGANIZATION IN INFECTED CELLS

1. Mitochondria and Symbiosomes A somewhat surprising feature of infected cells is the numerous mitochondria. These tend to be clustered at the periphery of the cells near the intercellular spaces (Fig. 2) (Goodchild and Bergersen, 1966); this presu-

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mably is related to their O2 requirement. In infected cells of soybean nodules, the mitochondria1volume per unit of cytoplasm is four times that in the uninfected cells (Newcomb et al., 1985). Methods for preparation of highly purified mitochondria from cowpea and soybean nodules have recently been developed (Rawsthorne and LaRue, 1986a; Day et al., 1986).

Fig. 2. An infected cell (left) and an uninfected, vacuolated cell (right) from a soybean nodule; magnification X 13500.SBM, symbiosome membrane; SBS, symbiosome space; BAC, bacteroid; MIT, mitochondrion. The white areas within the bacteroids are mainly polyp-hydroxybutyrate. The material within the SBM is generally not seen and is probably an artifact of fixation. Plasmodesmata can be seen connecting different cells (see text).

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Taxonomic relationships and some of the features of rhizobia have recently been summarized along with some details of the infection process (Sprent, 1989). Microsymbionts are transported to the inner tissues of the developing nodules by way of an infection thread. In some legume nodules the bacteria remain in the thread (Sprent, 1989) but, in general, they are released into a sac formed by the membrane surrounding the infection thread. The release process results in enclosure of bacteria in an “insideout” plasma membrane (Verma and Fortin, 1989), i.e. bacteria remain adjacent to the outer face of the membrane and this may influence transport processes, at least in the initial stages of development of the symbiosis. The bacteria undergo numerous morphological and biochemical changes, due in part to the low 0 2 environment (Gober and Kashket, 1989) and to exposure to dicarboxylic acids (Urban and Dazzo, 1982; Urban and Nelke, 1988; Reding and Lepo, 1989), and they are subsequently termed bacteroids. A detailed analysis of bacteroid differentiation in indeterminate alfalfa nodules has recently been published (Vasse et al., 1990). The membraneenclosed bacteroids constitute a fundamental unit of symbiotic nitrogen fixation, termed the “peribacteroid compartment” or “peribacteroid vacuole” or, most commonly, the “peribacteroid unit”. This terminology has recently been challenged as inappropriate, and suggestions for new terminology have been put forward (Roth et al., 1988; Roth and Stacey, 1989a). Major arguments for the new terminology are the potential for confusion of the “peribacteroid space” with the periplasmic space of bacteroids (see later) and the need to use (and publish) terms which correspond to other symbiotic systems, thereby encouraging physiological comparisons. The proposed terminology is gaining acceptance and will be used here; the new terms are defined in Table 11. Not all workers agree with this terminology (Smith and Smith, 1990), but the proposed use of “interfacial apoplast” for the symbiosome space will only add to the confusion and in the author’s opinion should be avoided. Symbiosomes, the symbiosome membrane (SBM), and the symbiosome space (SBS) in soybean nodules can be seen in Fig. 2. This organization is common to all legume nodules except that the number of bacteroids per symbiosome may vary, in a few cases, numbering only one per symbiosome TABLE I1 Terminology and definitions for symbiosomes New term and abbreviation

Previous term Definition

Symbiosome membrane (SBM) Peribacteroid membrane Symbiosome space (SBS) Peribacteroid space Symbiosome Peribacteroid unit

Plant-derived membrane surrounding the bacteroids in nodules Space between the bacteroids and the symbiosome membrane Bacteroids surrounded by the intact symbiosome membrane

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(e.g., peanut (Arachis hypopogaea (Bal et al., 1989)). In nodules with multiple bacteroids per symbiosome, symbiosomes in young nodules will also contain single bacteroids because bacteroids are released from the infection thread one at a time (e.g. Werner and Morschel, 1978; Roth and Stacey, 1989b). Obviously, the situation in mature nodules (Fig. 2) requires that bacteroids divide within the symbiosome. This process has been documented by Robertson and Lyttleton (1984) whose micrographs show that, at least in some cases, the SBM closes around the newly divided cells resulting in the production of a new, independent symbiosome. Formation of new symbiosomes may be facilitated by the physical association of the SBM and the lipopolysaccharide of the bacteroid outer membrane (Bradley et al., 1986). 2. The Symbiosome Membrane The exact origin of the SBM has been the subject of some debate, but space limitations preclude a complete review of all arguments (Mellor and Werner, 1987). Some authors have focused on the similarities of the SBM and plasma membranes, thereby implying a common origin (Verma et al., 1978; Blumwald et a l . , 1985). This position gains some support from the electron microscope evidence showing release of bacteria into membrane enclosures which are initially contiguous with the plasma membrane (Newcomb and McIntyre, 1981; Roth and Stacey, 1989b). Other authors conclude that the SBM is synthesized de novo from vesicles originating in the endoplasmic reticulum or from Golgi processing (e.g. Robertson et al., 1978a; Kijne and Planque, 1979; Mellor et af., 1985). The relative abundance of Golgi bodies in young nodules is consistent with this view. Both views have some merit and the SBM is probably a “mosaic” membrane unique to the legume nodule. Furthermore, there is evidence to support this in the patchwork appearance of the membrane when it is stained with phosphotungstic acid (PTA). In plant cells, PTA stains the plasma membrane specifically and intensely (Roland et a f . , 1972), and when used as a stain for nodule sections, stains only portions of the SBM (Verma et al., 1978; Robertson et al., 1978a; Roth and Stacey, 1989a); although Robertson et al. (1978a) rated the SBM as PTA-stainable (their Table I), they noted variation in stainability across a section, and their micrographs show a typical mosaic pattern. In unpublished studies, I have also noted only partial staining of the SBM in nodules and, in addition, found that purified SBM vesicles give an irregular pattern of staining with PTA. I suggest that the bulk of the SBM is initially derived from plasma membrane, but that, as symbiosomes divide, the additional membrane required is of a different composition. This suggestion is not radically different from previously proposed models (Robertson et d.,1978a; Roth and Stacey, 1989a); if, in fact, the SBM is a “mixture” of different membrane types, this may have an important impact on the physiology of symbiosomes.

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In 1978, two major studies demonstrated that intact symbiosomes could be isolated from legume nodules (Robertson et al., 1978b; Verma et al., 1978). Isolation of the SBM from symbiosomes permitted analysis of its chemical and physical properties by these groups and others. For example, several nodule-specific proteins have recently been localized in the SBM and, although these are host proteins, some appear to be under the control of bacteroids (Bisseling et al., 1983; Katinakis and Verma, 1985; Fortin et al., 1985, 1987; Werner etal., 1988; Mellor etal., 1989). There is, of course, great interest in the identity of the SBM-specificproteins but, to date, most have been described only as protein bands following gel electrophoresis. One of the SBM proteins is an ATPase, having been studied in preparations from soybean and lupin nodules. However, there is, as yet, not complete agreement on the properties of this important enzyme. For example, the highly interesting stimulation by NH4+ reported by Blumwald et al. (1985) has not been observed by others (Mellor et al., 1984; Bassarab et al., 1986). Also, the enzyme purified from lupin SBM was not stimulated by NH4+ (Domigan et al., 1988). Bassarab et al. (1986) have suggested the presence of two types of ATPase based on properties inconsistent with either pure plasma membrane or pure tonoplast ATPases. The enzyme purified from lupin SBM also has some atypical properties (Domigan et al., 1988). Perhaps these novel properties are due to the presence of multiple “types” of membrane within the SBM as suggested above; alternately, perhaps the membrane starting material in these studies was not pure SBM. Clearly, additional careful biochemistry is needed here. Other enzymes reported in the SBM from soybean nodules include a pyrophosphatase (Bassarab and Werner, 1989) and a protein kinase (Bassarab and Werner, 1987). Unfortunately, neither the ATPase nor these two enzymes seems like a good marker for the SBM because none of the enzymes can be easily distinguished from other similar enzymes in plant cells. Bassarab et al. (1989) have also recently reported the fatty acid composition of the SBM but, although the composition differs for that of the endoplastic reticulum and Golgi, it is not sufficiently unique to provide a convenient index of membrane purity. Monoclonal antibodies reacting with the SBM of pea nodules have been prepared, but the antibodies are not specific, apparently because the antigen is a glycoprotein component common to several different membrane types (Brewin et al., 1985; Bradley et al., 1988). The role of the SBM in regulating transport of carbon and nitrogen is discussed in later sections. In addition, the SBM probably acts to prevent movement of proteins into or out of the SBS. For example, a variety of proteases have been identified in nodules (Malik et al., 1981; Pfeiffer et a f . , 1983) and there is evidence that certain nodule proteases are active in hydrolysing bacteroid cell wall components (Pladys et al., 1986; Pladys and Rigaud, 1988). Thus, one function of the SBM may be to protect bacteroids

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from attack by host proteases. However, Rhizobium mutants which form nodules in which the SBM is absent have been reported (Shantharam et al., 1988), and some bacterial mutants which do not fix nitrogen in the nodule (Fix-) are lysed soon after infection leaving apparently intact membrane vesicles (Werner et al., 1984). 3. The Symbiosome Space The symbiosomespaceis analysed by rupture of purified symbiosomes,removal of bacteroids by low-speed centrifugation, and removal of the SBM by highspeed centrifugation. Analysis of this space fraction by gel electrophoresis reveals numerous proteins, some of which appear to be specificallylocalized in the SBS (e.g. Katinakis et al., 1988a,b).Most of these SBS proteinsremain to be identified, although some identities have been proposed. An example is a protease inhibitor (Garbers et d.,1988);it would be interesting if such an inhibitor is released by bacteroids as a protective measure. The localization of a form of a-mannosidasein the SBS has also been proposed (Melloretal., 1984;Kinnback et ul., 1987), but this has recently been questioned (Verma and Fortin, 1989; Streeter and Morre, 1990). An elaborate model of trehalose metabolism has been suggested based on the localization of trehalase in the SBS (Mellor, 1988), but this has also not been confirmed. Based on histochemical analysisof nodule slices, the presence of polysaccharide in the SBS has also been proposed (Andreeva et al., 1989). These reports on localization of proteins in the SBM and SBS raise the important issue of purity of the fractions studied. Contamination of symbiosomes with mitochondria has been noted (Herrada et al., 1989), contamination of host cytoplasm with SBS proteins has been noted (Bisseling et a f . , 1983), and contamination of SBS and SBM fractions with bacteroid periplasmic proteins has been noted (Katinakis et al., 1988a; Streeter and MorrC, 1990). Most of the papers published on symbiosome isolation demonstrate concern for purity of the fractions studied, but there are still many differences in what is being reported, suggesting that purity of fractions may vary between laboratories. For progress to continue in this area of research, convenient and reliable markers for the SBM and SBS are vitally needed. Until these are available, differences between reports will continue to make this literature seem confusing and inconclusive.

C. BACTEROIDS

Bacteroids can be isolated with a high degree of purity using density gradient systems which rupture the symbiosomes and result in the loss of the SBM and SBS. Percoll gradients offer the most advantages as first shown by Reibach et a f .(1981) and later modified by others (Day etal., 1986, McRae et al., 1989a). The metabolic and transport capabilities of isolated bacteroids

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have been studied in considerable detail. However, the spatial organization of these activities has been little studied. Rhizobia belong to a class of bacteria known as Gram-negative and the organization of the cell walls of these bacteria deserves brief comment. The outer membrane is very complex, containing structural and sensory components and, more pertinent here, open pores. These pores have exclusion limits of 600-800 Da, may exhibit some selectivity, but may be thought of as largely open passageways for metabolites (Nikaido and Vaara, 1985; Benz and Bauer, 1988). The inner membrane is a typical phospholipidbased membrane containing the proteins which control transport (Higgins et af., 1990). The space between the two membranes is referred to as the periplasmic space; its major feature is a rigid peptidoglycan layer which gives the bacterium its shape and which is anchored to the outer and inner membranes by lipoprotein bridges (Nikaido and Vaara, 1985). Within the periplasmic space are localized many proteins which bind specific substrates and transfer them to transport proteins in the inner membrane (Ames, 1988; Quiocho, 1990). Thus the cell wall of the Gram-negative bacterium is quite different from that found in plants, and especially noteworthy is the periplasmic space which serves the cell as an environment-sensing and metabolite-sorting area. The bacteroid cell wall has received almost no attention, in spite of its obvious potential importance. Systems for separation of inner and outer membranes and a few marker enzymes and distinguishing characteristics of the two membranes have been published (Robertson et af.,1978b;deMaagd and Lugtenberg, 1986). Also, it has been found that the lipid composition of the bacteroid inner membrane is altered (Miller and Tremblay, 1983), and the outer membrane contains less lipopolysaccharide (van Brussel er al., 1977) relative to cultured rhizobia. Very recent immunological characterization of the outer membrane has revealed bacterial/bacteroid differences in protein composition which could prove to be interesting and important (deMaagd et al., 1989a,b). Most pertinent here would be substrate recognition and transport activities localized in the periplasmic space, but there is virtually no evidence on these questions. Part of the reason for this is the difficulty in isolation of specific periplasmic proteins using conventional techniques (Glenn and Dilworth, 1979; Streeter, 1989a). In lieu of isolation proteins, it has recently been demonstrated that periplasmic enzymes can be assayed in intact bacteroids (Streeter, 1989a) and, using these techniques, the presence of aspartate aminotransferase activity has been demonstrated in the periplasmic space of bacteroids (Streeter and Salminen, 1990). Also, genetic studies of the dicarboxylic acid transport genes in rhizobia indicate than one gene product-presumably the transport protein-is probably localized in the cytoplasmic membrane (Ronson et al., 1987). The genetic evidence on the systems for dicarboxylate transport far exceeds the biochemical evidence on

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isolated proteins (there is none), and analysis of the bacteroid periplasmic space remains a fertile area for future research.

111. CARBON PROCESSING A. HOST FUNCTIONS

1. Conversion of Sugars to Organic Acids

Studies on the quantities of metabolites in nodules provide a useful background against which to consider transport and metabolism. Nodules contain very high concentrations of carbohydrates relative to other plant tissues and the major compound is generally sucrose (Antoniw and Sprent, 1978; Streeter, 1980, 1986; Kouchi and Yoneyama, 1986). Cyclitols (six-carbon ring structures related to myo-inositol) are also generally major constituents, especially in bacteroids, but their role in nodules is unknown (Phillips el al., 1984; Streeter, 1980, 1987). Trehalose, a glucose-glucose disaccharide common to fungi, is also a major carbohydrate in all legume nodules examined, and this sugar is known to be synthesized in the bacteroids (Salminen and Streeter, 1986). Perhaps the most important feature of carbohydrate composition is the rapid increase in concentration of all compounds just at the onset of nitrogen fixation; concentrations (except for fructose) become much higher than in the adjacent roots, but what initiates this rapid change in sink activitity is not known (Streeter, 1980). Another interesting feature occurs in the organic acids where, in addition to malate, malonate is a major constituent of some nodules (Antoniw and Sprent, 1978; Stumpf and Burris, 1981b; Kouchi and Yoneyama, 1986; Streeter, 1987), however, clover nodules appear to be an exception (Davis and Nordin, 1983). The occurrence of malonate in nodules is noteworthy because malonate is an inhibitor of succinate dehydrogenase. The concentration of malonate is much higher in the cytosol than in bacteroids of soybean nodules (Kouchi and Yoneyama, 1986; Streeter, 1987). Also, low concentration of malonate in the SBS has been reported, but the extent to which metabolite concentrations changed during the several hours required for symbiosome isolation was not estimated (Humbeck and Werner, 1987). The pathway for the biosynthesis of malonate has been reported in soybean nodules (Stumpf and Burris, 1981b). Amino acid composition of nodules has also been studied, and one of the most striking features is the high glutamate concentrations in bacteroids, a point returned to later. The “flow” of carbon through the various pools has been studied by providing labelled COzto shoots and following the time-dependent distribution of label in nodule metabolites. Sucrose is the most heavily labelled compound in the nodules after short time delays, and is clearly the primary source of reducing equivalents supplied by the plant (Reibach and Streeter,

142

JOHN G. STREETER

1983; Kouchi and Nakaji, 1985; Gordon et al., 1985). Some labelled sucrose can be found in bacteroids and the labelling of trehalose in bacteroids can be observed, but the major fate of incoming sucrose is conversion to organic acids (Kouchi, 1982; Reibach and Streeter, 1983). An important feature of these studies is the failure of the cyclitol and malonate pools to become significantly labelled, indicating that these “atypical” metabolites are probably not involved in the carbon nutrition of bacteroids (Reibach and Streeter, 1983; Kouchi and Nakaji, 1985). Two enzymes capable of sucrose hydrolysis are present with high activity in legume nodules, namely invertase (sucrose + fructose glucose) and sucrose synthase (sucrose + UDP + fructose + UDP-glucose). Both enzymes have been purified from soybean nodules (Morel1 and Copeland, 1984, 1985). Three independent reports indicate that invertase has much higher activity in the cortex than in the infected region (Streeter, 1982; Kouchi et al., 1988; Copeland e f al., 1989b); however, one report indicates higher concentration of sucrose synthase in the cortex (Copeland et al., 1989b), while the other indicates higher concentration of the enzyme in the infected region (Kouchi et al., 1988). Given the fact that the assembly of subunits of sucrose synthase is regulated by heme (Thummler and Verma, 1987), a location in the infected cells may be favoured. Sucrose synthase is probably the enzyme responsible for sucrose cleavage because its activity in nodules is well correlated with nitrogenase activity, whereas the activity of invertase is not (Anthon and Emerich, 1990); therefore, establishment of its exact localization deserves additional effort. The fact that there is active starch synthesis and turnover in nodules (Kouchi et al., 1985; Henson et al., 1986b) also may favour the involvement of sucrose synthase because its product, UDP-glucose might serve as a starch precursor. However, the enzyme required for interconversion of UDP-glucose and glucose-l-P, UDPG pyrophosphorylase, has very high activity in nodules (Salminen and Streeter, 1986; Copeland etal., 1989b). This enzyme has also recently been purified (Vella and Copeland, 1990). High activity of glycolytic enzymes in the host cytoplasm of nodules (“cytosol” fraction) is consistent with the rapid labelling of organic acids from sucrose (Reibach and Streeter, 1983; Smith, 1985). More detailed studies have recently indicated that the nodule cortex plays a significant role in the conversion of sugars to organic acids (Kouchi et al., 1988; Copeland et al., 1989b). These and other studies (Anthon and Emerich, 1990) also indicate the presence of enzymes of the pentose phosphate pathway, but is difficult to assess the importance of the pathway based on activity of enzymes. Some experiments with radioactive substrates indicate high pentose phosphate activity (Tajima and Yamamoto, 1984), whereas others do not (Laing et al., 1979). More important is the very high activity of phosphoenolpyruvate carboxylase ((PEPC);

+

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

phosphoenolpyruvate (PEP) + HC03- + oxalacetate inorganic phosphate (Pi)

143

+

in nodule cytosol. Although the dark fixation o f C 0 2 by nodulated roots had been studied earlier. the work of Lawrie and Wheeler (1975) involving “C02. demonstrated the magnitude of this activity for the first time. It is interesting that very high activity of carbonic anhydrase (COz H2 0 t-, HCOZ- + H’) in nodules relative to roots had been reported previously (Atkins. 1974). but the significance of this observation was not immedia.tely recognized. Labelling studies with intact nodules show rapid and substantial labelling of four-carbon organic acids, especially malate, and this activity is closely associated with nitrogen fixation activity of the nodules (Christeller et a / . , 1977; Coker and Schubert. 1981; Vance et a / . , 1983). In amide exporting nodules. aspartate and asparagine also become heavily labelled (Vance et rd.. lY83; Rosendahl et al., 1990), and it has been estimated that COz fixation by nodules may provide as much as 25% of the carbon consumed in synthesis of nitrogenous export products (Vance et af., 1983). In ureide exporting nodules, it has been estimated that about 14% of respired COz is recaptured (King e t a / . , 1986), and the importance of PEPC appears linked to malate synthesis and the maintenance of charge balance (Israel and Jackson. 1982; Gadal, 1983; Vance etal.. 1985). It is interesting that much of the [ “Clmalate synthesized as a result of I4CO2 fixation in soybean nodules is exported, and this area deserves further study. It should be noted that, contrary to the assumption of some authors, dark fixation of C02 does not result in increased availability of “energy” to nodules; it is actuaily an energy-consuming process and the key energy source remains sucrose import. The PEPC enzyme was first purified from soybean nodules (Peterson and Evans, 1979) and has since been purified from a number of legume nodules, both amide and ureide exporters (Vance and Stade, 1984; Deroche and Carrayol, 1989; Marczewski, 1989). These reports all indicate that the enzyme is not a highly regulated enzyme; thus, activity in the nodule presumably depends on PEP concentration. The concentration of COz has been estimated to be approximately 1% in the infected tissue of soybean nodules (Hunt et uf., 1988) and, at this high concentration, would not limit t h e activity of PEPC. Enzyme activity is much higher in amide-producing nodules, presumably because of the greater synthesis of the four-carbon amino acids used for nitrogen export (Sawhney et af., 1987). Also, enzyme activity is much higher in nodule cytosol than in roots (DeVries et al., 1980; Deroche et al., 1983) and is present in both infected and uninfected cells of the infected region of nodules (Vidal et u f . , 1986; Suganuma et a f . , 1987). Somewhat curiously, PEPC activity per gram of nodule can be influenced by the rhizobial genotype (Vance et a/., 1987); this implies that most of the enzyme may actually be present in infected cells. Analysis of PEPC distribu-

+

144

JOHN G. STREETER HEXOSE

O M

r

IMP or XMP

/PLASTIC

A IMP

NH; LU LN%NH;

E S

-

I '

FUM

n E

S

ASP

I

A

ASN

Fig. 3. Some interactions of plastids, bacteroids, and host cytoplasm in the metabolism of carbon and nitrogen in legume nodules. Metabolites: PEP, phosphoenolpyruvate; OAA. oxalacetate; MAL. malate; GLU. glutamate; 2 0 G . 2-oxoglutarate; GLN, glutamine; ASP, aspartate; FUM, fumarate; PYR, pyruvate; CIT, citrate; ASN, asparagine; AcCoA, acetyl coenzyme A; XMP, xanthine monophosphate; IMP, inosine monophosphate. Major enzymes include: PEPC, PEP carboxylase; PK, pyruvate kinase; MDH. malate dehydrogenase; ME, malic enzyme; GS. glutamine synthetase; GOGAT, glutamate synthase; AAT, aspartate aminotransferase; AS, asparagine synthetase; Nzase, nitrogenase. Note that complete reaction components and possible control of transport at the SBM are not shown. Carbon transferred to plastids and mitochondria may be in the form of malate, and NADH is not the actual electron donor to nitrogenase (see text). In addition, the recycling of FUM to OAA in plastids is only probable and has not been demonstrated experimentally.

tion using nodule fractions prepared with hydrolytic enzymes may be unreliable because large amounts of enzyme activity are lost during these procedures (Kouchi et a f . , 1988). The key position of PEPC in carbon metabolism in host cytoplasm is illustrated in Fig. 3. Conversion of oxalacetate to malate is accomplished by malate dehydrogenase, which is present at extremely high levels of activity in nodule cytosol and is correlated with nitrogenase activity (Nautiyal and Modi, 1987; Appels and Haaker, 1988). Detailed kinetic analysis of the enzyme from Pisum sativum nodules indicates that the nodule enzyme is much more efficient than the root enzyme in the conversion of oxalacetate to malate (Appels and Haaker, 1988). Respiratory activity of mitochondria isolated from legume nodules is high relative to roots and activity is stimulated by the addition of leghaemoglobin to reaction mixtures (Suganuma and Yamamoto, 1987; Suganuma et a f . ,

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

145

1987). Nodule mitochondria have similar respiratory control, much greater sensitivity to KCN, and much lower malic enzyme activity relative to mitochondria isolated from hypocotyls (Rawsthorne and LaRue, 1986a) or roots (Day et al., 1986; Day and Mannix, 1988). More importantly, nodule mitochondria appear to be “adapted” to a low 0 2 environment but are still not efficient in ATP production under microaerobic conditions (Rawsthorne and LaRue, 1986b). Because of these properties plus the potential for inhibition of succinate dehydrogenase by the high cytoplasmic concentration of malonate (Rawsthorne and LaRue, 1986b), I suggest that the principal function of nodule mitochondria may be the production of 2-oxoglutarate which is required in the initial steps of NH3 assimilation (see later). Nodule cytosol contains high activity of a NADP dependent isocitrate dehydrogenase, but a source of substrate for this enzyme has not been established (Henson ef al., 1986a; Nautiyal and Modi, 1987). The acetyl CoA oxalacetate -+ 2-oxoglutarate C 0 2 portion of the TCA cycle cannot be fully duplicated in the cytoplasm, and the operation of this portion of the mitochondrial metabolic system does not necessarily require the operation of the four-carbon portion of the cycle. Some ATP production outside of bacteroids would be required in order to maintain general metabolism in the infected cells, but the low O2plus high malonate in these cells is surely not conducive to the operation of the entire TCA cycle, and 2-oxoglutarate (possibly also succinate) may be exported by mitochondria in this unique environment. In summary, sucrose is released in large quantities from the phloem of vascular bundles imbedded in the inner cortex and is presumably transported symplastically through the inner cortex towards the infected cells. Mathematical models of this process indicate that diffusion will accommodate the needed fluxes (Sinclair and Goudriaan, 1981). Along the way sucrose is hydrolysed by sucrose synthase; this process as well as the subsequent conversion of hexose to triose may be accomplished in the inner cortex o r in the infected cells. Presumably it occurs in both places, but whether or not any sugar actually reaches the inner layers of infected cells is difficult to predict or to test experimentally. An important branch point occurs at PEP, most of the PEP being carboxylated to form oxalacetate and malate and a smaller portion being converted to pyruvate for consumption by mitochondria (Peterson and Evans, 1978) (Fig. 3 ) . That pyruvate must be supplied to mitochondria is consistent with the very low to nil activity of malic enzyme in these organelles (Rawsthorne and LaRue, 1986b; Day and Mannix, 1988). Mitochondria probably play a much more restricted role than they do in most plant cells because of the “non-oxidative” environment in which they must operate. The carbon nutrition needs of the bacteroids can be met without mitochondrial metabolism by coupling glycolysis to massive generation of four-carbon acids via PEPC. The principal function of mitochondria may be simply to generate a five-carbon acid.

+

+

146

JOHN G . STREETER

2. Transport to Bacteroids There are several places between the host cytoplasm and bacteroid cytoplasm where control of transport may be exercised. Most of the effort has been placed on describing metdbolite uptake systems of bacteroids, but in recent years analysis of the symbiosome membrane (SBM) as a selective barrier for transport has begun. Recent studies by Australian workers have emphasized comparisons between the uptake of metabolites by bacteroids and that by symbiosomes (Day et al., 1989). These studies show that sugars, oxoacids and glutamate were not readily transported into symbiosomes, whereas four-carbon acids (malate and succinate) were transported (Price et af., 1987; Udvardi et ul., 198%). Kinetic constants for the uptake of the dicarboxylic acids have also been published (Udvardi et af., 1988a). In contrast, symbiosomes from a Phaseolus vufgaris were found to absorb glucose at about 50% of the rate of succinate uptake (glutamate uptake was slow), and it was concluded that glucose could be a substrate for bacteroids in nodules (Herrada et al., 1989). As already pointed out, purity and intactness of the symbiosomes is a crucial issue here, and final answers on the uptake properties of symbiosomes will have to await the availability of convenient markers for the SBM and SBS. In addition, in these studies of uptake, it must be demonstrated that symbiosomes remain intact during the incubation with and removal of radioactive substrates. The selectivity of the SBM is an important issue because it is logical that the membrane should be an important mechanism for the host to control the activity of the microsymbiont. There is a little evidence to support this: Sen and Weaver (1980) found that nodules formed on two different legumes (Arachis hypogaea and Vigna urzguiculata)by the same strain of Rhizobium had very different nitrogen fixing activities, Arachis nodules having superior activity. One obvious difference between the two types of nodules is the number of bacteroids per symbiosome (one in Arachis, multiple in Vigna), suggesting that the SBM surface area per bacteroid may limit the availability of substrate. More detailed comparative analysis of systems like this should be useful.

B. BACTEROID FUNCTIONS

1. Mechanisms of Carbon Uptake Even though labelling studies indicate the transfer of some sugars to bacteroids (Reibach and Streeter, 1983; Romanov ef af., 1985; Kouchi and Yoneyama, 1986), it is important to note that isolated bacteroids do not absorb sugars efficiently. That is, apparent K,, values are high and V,,, values are low relative to those for other classes of compounds. This was first established for Rhizobium leguminosarum biovar viciae (Hudman and Glenn, 1980; Glenn and Dilworth, 1981; deVries et af., 1982), and was later

TABLE I11 Coniparisori of carhori uptake mutants of' rliizohia

Rhizobirim species (biovar) leguminosarum (viciae)

meliloti

trifolii leguminosaruni (phaseoli) japonicuni

Bacterial phenotype"

Phenotype in nodule'

Reference

Succinate uptake--'

Nod+ Fix-

Glenn and Brewin (1981)

Dct Dct DCpd(d) Dct- and Dctrcd('I) Dct DCt--' Dct Dct-' Dct -

Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+ Nod+

Arwas et al. (1985) Finan ef al. (1983) Finan et al. (1983) Engelke et al. (1987) Bolton et al. (1986) Hornez ef al. (1989) Watson er al. (1988) Ronson et al. (1981) LaFontaine et al. (1989)

Reduced succinate uptake'

Nod+ Fix""

FixFixFixred ("1 Fix- and Fixrcd(") FixFixFixFixFix(d)

Humbeck and Werner (1989)

"Dct- , dicarboxylate uptake negative; incapable of absorbing rnalate, succinate, etc. Mutants were generated with transposons unless otherwise indicated. 'Nod+, nodules are formed; Fix-, nodules do not fix nitrogen. 'Apparently a spontaneous Dct- mutant; only succinate uptake was studied. dRed, Reduced; i.e. reduced uptake of dicarboxylic acids and reduced fixation activity. 'Mutant produced with nitrosoguanidine. fExact genetic lesion unknown.

148

JOHN G . STREETER

also reported for other rhizobia (Saroso et al., 1984; Reibach and Streeter, 1984; Salminen and Streeter, 1987a). It is also important to note that, although cultured R. legitminosarum bv. viciae will take up sugars efficiently, bacteroids will not, indicating that fundamental changes in carbon transport and metabolism occur in response to the special environment in nodules (Hudman and Glenn, 1980; deVries et a [ . , 1982; SanFrancisco and Jacobson, 1986). In contrast to the situation for sugars, all rhizobia studied thus far absorb dicarboxylic acids rapidly with an active mechanism (Glenn et al., 1980; Reibach and Streeter, 1984; McRae et al., 1989b). Typically, apparent K , values are 100 FM or less and uptake rates are 30-50 times the uptake rates for sugars; also it has been demonstrated directly that uptake is against a concentration gradient (Reibach and Streeter, 1984; Salminen and Streeter, 1987a). Uptake of amino acids has been analysed in some studies and uptake rates are usually intermediate between the rates for sugars and dicarboxylic acids, but there appears to be major differences between species or rhizobia (Reibach and Streeter, 1984; McRae et a / . , 1989b). Numerous bacterial mutants with defects in dicarboxylate uptake have been obtained and most of these reports are summarized in Table 111. Most of the work has been done with the fast-growing species, but the overall results are remarkably consistent in showing that bacteroids incapable of dicarboxylate uptake do not fix nitrogen. This, in itself, does not prove that dicarboxylic acids are the sole source of reducing equivalents for bacteroids, but it does indicate that dicarboxylic acids play some essential role in the symbiotic system. An essential role in fixation is supported by recent evidence that Bradyrhizobium japonicum mutants capable of increased succinate uptake are also capable of increased nitrogen fixation in culture (Birkenhead et al., 1988; O’Gara et al., 1988); the phenotype of bacteroids has not yet been reported. Mutants with reduced dicarboxylate uptake and reduced nitrogen fixation were somewhat puzzling until the details of the dct genetic system were worked out. It is now known that there are three genes, only one of which is absolutely required for the transport of dicarboxylic acids, and this gene codes for the transport protein which is located in the bacteroid cytoplasmic (inner) membrane (Ronson et al., 1984, 1987). The other two genes are regulatory genes which are responsible for sensing the “metabolic environment” and activating the structural gene (Ronson et a[., 1987; Yarosh et al., 1989, Engelke et al., 1989; Birkenhead et al., 1990; Watson, 1990). Mutations in the regulatory genes have reduced dicarboxylate uptake and reduced nitrogen fixation, indicating that the regulatory genes are not absolutely required for symbiotic function. These mutants may be useful in understanding nodule function because they indicate that dicarboxylic acids are probably the major source of reduced carbon supporting nitrogen fixation. That is, if bacteroids could rely on a variety of metabolites for reducing equivalents, reduced dicarboxylate uptake should have little

TRANSPORT A N D METABOLISM OF CARBON AND NITROGEN

149

impact on nitrogen fixation; but the experimental evidence indicates that when dicarboxylate uptake is partially impaired, nitrogen fixation is also partially impaired. 2. Curhori Metabolism Distribution of enzymes of carbohydrate metabolism in bacteroids is consistent with a minor role for sugars in bacteroid function. For example, bacteroids appear to lack invertase, as first demonstrated by Robertson and Taylor (1973) and later confirmed by numerous other groups; however, bacteroids may have low sucrose synthase activity (Salminen and Streeter, 1987a). Low levels of glycolytic enzymes in bacteroids relative to host cytoplasm have also been demonstrated by numerous groups (Reibach and Streeter, 1983; Saroso etal., 1986; Salminen and Streeter, 1987; Copeland et ul., 1989b). Furthermore, activity of the Entner-Doudoroff pathway, a common mechanism for conversion of hexose to carboxylic acids in Gramnegative bacteria, is very low in bacteroids, although the pathway may be active in cultured rhizobia (Saroso et al., 1986; Salminen and Streeter, 1987a). Bacteroids also convert I4C labelled sugars to 14C02slowly relative to respiration of dicarboxylic acids (Salminen and Streeter, 1987a); this evidence is especially useful because it indicates overall activity of pathways in the cells. In contrast to the sluggish metabolism of sugars, the metabolism of dicarboxylic acids is rapid. Labelled acetate and pyruvate are rapidly incorporated into TCA cycle intermediates (Stovall and Cole, 1978), and there is substantial enzymatic capacity for the synthesis of acetyl CoA in bacteroids (Preston ef ul., 1989). Levels of TCA cycle enzymes are high in bacteroids (McKay et al.. 1989), especially malate dehydrogenase (Waters etal., 1985); also, the level of TCA enzymes is correlated with nitrogen fixing activity of nodules (Romanov et al., 1980b). The importance of the TCA cycle is also indicated by the finding that utilization of TCA intermediates by cultured bacteria is related to symbiotic effectiveness (Antoun et al., 1984). The accumulation in nodules of malonate, an inhibitor of succinate dehydrogenase, has been noted earlier, and it is interesting that succinate oxidation by bacteroids is largely insensitive to malonate (Thorne and Burris, 1940; Werner et al., 1982); mechanisms permitting this have not been reported. Again, the overall capacity for metabolism of dicarboxylic acids is illustrated by the rapid conversion of labelled compounds to COz relative to the slower respiration of amino acids and sugars by purified bacteroids incubated under microaerobic conditions (Salminen and Streeter, 1987b). Assuming that the four-carbon dicarboxylic acids are the principal (or only) source of reduced carbon for bacteroids and that these substrates are metabolized via the TCA cycle, then an enzyme required for the operation of the cycle would be malic enzyme (malate NAD(P) pyruvate + CO2 + NAD(P)H). Very recent studies indicate that this enzyme is present in

+

-

TABLE IV Comparison of carbon metabolism mutants of rhizobia Rhizobium species (biovar)

Bacterial phenotype

Lacks glucosedP DHb Lacks 2-oxoglutarate DHb Lacks succinate DHb Lacks phosphoglucose isomerase Lacks phosphoglucose isomerase leguminosarum Lacks fructokinase or glucokinase or pyruvate DHb (viciae) Lacks ribokinase or arabinonate dehydratase Lacks PEP carboxykinase leguminosarum No growth on glucose, fructose, or arabinose' (phaseoli) trifolii Lacks glucokinase, fructose uptake, pyruvate carboxylase

meliloti

"All mutants formed nodules; Fix+, active nitrogen fixation. bDH, dehydrogenase. 'Nitrosoguanidine mutants; exact lesion not defined.

Phenotype in nodule"

+

Reference

Fix Fix Fix Delayed fixation Fix+ All Fix+

Cervenansky and Arias (1984) Duncan and Fraenkel(l979) Gardiol et al. (1987) Arias et al. (1979) Guezzar et al. (1988) Glenn el al. (1984)

Both Fix+ Fix Limited fixation

Dilworth et al. (1986) Arwas et al. (1985) LaFontaine et al. (1989)

All Fix+

Ronson and Primrose (1979)

+

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

151

bacteroids with high relative activity (McKay ef af., 1988; Kimura and Tajima, 1989: Copeland et a f . , 1989). There are two forms of the enzyme present, one requiring NAD and the other requiring NADP. The NADPdependent enzyme may be the physiologically important one because it has a much higher affinity for malate; interestingly, this form of the enzyme is stimulated by ammonium (Copeland ef a f . , 1989a). A mutant lacking this enzyme will be very interesting and would be predicted to have impaired symbiotic effectiveness. Rhizobium mutants lacking various enzymes of carbon metabolism have been reported and most of these are summarized in Table IV. Although there are a few exceptions, this summary indicates that mutants lacking the capability of carbohydrate metabolism form nodules which are capable of nitrogen fixation, whereas mutants deficient in some step of the TCA cycle form nodules which do not fix nitrogen. Additional mutations for TCA cycle enzymes would be useful and, as already quoted, a malic enzyme mutant will be quite interesting. To this point, it has been possible to provide a reasonably coherent picture of nodule carbon metabolism; namely, conversion of sugars to dicarboxylic acids in the host cytoplasm and efficient, specific uptake and metabolism of these acids by bacteroids. However, another approach to this question is to test various metabolites for their ability to support respiration and nitrogenase activity by isolated bacteroids, and these studies are not completely consistent with the foregoing picture. Early studies indicated that organic acids are superior to sugars in supporting respiration of isolated bacteroids under aerobic conditions (Thorne and Burris, 1940; Tuzimura and Meguro, 1960). However, other studies indicate that, in bacteroids isolated from soybean or from Phaseofus vufgaris nodules, nitrogenase is stimulated by malate or succinate only at relatively high O2 concentrations (Trinchant et al., 1981): in fact, dicarboxylic acid may even be inhibitory to nitrogenase activity (Bergersen and Turner, 1990). Surprisingly, glucose, when supplied at very low 0 2 concentrations (a3 kPa in the gas phase) gives the highest nitrogenase activities (Trinchant el a f . , 1981; Gadzhi-zade et al., 1985; Guerin et a f . , 1990). It is important to note that dicarboxylic acids were found to support respiration in spite of their relatively poor support of nitrogenase activity (Trinchant et a f . ,1981; Bergersen and Turner, 1990). It is difficult to reconcile these reports with the extensive evidence indicating the importance of dicarboxylic acids in supplying reducing equivalents to bacteroids. The above studies have employed rather high substrate concentrations ( 1 0 m ~to as much as 5 0 m ~sugar): the sugar versus acid comparisons need to be extended to lower (a1mM) substrate concentrations. Many of these studies have not employed haemoglobin in assay mixtures to control free 0 2 concentration, and this technique has been shown to have a major influence on the utilization of dicarboxylic acids by bacteroids (Tajima et al., 1985, 1986). Also, bacteroids are capable of significant

152

JOHN G . STREETER

nitrogenase activity supported only by endogenous substrates (e.g. Peterson and LaRue, 1981; Miller et al., 1988; Bergersen and Turner, 1990); it would be interesting to test the effect of sugars on isolated bacteroids using a mutant incapable of metabolizing sugars. The stimulation of nitrogenase by sucrose (Trinchant et a[., 1981) is especially curious in view of all of the evidence showing that bacteroids have limited capacity for sucrose hydrolysis. Regardless of how these studies are ultimately interpreted, they have been useful in emphasizing the importance of strict control of O2concentration in the analysis of effects of various metabolites on respiratory and nitrogenase activities of isolated bacteroids (Trinchant et al., 1981; Peterson and LaRue, 1981). Substrates other than sugars and dicarboxylic acids have been proposed as important sources of reduced carbon for bacteroids. For example, Peterson and LaRue (1981) noted that certain aldehydes and alcohols stimulated nitrogenase and respiratory activity of bacteroids above levels supported by endogenous substrates. Although the presence of enzymes for formation and metabolism of these compounds have been demonstrated in bacteroids (Tajima and LaRue, 1982; Peterson and LaRue, 1982), a complete system for using reducing equivalents from this source has not been established. Lactate is present in stem nodules of Sesbania rostruta and could support nitrogenase activity in bacteroids isolated from these nodules (Trinchant and Rigaud, 1987); however, lactate has not been found effective with bacteroids from other sources (Peterson and LaRue, 1981; Trinchant and Rigaud, 1987). Finally a few amino acids have been considered as sources of reducing power for bacteroids from soybean nodules. Glutamate has been reported to stimulate O2 uptake, but was ineffective in supporting nitrogenase (Bergersen and Turner, 1988). Very recently, high levels of enzymes of proline metabolism and the distribution of enzymes in host and bacteroid cytoplasms suggested the possibility of active proline utilization by bacteroids (Kohl et al., 1988); it is not yet possible to evaluate the importance of this potential mechanism. McDermott et al. (1989) have promoted the possibility of a malate/aspartate shuttle mechanism, but this is not attractive because bacteroids lacking aspartate aminotransferase, an enzyme required for a functional shuttle, are Fix+ (Zlotnikov et al., 1984). The thesis in this chapter is that the NAD(P)H and ATP required to operate nitrogenase are not transferred from the host to the bacteroids but are generated in bacteroids from the reduced carbon supplied. For ATP synthesis to proceed. coupling of electron transfer to 0 2 is required, and this specialized subject will not be reviewed in detail; the recent review by O’Brian and Maier (1989) is recommended. The first major study of cytochromes (now over 20 years old) showed that several cytochromes are unique to bacteroids (Appleby, 1969). With the availability of new techniques for the analysis of bacterial genes, new progress is now being made in understanding which of these compounds is essential for bacteroid function

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

153

(Nautiyal et al., 1989; Thony-Meyer et al., 1989). What is most important here is that several bacteroidal cytochromes have very high affinity for O2 and thus appear to be well equipped for ATP synthesis in the microaerobic environment of the nodule (O'Brian and Maier, 1989).

IV. NITROGEN PROCESSING A.

BACTEROID FUNCTIONS

Nitrogenase, the enzyme of nitrogen fixation Nz

+ 8H+ + 8e- + 16ATP-

2NH3

+ H2 + 16ADP + 16Pi

is found only in micro-organisms. In rhizobia, the enzyme is generally expressed only in the symbiotic state, although for some rhizobial species, induction of the enzyme is possible in vitro. The large body of literature on this highly important enzyme is not reviewed here; some aspects of the subject, including nifgenetics in Klebsiellapneumoniae have been reviewed previously (Yates, 1980; Orme-Johnson, 1985). One interesting aspect of this subject in rhizobia is that the mechanism by which electrons are supplied to the enzyme complex is still not understood. Genetic analysis should resolve this question, and recent evidence for Rhizobium meliloti indicates a requirement for ferredoxin (Klipp et al., 1989). A ferredoxin has been purified from Bradyrhizobium juponicum (Carter et al., 1980), but it now appears that ferredoxin may not be required in this organism (Ebling et al., 1988). Thus, mechanisms for electron transfer may vary among rhizobia; a novel dehydrogenase has recently been identified in the cytoplasmic membrane of R. leguminosarum bv viciae bacteroids and this may constitute still another mechanism (Lankhorst et al., 1988). It has been known for some time that the levels of glutamine synthetase glutamate

+ ATP + NH4'+

glutamine + ADP

+ Pi

are very low in bacteroids (Dunn and Klucas, 1973; Brown and Dilworth, 1975; Robertson et al., 1975). This may be due to the low 0 2 concentrations in nodules and possibly also to elevated K+ concentration (Upchurch and Elkan, 1978; Gober and Kashket, 1987). Genetic studies indicate that R. meliloti has three distinct glutamine synthetases and that none of the isoforms is required for nitrogen fixation to occur in bacteroids (Kumar and Rao, 1986; Somerville et al., 1989; deBruijn et al., 1989). The enzyme is also not required for fixation by R. leguminosarum bv. phaseoli bacteroids (Morett et al., 1985). Also, bacteroids appear to have low levels of glutamate synthase 2-oxoglutarate + glutamine

+ NADH+

2glutamate

+ NAD

154

JOHN G . STREETER

and this enzyme is not needed for Fix+ bacteroids (Lewis et al., 1990). In contrast to the above two enzymes, levels of glutamate dehydrogenase

+

NH4+ NADH

+ 2-oxoglutarate + glutamate + NAD

in bacteroids may be substantial (Brown and Dilworth, 1975; Awonaike et af., 1981); however, this enzyme is probably not essential for Fix+ bac-

teroids (Lane et af., 1986). These results, in sum, indicate that bacteroids need not assimilate any of the ammonium that they synthesize from N2 and that amino nitrogen from the host cytoplasm must be available to them. Evidence that bacteroids readily release ammonium (ammonia, depending on the pH) is consistent with the low activity of ammonium assimilating enzymes in the bacteroids (Bergersen, 1965). Cultured rhizobia contain an ammonium permease as indicated by the active transport of [ ''C]methylammonium (Gober and Kashket, 1983; O'Hara et af., 1985), and uptake is stimulated by microaerobic conditions (Gober and Kashket, 1983). However, several attempts to measure permease activity in bacteroids have been unsuccessful, leading to the suggestion that transfer of ammonium to the host is via passive diffusion (O'Hara et af., 1985; Jin et al., 1988; Howitt et af., 1986). Estimates of ammonium concentration in the host cytoplasm may be confounded by the rapid build-up of ammonium following nodule maceration (Streeter, 1989b). When this build-up was avoided, ammonium was essentially undetectable in the cytosol and this is consistent with transfer of ammonium from bacteroids to host by diffusion (Streeter, 1989b). B. HOST FUNCTIONS

1. Initial Steps of NH4+ Assimilation

Very high levels of glutamine synthetase were reported in nodule cytosol in early studies of legume nodules (Dunn and Klucas, 1973; Robertson et al., 1975). In fact, it has been estimated that 2% of the cytosolic protein is glutamine synthetase in soybean nodules (McParland et a [ . , 1976). The use of radioactive "N2 indicated that glutamine is the first major organic product to be formed (Meeks et d . ,1978). These studies have led to acceptance of the idea that ammonium released by bacteroids is initially assimilated into the amino acid glutamine. Glutamine synthetase has been purified and characterized (McParland et af., 1976; McCormick et al., 1982), and the activity of the enzyme in nodules is well correlated with the level of nitrogen fixation during plant development (Stripf and Werner, 1978; SenguptaGopalan and Pitas, 1986). Two forms of glutamine synthetase have been identified in nodules; one form is found in both nodules and other plant parts, whereas the other is nodule specific (Cullimore et al., 1983). The enzyme is a complex molecule composed of eight subunits, and four subunit types are known to occur in plants. Thus, the glutamine synthetase in nodules may have several elec-

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trophoretic forms depending on the types of subunits which are assembled to form the active enzyme (Cai and Wong, 1989; Bennett and Cullimore, 1989). The major subunit type in nodules is the y form, and the gene coding for this subunit is expressed specifically in the infected cells (Bennett and Cullimore, 1989; Forde et al., 1989). Expression of the glutamine synthetase genes is triggered by NH4+ in soybean nodules (Hire1 e t a f . , 1987), but this is not the case in Phaseolus vulgaris nodules (Cock et al., 1990). A curious and unresolved aspect of glutamine synthetase is that inhibition of the form of the enzyme which is not specific to nodules actually increases the amount of nitrogen fixed (Knight and Langston-Unkefer, 1988). Glutamate dehydrogenase is present in the host cytoplasm, but it is probably not a major route for the assimilation of the NH4+ from bacteroids. The enzyme does not have a high affinity for the key substrate (Stone et a f . , 1979), and its activity in nodules does not depend on the availability of ammonium (Atkins et a f . ,1984a; Egli et a/.,1989). In contrast, glutamate synthase activity is correlated with ammonium synthesis by bacteroids (Atkins et al., 1984b; Egli et al., 1989), and even more convincing, in vivo labelling results indicate that NH4+ is incorporated into glutamate via glutamate synthase (Ohyama and Kumazawa, 1980ab; Ta et al., 1986). The enzyme has been purified from several legume nodules (Boland and Benny, 1977; Suzuki et al., 1984; Chen and Cullimore, 1988; Anderson et a f . ,1989). Reminiscent of glutamine synthetase, two isoenzymes of glutamate synthase have been reported in nodules and one form is nodule specific; both forms are localized in plastids (Shelp and Atkins, 1984; Chen and Cullimore, 1989). Activity of the nodule-specific form is correlated with nitrogenase activity (Chen and Cullimore, 1988). Overall, there is a strong consensus that ammonium diffusing through the symbiosome membrane (SBM) is rapidly assimilated into glutamine as a result of the very high activity of cytosolic glutamine synthetase; glutamine would then be transported into plastids for incorporation into glutamate (Fig. 3). This metabolism presumably occurs in the plastids of infected cells inasmuch as Aruchis nodules lack uninfected cells in the infected region (Sen et a f . , 1986). Glutamate formed by the plastid glutamate synthase would then have to be “recycled” to the cytoplasm to support the continued operation of glutamine synthetase. The picture presented in Fig. 3 may have to be modified somewhat based on recent evidence for the presence of some glutamine synthetase in plastids (Vezina and Langlois, 1989; Brangeon et a f . ,1989). More work is needed on isolated plastids including an analysis of NH4+ and glutamine/glutamate transport. 2. Asparagine Synthesis Asparagine, a four-carbon amide amino acid, is the principal export product of a large number of legume nodules (see Section 1I.A). Asparagine synthesis occurs via a glutamine dependent amidation of aspartate

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JOHN G. STREETER

aspartate

+ glutamine + ATP+

asparagine

+ glutamate + A D P + Pi

This conclusion is well supported by in vivo evidence from 14C and ‘’N labelling studies (Fujihara and Yamaguchi, 1980a; Huber and Streeter, 1985; Minamisawa et al., 1986; Ta et al., 1988a). However, recent results for alfalfa nodules indicate that glutamine may not be required in these nodules (Snapp and Vance, 1986; Ta et af., 1988b); in this case, NH4’ would substitute for glutamine in the above reaction. Asparagine synthetase studied in vitvo has a much higher affinity for glutamine (Scott et al., 1976; Huber and Streeter, 1985, Ta et al., 1989), so that glutamine is the favoured substrate based on these studies. If the NH4+concentration in alfalfa nodule cytosol is as low as that in soybean nodule cytosol (Streeter, 1989b), it is difficult to see how aparagine synthetase would be effective in the direct assimilation of ammonium. Although significant asparagine synthesis occurs in ureide producing soybean nodules (Streeter, 1979), it is interesting that virtually no asparagine synthesis occurs in the nodules of another ureide producing legume, namely cowpea (Atkins et al., 1988). The aspartate required for asparagine synthetase is produced by aspartate aminotransferase oxalacetate + glutamate

-

aspartate + 2-oxoglutarate

High levels of this enzyme are found in nodules and particularly in asparagine exporting nodules (Reynolds and Farnden, 1979; Sawhney et al., 1987). Ryan et al. (1972) first purified the enzyme from soybean nodules and noted the presence of two isoenzymes; this has subsequently been confirmed for the enzyme from amide producing nodules (Reynolds et al., 1981; Griffith and Vance, 1989). Although asparagine synthetase is cytoplasmic, aspartate aminotransferase is probably located in plastids (Boland et al., 1982; Shelp and Atkins, 1984). Thus, some of the glutamate formed by glutamate synthase in plastids presumably is recycled to 2-oxoglutarate via aspartate aminotransferase and the resulting aspartate would be exported to the cytoplasm for the final step in the synthesis of the asparagine exported from the nodule (Fig. 3). As long as there is no net consumption of 2oxoglutarate or glutamate in plastids, then there would be no requirement for net import of 2-oxoglutarate from mitochondria. That is, the sum of the two reactions oxalacetate + glutamate + aspartate 2-oxoglutarate and 2-oxoglutarate + glutamine+ glutamate + glutamate is oxalacetate + glutamine -+ aspartate glutamate

+

+

The two reactants would be imported into plastids and the two products exported; glutamate would “pick up” another NH4+ and be returned to the

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

157

0

URIC

ACID

COe

Of! b ,

ALL ANT0 IN

ALLANTOIC ACID

Fig. 4. Structures of allantoin and allantoic acid, major export products of many legume nodules. Also shown is the reaction catalysed by uricase, an enzyme requiring molecular oxygen and restricted to uninfected cells in the nodule.

plastid. The overall result of reactions in plastids and host cytoplasm is the synthesis of asparagine from oxalacetate and ammonium with no net requirement for five-carbon acid. 3. Ureide Synthesis Ureides are purine derivatives which yield urea upon hydrolysis. The two ureides exported from some legume nodules are allantoin and allantoic acid (e.g. Streeter, 1979;Schubert, 1981),and thestructureofthesecompounds is shown in Fig. 4. In early studies, the de nova synthesis of ureides was demonstrated from 14Clabelled precursors using in vitro systems (Atkinsetal., 1982; Boland and Schubert, 1983) and from IsN1 using intact nodules (Fujihara and Y amaguchi, 1980b). Early studies also demonstrated a correlation of some of the enzymes of ureide synthesis with nitrogen fixation activity of nodules (Schubert, 1981; Reynolds et al., 1982). Evidence available to date indicates that the initial steps of the pathway in nodules are the same as those for purine synthesis in animals, and these steps are summarized in Fig. 5 . In Fig. 5 some of the biochemical details which are available elsewhere (Schubert, 1986; Mitchell et a[., 1986; Reynolds et al., 1988) have been deliberately omitted. The details which are essential here are as follows.

(1) Nitrogen inputs come from two glutamines which are converted to glutamate, from glycine, and from aspartate which yields fumarate.

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JOHN G . STREETER

PURINE SYNTHESIS IN

OUT

Ribose-5-P

5 ATP

7 AMP, 4ADP

2 glutamlne --L

2 glutamate

rnethenyl FH4 FH4

c

coo Y aspartate

y

-

fumarate

7

formyl FH,

c FH4

c

glycine

\r

(7’) 0

Adenylic Acid

II

A

N‘

Guanylic Acid

\N

I ri bow P INOSlNlC ACID

-

Fig. 5. Inputs required in the synthesis of purines, specifically the steps from ribose 5-phosphate to inosinic acid. The reactants are not shown in the order of their involvement in the pathway. FH4, tetrahydrofolate.

(2) The enzymes of the pathway are located in plastids (Boland et al., 1982, Boland and Schubert, 1983) which affords convenient recycling of fumarate and glutamate by systems which are also required for asparagine synthesis (Fig. 3). (3) Carbon is supplied by tetrahydrofolate derivates, COZ, plus glycine, which is “consumed”; the details of this portion of the pathway have recently been worked out (Mitchell et al., 1986; Reynolds et al., 1988). (4) Xanthosine monophosphate or inosinic acid (inosine monophosphate, IMP) is the product exported from the plastid (Shelp and Atkins, 1983; Schubert, 1986). Between IMP and allantoin are several additional steps which differ in legume nodules and animals. First, .IMP is converted to xanthine

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159

monophosphate before dephosphorylation, bypassing the formation of hypoxanthine (Boland and Schubert, 1983; Shelp and Atkins, 1983). Secondly, xanthine is converted to uric acid via a NADH-dependent dehydrogenase instead of via an oxidase (Triplett et af., 1980, 1982); this is important because it avoids a step requiring molecular oxygen. This enzyme is localized in the host cytoplasm (Triplett et af., 1980), and is probably concentrated in the uninfected cells (Nguyen et al., 1986). In unpublished studies, we have found low concentrations of xanthine and only trace quantities of uric acid in soybean nodules. The localization of xanthine dehydrogenase in uninfected cells would be consistent with this observation because
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JOHN G . STREETER

duction of IMP in infected cells is indicated by Schubert's (1986) interesting analysis of the distribution of enzymes reported by Shelp et al. (1983). 4. Miscellaneous Subjects The route for transport of fixation products from the infected region to the vascular bundles has not been resolved and is very difficult to analyse experimentally. This question is not a trivial one because large amounts of carbon and nitrogen are transported out of nodules (Streeter, 1979; Kouchi and Higuchi, 1988). The best evidence on the transport route consists of data on plasmodesmatal frequency between various cell types in soybean nodules. Selker (1988) found the average number of plasmodesmata per micrometre of wall to be 0.46 for uninfected-uninfected cell pairs, 0.28 for uninfected-infected cell pairs, and 0.03 for infected-infected cell pairs. These results coupled with the evidence that all infected cells are in contact with at least one uninfected cell (Selker and Newcomb, 1985) indicate that symplastic movement of solutes from infected cells to uninfected cells and thence through the rays of uninfected cells to the vascular bundles is favoured over transport through infected cells. In fact, there is apparently little symplastic exchange between infected cells without transit through uninfected cells, suggesting that significant control of transport among tissues within the nodule may reside in the uninfected cells. Soon after the discovery that ureides are the major transport form of nitrogen in some nodules, it was realized that ureide concentration in the xylem of stems might be a sensitive indicator of fixation activity in nodules (McClure et a/., 1980; Herridge, 1982). Other more recent studies have refined these techniques so that collection and analysis of ureides from field-grown plants is now rather routine (vanBerkum et al., 1985; Herridge, 1984; Yoneyama et a/., 1985; Argillier et al., 1989). Herridge has contributed most to defining conditions under which ureide analysis will provide the most accurate estimate of true nitrogen fixing activity, and the most recent contribution provides comprehensive methodological descriptions along with standard curves for converting nitrogen composition to actual fixation rates (Herridge and Peoples, 1990). Finally, an anomaly which may ultimately tell us something about nitrogen assimilation in nodules is the marked enrichment of "N which occurs in some nodules under natural conditions. The phenomenon was first reported 10 years ago in soybean nodules (Shearer e t a / ., 1980), and studies have expanded to include numerous comparisons of legume species and Rhizobium strains (Yoneyama et al., 1986; Shearer and Kohl, 1989). There are differences among strains and species, but none of these differences seem to correspond to other known criteria for classification of legume nodules (e.g. enrichment occurs in some amide-producing and some ureideproducing nodules). Recently, soybean nodules were subdivided by physical and chemical methods and fractions analysed for "N enrichment (Kohl et

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161

a / ., 1989). Unfortunately, no patterns which would explain the phenomenon

emerged. and the basis for the isotopic fractionation which occurs in some nodules remains unexplained.

V.

RESTRICTIONS IMPOSED BY MICROAEROBIC CONDITIONS A. T H E OXYGEN REGULATION SYSTEM

The early evidence for a very low oxygen concentration in the infected region of nodules (Tjepkema and Yocum, 1974) led to a search for some mechanism for restricting the entry of oxygen into nodules. These efforts have revealed that there is a barrier to the diffusion of gases located in the inner cortex of nodules and, most remarkably, that the barrier is variable (see e.g. Sheehy etal.. 1983; Hunt etal., 1987; King etal., 1988; Davey and Simpson. 1989). A barrier has been demonstrated in all nodule types examined, and the barrier can be manipulated by a variety of techniques, leading to an increase or decrease in oxygen flux and nitrogenase activity (Hunt et al.. 1987; Witty et al., 1987). Mechanisms controlling the operation of the barrier are not known (Dakora and Atkins, 1989). As already noted, the infected cells in nodules contain a haemoprotein call leghaemoglobin; there is a very large body of literature on this subject which cannot be reviewed here (see e.g. O’Brian and Maier, 1989). The function of leghaemoglobin is to provide a large reservoir of oxygen while, at the same time, maintaining a low free 0: concentration in the bacteroidcontaining cells. In this way nitrogenase, which is extremely sensitive to oxygen, can be protected while at the same time making oxygen available for consumption in hacteroids (Bergersen et al., 1973; Wittenberg et al., 1974; Appleby eta/.. 1975). The leghaemoglobin is in the host cytoplasm, outside of the symbiosome membrane (Verma and Bal, 1976). Operating in conjunction with the diffusion barrier, leghaemoglobin can be thought of as a sink and buffer for 0,;if it were not present it would be difficult to ensure an adequate supply of oxygen in the interior of the nodule. Although unrelated to the function of the leghaemoglobin, systems for scavenging oxygen radicals and peroxides are also present in nodules (Dalton et al., 1986; Puppo et a / ., 1987). B.

IMPACT OF LOW O2 ON METABOLISM

Although there is relatively little experimental evidence to review in this section, the topic is given a major heading because an understanding of the impact of low 0, concentration on metabolism will greatly advance our understanding of factors limiting nodule efficiency. The operation of

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JOHN G . STREETER

mitochondria under low O2conditions has already been discussed briefly. A key feature is very low malic enzyme activity relative to mitochondria from other sources (Rawsthorne and LaRue, 1986a; Day and Mannix, 1988); this indicates a requirement for an external pyruvate supply and could represent a point at which mitochondrial activity is controlled. This pyruvate requirement, in turn, emphasizes the pivotal role of phosphoenolpyruvate (PEP) and the relative activity of PEP carboxylase and pyruvate kinase (Fig. 3). The inhibition of pyruvate kinases by NH4+ could be significant (Peterson and Evans, 1978), unless NH4+concentration in the cytosol is so low that the control is inoperative (Streeter ,1989b). Although fumarate reductase could not be detected in nodule mitochondria (Rawsthorne and LaRue, 1986b), other models for the function of these normally aerobic organelles under microaerobic conditions should be considered (Vanlerberghe et al., 1989). Inhibition of “normal” mitochondria1 function may even be an important mechanism to insure the availability of large supplies of four-carbon acids for consumption by bacteroids. Mitochondria may “suffer” from another problem which appears to influence bacteroids, namely high NADHiNAD ratios. Recent studies have provided quantitative evidence for high NADH concentrations in anaerobically isolated bacteroids relative to cultured rhizobia or bacteroids isolated under aerobic conditions (Tajima and Kouzai, 1989; Salminen and Streeter, 1990). This is important because 2-oxoglutarate dehydrogenase is inhibited by high NADH/NAD (Jackson and Dawes, 1976;Heckert etal., 1989). This has been confirmed in Bradyrhizobiumjaponicum bacteroids, the apparent NADH/ NAD ratios being sufficient to inhibit the enzyme about 50% (Salminen and Streeter, 1990). This impediment in the TCA cycle probably explains the substantial labelling of glutamate in bacteroids supplied with 14C labelled substrates (Salminen and Streeter, 1987b) or lSN2(Ohyama and Kumazawa, 1980b). Bypass of the 2-oxoglutarate dehydrogenase step via glutamate, 4aminobutyrate and succinic semialdehyde was not possible because of the lack of glutamate decarboxylase (Salminen and Streeter, 1990). Another negative impact of the microaerobic environment in nodules is the diversion of reduced carbon into products for which there is no apparent role in nodules. One example of this is the disaccharide a,&-trehalosewhich is synthesized by bacteroids, often in significant quantities (Reibach and Streeter, 1983; Phillips et al., 1984; Salminen and Streeter, 1986). What is most curious about this phenomenon is that much of the trehalose synthesized in bacteroids is released to the host cytoplasm where it is hydrolysed by highly active trehalase (Streeter, 1982, 1985). This apparently “futile cycle” may be a consequence of the microaerobic conditions because it has recently been discovered that the accumulation of trehalose in cultured rhizobia can be triggered by low 0 2 in the gas phase (Hoelzle and Streeter, 1990). A better known and more puzzling example is the marked accumulation of poly-P-hydroxybutyric acid in some bacteroids. The accumulation of the

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163

polymer is undoubtedly linked to the microaerobic environment because low 0 2 induces its accumulation in cultured Azotobacter (Jackson and Dawes, 1976; Ward et a f . , 1977), Azospiriffum (Nur et a f . , 1982), and Rhizobium (DeVries et a f . , 1986). Following the initial observations on its dramatic accumulation in soybean nodules (Wong and Evans, 1971), other studies provided more anatomical and chemical documentation (Werner and Morschel, 1978; Karr etal., 1983). Some workers have suggested a vital role for poly-p-hydroxybutyrate in bacteroids, emphasizing its high concentration and accumulation during the onset of nitrogen fixation or during periods of dark stress (Kretovich et a f . , 1977; Romanov et a f . , 1980a; McDermott et a f . , 1989). However, accumulation of the polymer occurs during the onset of nitrogen fixation and the concentration then becomes static (Wong and Evans, 1971; Karr et a f . , 1983, 1984); this pattern is consistent with induction of polymer synthesis by the low 0 2 environment. High correlations between nitrogenase activity and polymer content are obtained only when young nodules are considered (Karr et a f . , 1984). Also poly-p-hydroxybutyrate was used by bacteroids only when nodules were severely stressed for carbohydrate (Wong and Evans, 1971). It is noteworthy that many rhizobial symbionts do not accumulate polyp-hydroxybutyrate. This argues against a “universal” role for the polymer, but also raises the question of why the response to microaerobic conditions is not uniform among rhizobia. Perhaps certain rhizobial species cannot synthesize the polymer; but, to my knowledge, this has not been examined.

VI.

SUMMARY AND SUGGESTIONS FOR FUTURE WORK

Re-reading of the literature cited above leaves one with the impression that excellent progress has been made in the last 10 years in elucidating the inner workings of legume nodules. With respect to nitrogen, we now know many of the details regarding the synthesis of the two principal export productsasparagine and ureides. The progress on ureide synthesis has been more impressive because of the complexity of the pathway. Of course, regulatory aspects are still largely open questions. With regard to carbon metabolism, it is time to conclude that the major sources (if not only significant sources) of reducing equivalents for bacteroids are the four-carbon dicarboxylic acids. The evidence supporting this conclusion is substantial: (1) labelling patterns in the cytosol from incoming labelled sucrose; (2) enzyme activities in the cytosol for the conversion of hexose to four-carbon acids; (3) studies with labelled substrates supplied to purified bacteroids, especially comparative rates of conversion of substrates to C02;

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JOHN G. STREETER

(4) levels of enzyme activities in bacteroids, ( 5 ) Rhizobium mutants lacking various substrate uptake activity; (6) Rhizobium mutants lacking various substrate metabolism activity; (7) relative ability of metabolites to support respiration and/or nitrogenase activity of bacteroids; and (8) kinetic parameters for uptake of various metabolites. Without wishing to suppress further speculation on this subject, it is suggested that workers who promote the importance of other possible metabolites or mechanisms consider the above as a check-list which ultimately needs to be satisfied by experimental evidence. Legume nodules as experimental systems for the study of metabolism and transport are simultaneously exciting and maddening. With two independent (but interdependent) organisms and a myriad of tissue-specific and cellular compartments, the opportunities for future research seem almost endless. The needs which have become most obvious during the assembly of this review are as follows. (1) Factors which may influence the unloading and loading of vascular bundles must be elucidated. It is interesting to note that the vascular system is outside the cortical diffusion barrier and, therefore, presumably operates by “aerobic rules”. Is xylem export really limited by phloem import; if export rate could be improved, would overall nodule efficiency be improved? Why is the nodule such an active sink for carbohydrate, accumulating seemingly luxury concentrations shortly after the onset of nitrogen fixation? (2) The fundamental unit of the symbiotic nodule, the symbiosome, must be understood in greater detail. Much of the previous work may have been done with impure or damaged preparations, and convenient markers for the symbiosome membrane and symbiosome space are desperately needed in order to move this work forward. The extent to which transport of metabolites is regulated by the symbiosome membrane is a question of unsurpassed importance and a question which is still largely open. (3) More work with purified bacteroids is needed with emphasis on possible control of transport by periplasmic proteins and by transport activities in the cytoplasmic membrane, none of which have yet been studied in isolation. Because recent studies have documented the difference between anaerobically and aerobically isolated bacteroids, future work with intact bacteroids should employ anaerobically isolated bacteroids and microaerobic assay conditions in order to simulate the nodule environment. (4) Plastids have emerged in recent years as key organelles, but there has been very little work done with purified plastids. First, a system for purification which avoids the undesirable osmotic effects of high sucrose concentration needs to be devised (Percoll?). Studies on the metabolic and

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

165

transport capabilities of purified plastids using standard enzymological and radioisotope techniques will be interesting and useful. (5) The way in which the O2concentration in nodules is regulated is still a "black box", and the operation of the variable gas diffusion barrier needs to be explained. We need to be increasingly alert to the possibility that nearly every process or phenomenon which we study may be directly or indirectly influenced by the microaerobic environment.

ACKNOWLEDGEMENTS I thank David Emerich, Seppo Salminen, and Janet Sprent for helpful suggestions regarding the organization and content of the manuscript and Robert Whitmoyer for micrographs.

REFERENCES Ames, G . F.-L. (1988). Structure and mechanism of bacterial periplasmic transport systems. Journal of Bioenergetics and Biomemhranes 20, 1-18. Anderson, M. P . , Vance. C. P.. Heichel, G . H. and Miller, S. S. (1989). Purification and characterization of NADH-glutamate synthase from alfalfa root nodules. Plant Physiology. 90, 351-358. Andreeva, I. N.. Kozlova. G . I . , Livanova, G . I . , Zhiznevskaya, G. Ya. and Izmailov, S. F. (1989). Electron microscope study of the peribacteroid space in legume nodules. Fiziologiya Rastenii 36, 55 1-560. Anthon, G . E. and Emerich, D . W. (1990). Developmental regulation of enzymes of sucrose and hexose metabolism in effective and ineffective soybean nodules. Plant Physiology 92, 346-35 1 . Antoniw, L. D . and Sprent. J . I. (1978). Primary metabolites of Phaseolus vulgaris nodules. Phytocheniistry 17, 675-678. Antoun, H., Bordeleau, L. M . and Sauvageau, R. (1984). Utilization of the tricarboxylic acid cycle intermediates and symbiotic effectiveness in Rhizobium meliloti. Plant Soil 77, 29-38. Appels, M. A . and Haaker, H . (1988). Identification of cytoplasmic nodule associated forms of malate dehydrogenase involved in the symbiosis between Rhizobium leguminosarum and Pisum sativum. European Journal of Biochemistry 171, 515-522. Appleby, C. A . (1969). Electron transport systems of Rhizobium japonicurri. 1. Haemoprotein P-450, other co-reactive pigments. cytochromes and oxidases in bacteroids from N2-fixing root nodules. Biochimica et Biophysica Acta 172, 71-87. Appleby, C. A . . Turner, G. L. and Macnicol. P. K. (1975). Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation in Rhizobium. Biochimica et Biophysics Acta 387, 461-474. Argillier, C.. Drevon, J.-J., Zengbe, M . and Salsac, L. (1989). Relation between nitrogenase activity and stem or xylem sap ureide content of soybean plants (Glycine max L. Merr). Plant Science 61, 3 7 4 2 .

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Arias, A., Cervenansky, C., Gardiol, A. and Martinez-drets, G. (1979). Phosphoglucose isomerase mutant of Rhizobium meliloti. Journal of Bacteriology 137, 409-4 14. Arwas, R., McKay, I. A., Rowney, F. R. P., Dilworth, M. J. and Glenn, A. R. (1985). Properties of organic acid utilization mutants of Rhizobium leguminosarum strain 300. Journal of General Microbiology 131,2059-2066. Atkins, C. A. (1974). Occurrence and some properties of carbonic anhydrases from legume root nodules. Phytochemistry 13, 93-98. Atkins, C. A., Ritchie, A., Rowe, P. B., McCairns, E. and Sauer, D. (1982). Denovo purine synthesis in nitrogen-fixing nodules of cowpea (Vigna unguiculata [L.] Walp.) and soybean (Clycine rnax [L.] Merr.) Plant Physiology 70, 55-60. Atkins, C. A., Pate, J. S. and Shelp, B. J. (1984a) Effects of short-term N2 deficiency on N metabolism in legume nodules L. Plant Physiology 76, 705-710. Atkins. C. A., Shelp, B. J . , Storer, P. J. and Pate, J. S. (1984b) Nitrogen nutrition and the development of biochemical functions associated with nitrogen fixation and ammonia assimilation of nodules on cowpea seedlings. Planta 162, 327-333. Atkins, C. A., Storer, P. J. and Pate, J. S. (1988). Pathways of nitrogen assimilation in cowpea nodules studied using "Nz and allopurinol. Plant Physiology 86, 204-207. Awonaike, L. O., Lea, P. J. and Miflin, B. J. (1981). The location of the enzymes of ammonia assimilation in root nodules of Phaseolus vulgaris L. Plant Science Letters 23, 189-195. Bal, A. K., Hameed. S. and Jayaram, S. (1989). Ultrastructural characteristics of the host-symbiont interface in nitrogen-fixing peanut nodules. Protoplasma 150, 19-26. Bassarab, S . and Werner, D. (1987). Ca2+-dependentprotein kinase activity in the peribacteroid membrane from soybean root nodules. Journal of Plant Physiology 130, 233-241. Bassarab, S. and Werner, D. (1989). Mg2+dependent pyrophosphatase, a tonoplast enzyme in the peribacteroid membrane of Glycine max root nodules. Symbiosis 7,81-94. Bassarab, S., Mellor, R. B. and Werner, D. (1986). Evidence for two types of Mg++-ATPase in the peribacteroid membrane from Glycine rnax root nodules. Endocytobiosis and Cell Research 3, 189-196. Bassarab, S., Schenk, S. U . and Werner, D. (1989). Fatty acid composition of the peribacteroid membrane and the ER in nodules of Glycine rnax varies after infection by different strains of the microsymbiont Bradyrhizobium japonicum. Botanica Acta 102, 196-201. Bennett, M. J . and Cullimore, J. V. (1989). Glutamine synthetase isoenzymes of Phaseolus vulgaris L. : subunit composition in developing root nodules and plumules. Planta 179, 433-440. Benz, R. and Bauer, K. (1988). Permeation of hydrophilic molecules through the outer membrane of gram-negative bacteria. Review on bacterial porins. European Journal of Biochemistry 176, 1-19. Bergersen, F. J. (1965). Ammonia - an early stable product of nitrogen fixation by soybean root nodules. Australian Journal of Biological Science 18, 1-9. Bergersen, F. J. and Turner, G. L. (1988). Glutamate as a carbon source for N2-fixing bacteroids prepared from soybean root nodules. Journal of General Microbiology 134, 24413448. Bergersen, F. J. and Turner, G. L. (1990). Bacteroids from soybean root nodules:

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167

Respiration and Nl-fixation in flow-chamber reactions with oxyleghaemoglobin. Proceedings of the Royal Society of London. Series B 238. 295-320. Bergersen, F. J . . Turner, G. L. and Appleby, C . A . (1973). Studies of the physiological role of leghaemoglobin in soybean root nodules. Biochimica et Biophysics Acta 292, 271-282. Birkenhead. K.. Manian, S. S. and O’Gara, F. (1988). Dicarboxylic acid transport in Bradyrhizobium japonicutn: Use of Rhizobium meliloti dct genes to enhance nitrogen fixation. Journal of Bacteriology 170, 184-189. Birkenhead, K., Noornan, B., Reville. W. J . , Boesten, B., Manian, S. S. and O’Gara. F. (1990). Carbon utilization and regulation of nitrogen fixation genes in Rhizobiuni meliloti. Molecular Plant-Microbe Interactions 3, 167-173. Bisseling. T., Been, C.. Klugkist, J.. vanKammen, A. and Nadler, K. (1983). Nodule-specific host proteins in effective and ineffective root nodules of Pisum sativum. EMBO Journal 2,961-966. Blumwald, E., Fortin, M . G., Rea, P. A., Verma, D. P. S. and Poole. R . J. (1985). Presence of host-plasma membrane type H+-ATPase in the membrane envelope enclosing the bacteroids in soybean root nodules. Plant Physiology 78, 665-672. Boland. M. J . and Benny, A . G. (1977). Enzymes of nitrogen metabolism in legume nodules. Purification and properties of NADH-dependent glutamate synthase from lupin nodules. European Journal of Biochemistry 79, 355-362. Boland, M. J. and Schubert, K. R . (1983). Biosynthesis of purines by a proplastid fraction from soybean nodules. Archives of Biochemistry and Biophysics 220. 179- 187. Boland, M. J . , Hanks, J . F . , Reynolds, P. H . S., Blevins, D. G., Tolbert, N. E. and Schubert. K. R. (1982). Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules. Planta 155, 45-5 1. Bolton. E . , Higgisson, B., Harrington, A . and 0 ’ G a r a . F . (1986). Dicarboxylic acid transport in Rhizobium meliloti: isolation of mutants and cloning of dicarboxylic acid transport genes. Archives of Microbiology 144, 142-146. Bradley, D . J.. Butcher, G. W.. Galfre, G., Wood, E. A . and Brewin, N. J. (1986). Physical association between the peribacteroid membrane and lipopolysaccharide from the bacteroid outer membrane in Rhizobium-infected pea root nodule cells. Journal of Cell Science 85, 47-61. Bradley, D. J.. Wood, E. A , , Larkins, A . P., Galfe, G., Butcher, G. W . and Brewin, N. J. (1988). Isolation of monoclonal antibodies reacting with peribacteroid membranes and other components of pea root nodules containing Rhizobium leguniinosarum. Planta 173, 149-160. Brangeon, J., Hirel, B. and Forchioni. A. (1989). Immunogold localization of glutamine synthetase in soybean leaves, roots, and nodules. Protoplasma 151, 88-97. Brewin, N. J., Robertson, J. G., Wood, E. A , , Wells, B., Larkins, A. P., Galfre, G. and Butcher, G . W. (1985). Monoclonal antibodies to antigens in the peribacteroid membrane from Rhizobium-induced root nodules of pea cross-react with plasma membranes and Golgi bodies. EMBO Journal 4, 605-611. Brown, C. M. and Dilworth, M. J. (1975). Ammonia assimilation by Rhizobium cultures and bacteroids. Journal of General Microbiology 86, 3 9 4 8 . Cai, X. and Wong, P. P. (1989). Subunit composition of glutamine synthetase isozymes from root nodules of bean (Phaseolus vulgaris L.). Plant Physiology 91, 105G1062.

168

JOHN G. STREETER

Carter, K. R., Rawlings, J., Orme-Johnson, W. H., Becker, R. R.. and Evans, H. J. (1980). Purification and characterization of a ferridoxin from Rhizobium japonicum bacteroids. Journal of Biological Chemistry 255, 4213-4223. Cervenansky, C. and Arias, A. (1984). Glucose 6-phosphate dehydrogenase deficiency in pleiotropic carbohydrate-negative mutant strains of Rhizobium rneliloti. Journal of Bacteriology 160,. 1027-1030. Chen, F.-L. and Cullimore, J. V. (1988). Two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L. Purification, properties and activity changes during nodule development. Plant Physiology 88, 1411-1417. Chen. F.-L. and Cullimore, J. V. (1989). Location of two isoenzymes of NADHdependent glutamate synthase in root nodules of Phaseolus vulgaris L. Plarzta 179,441447. Christeller, J. T., Laing, W. A. and Sutton, W. D. (1977). Carbon dioxide fixation by lupin root nodules. I . Characterization, association with phosphoenolpyruvate carboxylase, and correlation with nitrogen fixation during nodule development. Plant Physiology 60, 47-50. Cock, J. M., Mould, R. M.. Bennett, M. J. and Cullimore, J. V. (1990). Expression of glutamine synthetase genes in roots and nodules of Phaseolus vulgaris following changes in the ammonium supply and infection with various Rhizobium mutants. Plant Molecular Biology 14, 549-560. Coker. G. T. and Schubert. K. R. (1981). Carbon dioxide fixation in soybean roots and nodules. I. Characterization and comparison with N2 fixation and composition of xylem exudate during early nodule development. Planr Physiology 67, 691-696. Copeland. L., Quinnell, R. G. and Day. D. A . (1989a). Malic enzyme activity in bacteroids from soybean nodules. Journal of General Microbiology 135,20052011. Copeland, L., Vella, J. and Hong, Z . (1989b). Enzymes of carbohydrate metabolism in soybean nodules. Phytochemistry 28, 57-61. Cullimore, J. V.. Lara, M., Lea, P. J . and Miflin. B. J. (1983). Purification and properties of two forms of glutamine synthetase from the plant fraction of Phaseolus root nodules. Planta 157, 245-253. Dakora, F. D. and Atkins, C. A. (1989). Diffusion of oxygen in relation to structure and function in legume root nodules. Australian Journal of Plant Physiology 16, 131-140. Dalton, D. A.. Russell. S . A., Hanus, F. J.. Pascoe. G. A. and Evans, H. J . (1986). Enzymatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules. Proceedings of the National Academy of Science U S A 83, 381 1-3815. Davey, A. G. and Simpson, R. J. (1989). Changes in nitrogenase activity and nodule diffusion resistance of subterranean clover in response to p O z . Journal of Experimental Botany 40, 149-158. Davis. L. C. and Nordin, P. (1983). Sugar and organic acid constituents in white clover. Plant Physiology 72, 1051-1055. Day, D. A. and Mannix. M. (1988). Malate oxidation by soybean nodule mitochondria and the possible consequences for nitrogen fixation. Plant Physiology and Biochemistry 26, 567-573. Day, D. A., Price, G. D. and Gresshoff, P. M (1986). Isolation and oxidative properties of mitochondria and bacteroids from soybean root nodules. Protoplasma 134, 121-129. Day, D. A , , Price, G. D. and Udvardhi, M . K. (1989). Membrane interface of the

TRANSPORT A N D METABOLISM OF CARBON A N D NITROGEN

169

Bradyrhizobium japonicum-Glycine max symbiosis: Peribacteroid units from soybean nodules. Australian Joirrnaf of Plant Physiology 16, 69-84. deBruijn, F. J.. Rossbach, S.. Schneider, M . , Ratet, P., Messmer, S., Szeto, W. W., Ausubel, F. M. and Schell. J. (1989). Rhizobium meliloti 1021 has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation. Journal of Bacteriology 171, 16731682. deFaria, S. M . , Lewis. G . P . , Sprent. J . I . and Sutherland, J. M. (1989). Occurrence of nodulation in the Leguminosae. New Phytologist 111, 607-619. deMaagd. R. A. and Lugtenberg, B. (1986). Fractionation of Rhizobium leguminosarum cells into outer membrane. cytoplasmic membrane, periplasmic and cytoplasmic components. Journal of Bacteriology 167, 1083-1085. deMaagd, R. A., deRijk, R., Mulders, I . H . M. and Lugtenberg, B. J. J. (198%). Immunological characterization of Rhizobiiim leguminosarum outer membrane antigens by use of polyclonal and monoclonal antibodies. Journal of Bacteriology 171. 1136-1142. deMaagd, R. A . , Wientjes, F. B. and Lugtenberg, B. J. J . (1989b). Evidence for divalent cation (Ca'+)-stabilized oligomeric proteins and convalently bound protein-peptidoglycan complexes in the outer membrane of Rhizobium leguminosarum. Journal of Bacteriology 171, 3989-3995. Deroche, M.-E. and Carrayol. E. (1989). Some properties of legume nodule phosphoenolpyruvate carboxylase. Plant Physiology and Biochemistry 27, 379-386. Deroche, M.-E., Carrayol. E. and Jolivet, E. (1983). Phosphoenolpyruvate carboxylase in legume nodules. Physiologie Vtgttale 21, 1075-1081. d e v i s e r , R. and Poorter, H. (1984). Growth and root nodule nitrogenase activity of Pisum sativum as influenced by transpiration. Physiologia Plantarum 61, 637-642. devries, G . E., In't Veld, P. and Kijne, J. W. (1980). Production of organic acids in Pisum sativurn root nodules as a result of oxygen stress. Plant Science Letters 20, 115-123. deVries, G. E., vanBrusse1, A . A. N. and Quispel, A. (1982). Mechanism and regulation of glucose transport in Rhizobium leguntinosarum. Journal of Bacteriology 149, 872-879. devries, W., Stam, H., Duys, J. G.. Ligtenberg. A. J. M., Simons. L. H. and Stouthamer, A. H. (1986). The effect of the dissolved oxygen concentration and anabolic limitations on the behavior of Rhizobium ORS571 in chemostat cultures. Antonie van Leeuwenhoek 52, 85-96. Dilworth, M. J . , Arwas, R., McKay. I . A., Saroso, S. and Glenn, A . R. (1986). Pentose metabolism in Rhizobiurn leguminosarum MNF300 and in cowpea Rhizobium NGR234. Journal of General Microbiology 132,2733-2742. Domigan. N. M., Farnden, K. J. F., Robertson, J . G . and Monk, B. C. (1988). Characterization of the peribacteroid membrane ATPase of lupin root nodules. Archives of Biochemistry and Biophysics 264, 564573. Duncan, M. J. and Fraenkel, D. G . (1979). a-Ketoglutarate dehydrogenase mutant of Rhizobium meliloti. Journal of Bacteriology 37, 415419. Dunn. S. D. and Klucas, R. V. (1973). Studies on possible routes of ammonium assimilation in soybean root nodule bacteroids. Canadian Journal of Microbiology 19, 1493-1499. Ebling, S . , Noti. J. D. and Hennecke, H. (1988). Identification of a new Bradyrhizobium japonicurn gene @A) encoding a ferridoxin like protein. Journal of Bacteriology 170, 1999-2001.

170

JOHN G . STREETER

Egli, M. A., Griffith, S. M., Miller, S. S . , Anderson, M. P. and Vance, C. P. (1989). Nitrogen assimilating enzyme activities and enzyme protein during development and senescence of effective and plant gene-controlled ineffective alfalfa nodules. Plant Physiology 91, 898-904. Engelke, T.. Jagadisn. M. N. and Piihler, A . (1987). Biochemical and genetical analysis of Rhizobium meliloti mutants defective in Cj-dicarboxylate transport. Journol of General Microbiology 133, 3019-3029. Engelke, T., Jording, D.. Kapp, D. and Piihler, A. (1989). Identification and sequence analysis of the Rhizobium meliloti dctA gene encoding the C4dicarboxylate carrier. Journal of Bacteriology 171, 5551-5560. Finan. T. M., Wood, J. M. and Jordan, D. C. (1983). Symbiotic properties of C4-dicarboxylicacid transport mutants of Rhizobium leguminosarum. Journal of Bacteriology 154. 1403-1413. Forde. B. G., Day, H. M., Turton, J. F., Wen-jun. S . , Cullimore, J. V. and Oliver, J. E. (1989). Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants. Plant Cell 1, 391-401. Fortin. M. G . , Zelechowska, M. and Verma, D. P. S. (1985). Specific targeting of membrane nodulins to the bacteroid-enclosing compartment in soybean nodules. EMBO Journal 4, 3041-3046. Fortin, M. G., Morrison, N. A. and Verma, D. P. S. (1987). Nodulin 26, a peribacteroid membrane nodulin is expressed independently of the development of the peribacteroid compartment. Niicleic Acids Research 15, 813-824. Fujihara, S. and Yamaguchi, M. (1980a). Asparagine formation in soybean nodules. Plant Physiology 66, 139-141. Fujihara, S . and Yamaguchi, M. (1980b). Nitrogen fixation and allantoin formation in soybean plants. Agricultural and Biological Chemistry 44, 2569-2573. Gadal, P. (1983). Phosphoenolpyruvate carboxylase and nitrogen fixation. Physiologic V6g6tale 21. 1069-1074. Gadzhi-zade, B. R., Fedulova, N. G., Topunov, A. F., Romanov, V. I. and Kretovich, V. L. (1985). Influence of pO2 on utilization of different carbon sources for nitrogen fixation by the bacteroids dhizobiurn lupini. Mikrobiologiya 54, 804-809. Garbers. C., Meckbach, R.. Mellor, R. B . and Werner, D . (1988). Protease (Thermolysin) inhibition activity in the peribacteroid space of Glycine max root nodules. Journal of Plant Physiology 132, 442445. Gardiol. A. E., Truchet. G. L. and Dazzo, F. B. (1987). Requirement of succinate dehydrogenase activity for symbiotic bacteroid differentiation of Rhizobium rneliloti in alfalfa nodules. Applied and Environmental Microbiology 53, 19471950. Glenn, A. R. and Brewin, N. J . (1981). Succinate-resistant mutants of Rhizobium leglrniinosarum. Journal of General Microbiology 126, 237-241. Glenn, A. R. and Dilworth, M. J. (1979). An examination of Rhizobium leguminosarum for the production of extracellular and periplasmic proteins. Journal of General Microbiology 112, 405-409. Glenn, A. R. and Dilworth, M. J. (1981). The uptake and hydrolysis of disaccharides by fast- and slow-growing species of Rhizobium. Archives of Microbiology 129, 233-239. Glenn, A. R., Poole, P. S. and Hudman, J. F. (1980). Succinate uptake by free-living and bacteroid forms of Rhizobium leguminosarum. Journal of General Microbiology 119, 267-271. Glenn, A. R., McKay, I. A., Arwas. R. and Dilworth, M . J. (1984). Sugar metabo-

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

171

lism and the symbiotic properties of carbohydrate mutants of Rhizobium leguminosarum. Journal of General Microbiology 130, 239-245. Gober, J. W. and Kashket, E. R. (1983). Methylammonium uptake by Rhizobium sp. strain 32H1. Journal of Bacteriology 153, 1196-1201. Gober, J. W. and Kashket, E. R. (1987). K + regulates bacteroid-associated functions of Bradyrhizobium. Proceedings of the National Academy of Science USA 84,465M654. Gober, J. W. and Kashket, E. R. (1989). Role of DNA superhelicity in regulation of bacteroid-associated functions of Bradyrhizobium sp. Strain 32H1. Applied and Environmental Microbiology 55, 1420-1425. Goodchild, D. J . and Bergersen, F. J. (1966). Electron microscopy of the infection and subsequent development of soybean nodule cells. Journal of Bacteriology 92, 204-213. Gordon, A. J., Ryle, G. .IA., . Mitchell, D. F. and Powell, C. E. (1985). The flux of ''C-labeled photosynthate through soyabean root nodules during N2 fixation. Journal of Experimental Botany 36, 756-769. Griffith, S. M. and Vance, C. P. (1989). Aspartate aminotransferase in alfalfa root nodules. I. Purification and partial characterization. Plant Physiology 90, 1622-1629. Guerin, V., Trinchant, J.-C. and Rigaud, J. (1990). Nitrogen fixation (C2Hz reduction) by broad bean (Vicia faba L.) nodules and bacteroids under waterrestricted conditions. Plant Physiology 92, 595-601. Guezzar, M. E., Hornez, J.-P., Courtois, B . and Derieux, J.-C. (1988). Study of a fructose-negative mutant of Rhizobium meliloti. FEMS Microbiology Letters 49,429-434. Hanks, J. F., Tolbert, N. E. and Schubert. K. R. (1981). Localization of enzymes of ureide biosynthesis in peroxisomes and microsomes of nodules. Plant Physiology 68, 65-69. Hanks, J. F., Schubert, K. and Tolbert, N. E. (1983). Isolation and characterization of infected and uninfected cells from soybean nodules. Role of uninfected cells in ureide synthesis. Plant Physiology 71, 869-873. Heckert, L. L., Butler, M. H., Reimers, J. M . , Albe, K. R. and Wright, B. E. (1989). Purification and characterization of the 2-oxoglutarate dehydrogenase complex from Dictyostelium discoideum. Journal of General Microbiology 135, 155-161. Henson, C. A., Duke, S. H. and Collins, M. (1986a). Characterization of NADP+isocitrate dehydrogenase from the host plant cytosol of lucerne (Medicago sativa) root nodules. Physiologia Plantarum 67, 538-544. Henson, C. A., Duke, S. H. and Koukkari, W. L. (1986b). Rhythmicoscillationsin starch concentrations and activities of amylolytic enzymes and invertase in Medicago sativa nodules. Plant Cell Physiology 27, 233-242. Herrada, G., Puppo, A. and Rigaud, J. (1989). Uptake of metabolites by bacteroidcontaining vesicles and by free bacteroids from French bean nodules. Journal of General Microbiology 135, 3165-3171. Herridge, D. F. (1982). Use of the ureide technique to describe the nitrogen economy of field-grown soybeans. Plant Physiology 70,7-11. Herridge, D. F. (1984). Effects of nitrate and plant development on the abundance of nitrogenous solutes in root-bleeding and vacuum-extracted exudates of soybean. Crop Science 25, 172-179. Herridge, D. F. and Peoples, M. B. (1990). Ureide assay for measuring nitrogen fixation by nodulated soybean calibrated by I5Nmethods. Plant Physiology 93, 495-503.

172

JOHN G. STREETER

Heytler, P. G. and Hardy R . W. F. (1984). Calorimetry of nitrogenase-mediated reductions in detached soybean nodules. Plant Physiology 75, 304-310. Higgins, C. F., Gallagher. M. P., Hyde. S. C., Mimmack, M. L. and Pearce, S. R. (1990). Periplasmic binding protein-dependent transport systems: the membrane-associated components. Philosophical Transactions of the Royal Society of London, Series B 326, 353-365. Hirel. B., Bouet, C., King, B., Layzell, D., Jacobs, F. and Verma, D. P. S. (1987). Glutamine synthetase genes are regulated by ammonia provided externally or by symbiotic nitrogen fixation. EMBO Joirrnal6, 1167-1171. Hoelzle, I. and Streeter. J. G. (1990). Increased accumulation of trehalose in rhizobia cultured under 1o/c oxygen. Applied and Environmental Microbiology 56, 3213-3215. Hornez, J.-P., Guezzar, M. E. and Derieux, J.-C. (1989). Succinate transport in Rhizobium meliloti: Characteristics and impact on symbiosis. Current Microbiology 19. 207-212. Howitt, S. M . , Udvardi. M. K., Day, D. A. and Gresshoff, P. M. (1986). Ammonia transport in free-living and symbiotic Rhizobium sp. ANU289. Journal of General Microbiology 132, 257-261. Huber, T. A. and Streeter, J. G . (1984). Asparagine biosynthesis in soybean nodules. Plant Physiology 74, 605-610. Huber. T. A. and Streeter, J. G. (1985). Purification and properties of asparagine synthetase from soybean root nodules. Plant Science 42, 9-17. Hudman, J. F. and Glenn, A. R . (1980). Glucose uptake by free living and bacteroid forms of Rhizobium leguminosarum. Archives of Microbiology 128, 72-77. Humbeck. C. and Werner, D. (1987). Separation of malate and malonate pools by the peribacteroid membrane in soybean nodules. Endocytobiosis and Cell Research 4, 185-196. Humbeck. C. and Werner, D. (1989). Delayed nodule development in a succinate transport mutant of Bradyrhizobiurn japonicum. Journal of Plant Physiology 134, 276283. Hunt, S . , King, B. J., Canvin, D. T. and Layzell, D. B. (1987). Steady and nonsteady state gas exchange characteristics of soybean nodules in relation to the oxygen diffusion barrier. Plant Physiology 84, 164-172. Hunt, S., Gaito, S. T. and Layzell, D. B. (1988). Model of gas exchange and diffusion in legume nodules. 11. Characterization of the diffusion barrier and estimation of the concentrations of COz, H2, and N2 in the infected cells. Planta 173, 128-141. Israel, D. W. and Jackson, W. A. (1982). Ion balance, uptake, and transport processes in N2-fixing and nitrate- and urea-dependent soybean plants. Plant Physiology 69. 171-178. Jackson, F. A. and Dawes, E. A . (1976). Regulation of the tricarboxylic acid cycle and poly-B-hydroxybutyrate metabolism in Azotobacter biejerinckii grown under nitrogen or oxygen limitation. Journal of General Microbiology 97, 303-3 12. Jin, H. N . , Glenn, A. R. and Dilworth, M. J. (1988). Ammonium uptake by cowpea Rhizobium strain MNF 2030 and Rhizobium trifolii MNF 1001. Archives of Microbiology 149, 3013-31 1. Karr, D. B., Waters, J. K. and Emerich, D. W. (1983). Analysis of polyR-hydroxybutyrate in Rhizobiurn japonicum bacteroids by ion-exclusion highpressure liquid chromatography and UV detection. Applied and Environmental Microbiology 46, 1339-1344. Karr, D. B., Waters, J. K . , Suzuki, F. and Emerich, D. W. (1984). Enzymes of the

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

173

poly-8-hydroxybutyrate and citric acid cycles of Rhizobium japonicum bacteroids. Plant Physiology 75, 1158-1162. Katinakis, P. and Verma, D. P. S. (1985). Nodulin-24 gene of soybean codes for a peptide of the peribacteroid membrane and was generated by tandem duplication of a sequence resembling an insertion element. Proceedings of the National Academy of Science USA 82. 41574161. Katinakis, P., Klein-Lankhorst, R. M.. Louwerse. J . , vanKammen. A . and vandenBos, R. C. (1988a). Bacteroid-encoded proteins are secreted into the peribacteroid space by Rhizobiitm leguminosariim. Plant Molecular Biology 11, 183-190. Katinakis, P., vanKammen, A . and vandenBos, R . C. (1988b). Protein composition of the peribacteroid space in root nodules of Pisum sativum and Vicia faba induced by Rhizobium leguminosarum biovar viciae. Letters in Applied Microbiology 7, 115-118. Kijne. J . W. and Planque. K. (1979). Ultrastructural study of the endomembrane system in infected cells of pea and soybean root nodules. Physiological Plant Pathology 14, 339-345. Kimura, I . and Tajima, S. (1989). Presence and characteristics of NADP-malic enzyme in soybean nodule bacteroids. Soil Science and Plant Nutrition 35, 271-279. King, B. J., Layzell, D. B. and Canvin. D . T . (1986). The role of dark carbon dioxide fixation in root nodules of soybean. Plant Physiology 81, 200-205. King, B. J . . Hunt, S., Weagle, G. E., Walsh. K. B . , Pottier, R. H., Canvin, D. T. and Layzell, D. B. (1988). Regulation of 0 2 concentration in soybean nodules observed by in siru spectroscopic measurement of leghemoglobin oxygenation. Plant Physiology 87. 296-299. Kinnback, A.. Mellor, R. B. and Werner D. (1987). Alpha-mannosidase I1 isoenzyme in the peribacteroid space of Clycine max root nodules. Journal of Experimental Botany 38, 1373-1377. Klipp, W., Reiliinder, H . , Schliiter, A . , Krey, R. and Piihler, A . (1989). The Rhizobiunz meliloti fdxN gene encoding a ferredoxin-like protein is necessary for nitrogen fixation and is cotranscribed with nifA and niJB. Molecular and General Genetics 216, 293-302. Knight, T. J . and Langston-Unkefer, P. J. (1988). Enhancement of symbiotic dinitrogen fixation by a toxin-releasing plant pathogen. Science 241,951-954. Kohl, D. H . , Schubert, K. R . , Carter, M. B.. Hagedorn, C. H. and Shearer, G . (1988). Proline metabolism in N2-fixing root nodules: Energy transfer and regulation of purine synthesis. Proceedings of the National Academy of Science 85, 2036-2040. Kohl, D. H., Reynolds, P. H. S. and Shearer, G. (1989). Distribution of I5N within pea. lupin. and soybean nodules. Plant Physiology 90, 4 2 M 2 6 . Kouchi, H. (1982). Direct analysis of 13C abundance in plant carbohydrates by gas chromatography-mass spectrometry. Journal of Chromatography 241, 305-323. Kouchi, H. and Higuchi, T. (1988). Carbon flow from nodulated roots to the shoots of soybean (Glycine max L. Merr.) plants: An estimation of the contribution of current photosynthate to ureides in the xylem stream. Journal of Experimental Botany 39, 1015-1023. Kouchi. H. and Nakaji, K. (1985). Utilization and metabolism of photoassimilated I3Cin soybean roots and nodules. Soil Science and Plant Nutrition 31,323-334. Kouchi, H., and Yoneyama, T. (1986). Metabolism of ['3C]-labelled photosynthate in plant cytosol and bacteroids of root nodules of Clycine max. Physiologia Plantarum 68.238-244.

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JOHN G . STREETER

Kouchi, H., Nakaji, K., Yoneyama, T. and Ishizuka, J. (1985). Dynamics of carbon photosynthetically assimilated in nodulated soya bean plants under steadystate conditions. Annals of Botany 56, 333-346. Kouchi, H., Fukai, K., Katagiri, H., Minamisawa, K. and Tajima, S. (1988). Isolation and enzymological characterization of infected and uninfected cell protoplasts from root nodules of Glycine max. Physiologia Plantarum 73, 327-334. Kouchi, H., Tsukamoto, M. andTajima, S. (1989). Differential expression ofnodulespecific (nodulin) genes in the infected, uninfected and cortical cells of soybean (Glycine max) root nodules. Journal of Plant Physiology 135,608-617. Kretovich, W. L., Romanov, V. I., Yushkova. L. A . , Shramko, V. I. and Fedulova, N. G. (1977). Nitrogen fixation and poly-B-hydroxybutyric acid content in bacteroids of Rhizobium lupini and Rhizobium leguminosarum. Plant Soil 48, 291-302. Kumar, P. S. and Rao, S. L. N. (1986). Identification and characterization of three forms of glutamine synthetase unique to rhizobia. Current Microbiology 14, 113-116. LaFontaine, P. J., LaFreniere, C. and Antoun, H. (1989). Some properties of carbohydrate and C4-dicarboxylic acid utilization negative mutants of Rhizobium leguminosarum biovar phaseoli strain P121. Plant Soil 120, 195-201. Lafontaine, P. J., Benhamou, N. and Antoun, H. (1990). The occurrence of unusual laminated structures rich in B-1, 4-glucans in plastids of Phaseolus vulgaris root-nodule cells infected by an ineffective C4-dicarboxylic-acid mutant of Rhizobium leguminosarum bv phaseoli. Planta 180, 312-323. Laing, W. A., Christeller, J. T. and Sutton, W. D. (1979). Carbon dioxide fixation by lupin root nodule. 11. Studies with “C-labeled glucose, the pathway of glucose catabolism, and the effects of some treatments that inhibit nitrogen fixation. Plant Physiology 63, 450-454. Lane, M., Meade, J., Manian, S. S. and O’Gara, F. (1986). Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh)in Rhizobium japonicum. Archives of Microbiology 144,29-34. Lankhorst, R. M. K., Katinakis, P., vanKammen, A. and van den Bos, R. C. (1988). Identification and characterization of a bacteroid-specific dehydrogenase complex in Rhizobium leguminosarum PRE. Applied and Environmental Microbiology 54, 3008-3013. Lawrie, A. C. and Wheeler, C. T. (1975). Nitrogen fixation in the root nodules of Viciafaba L. in relation to the assimilation of carbon. 11. The dark fixation of carbon dioxide. New Phytologist 74, 437445. Lewis, T. A., Gonzalez, R. and Botsford, J. L. (1990). Rhizobium meliloti glutamate synthase: Cloning and initial characterization of the glt locus. Journal of Bacteriology 172, 2413-2420. Lucas, K., Boland, M. J. and Schubert, K. R. (1983). Archives of Biochemistry and Biophysics 226, 190-197. Malik, N. S. A.. Pfeiffer, N. E., Williams, D. R. and Wagner, F. W. (1981). Peptidohydrolases of soybean root nodules. Identification, separation, and partial characterization of enzymes from bacteroid-free extracts. Plant Physiology 68, 386-392. Marczewski, W. (1989). Kinetic properties of phosphenolpyruvate carboxylase from lupin nodules and roots. Physiologia Plantarum 76, 539-543. McClure, P. R., Israel, D. W. and Volk, R. J. (1980). Evaluation of the relative ureide content of xylem sap as an indicator of N2 fixation in soybeans. Plant Physiology 66, 72G725.

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

175

McCormick, D. K.. Farnden, K. J. F. and Boland, M. J. (1982). Purification and properties of glutamine synthetase from the plant cytosol fraction of lupin nodules. Archives of Biochemistry and Biophysics 218, 561-571. McDermott, T. R., Griffith, S. M . , Vance, C. P. and Graham, P. H. (1989). Carbon metabolism in Bradyrhizobium japonicum bacteroids. FEMS Microbiology Reviews 63, 327-340. McKay, I. A., Dilworth, M. J. and Glenn, A. R. (1988). C4-dicarboxylate metabolism in free-living and bacteroid forms of Rhizobium leguminosarum MN3841. Journal of' General Microbiology 134, 1433-1440. McKay, I. A., Dilworth, M. J . and Glenn, A. R. (1989). Carbon catabolism in continuous cultures and bacteroids of Rhizobium leguminosarum MNF 3841. Archives of Microbiology 152, 606610. McParland, R. H . , Guevara, J. G . , Becker, R. R. and Evans, H. J. (1976). The purification and properties of the glutamine synthetase from the cytosol of soya-bean root nodules. Biochemistry Journul 153. 597406. McRae. D. G., Miller, R. W. and Berndt, W. B. (1989a). Viability of alfalfa nodule bacteroids isolated by density gradient centrifugation. Symbiosis 7,67-80. McRae, D. G., Miller, R. W., Berndt. W. B. and Joy, K. (1989b). Transport of C4-dicarboxylates and amino acids by Rhizobium meliloti bacteroids. Molecular Plant-Microbe Interactions 2, 273-278. Meeks. J. C.. Wolk. C. P . , Schilling, N., Shaffer, P. W., Avissar, Y. and Chien, W . 4 . (1978). Initial organic products of fixation of ["Nldinitrogen by root nodules of soybean (Glycine max). Plant Physiology 61,980-983. Mellor, R . B. (1988). Distribution of trehalase in soybean root nodule cells: Implications for trehalose metabolism. Journal of Plant Physiology 133, 173-177. Mellor, R. B. and Werner, D. (1987). Peribacteroid membrane biogenesis in mature legume root nodules. Symbiosis 3, 75-100. Mellor, R. B., Morschel, E. and Werner, D. (1984). Legume root response to symbiotic infection. Enzymes of the peribacteroid space. Zietschrift fur Naturforschung, Teil C 39, 123-125. Mellor. R. B., Christensen, T. M. I . E., Bassarab, S. and Werner, D. (1985). Phospholipid transfer from ER to the peribacteroid membrane in soybean nodules. Zietschrift fur Naturforschung, Teil C 40, 73-79. Mellor, R. B., Garbers, C., and Werner, D. (1989). Peribacteroid membrane nodulin gene induction by Bradyrhizobium japonicum mutants. Plant Molecular Biology 12, 307-315. Miller, R. W. and Tremblay, P. A. (1983). Cytoplasmic membrane of Rhizobium meliloti bacteroids. I. Alterations in lipid composition, physical properties, and respiratory proteins. Canadian Journal of Biochemistry 61, 1334-1340. Miller, R. W., McRae, D. G., Al-Jobore, A. and Berndt. W. B. (1988) Respiration supported nitrogenase activity of isolated Rhizobium meliloti bacteroids. Journal of Cellular Biochemistry 38, 35-49. Minamisawa, K . . Arima, Y. and Kumazawa, K. (1986). Characteristics of asparagine pool in soybean nodules in comparison with ureide pool. Soil Science and Plant Nutrition 32, 1-14. Minchin, F. R. and Pate, J. S. (1974). Diurnal functioningof the legume root nodule. Journal of Experimental Botany 85, 295-308. Mitchell, M. K . , Reynolds, P. H. S. and Blevins, D. G. (1986). Serine hydroxymethyltransferase from soybean root nodules. Purification and kinetic properties. Plant Physiology 81, 553-557. Morell, M. and Copeland, L. (1984). Enzymes of sucrose breakdown in soybean nodules. Alkaline invertase. Plant Physiology 74, 1030-1034.

176

JOHN G . STREETER

Morell, M. and Copeland, L. (1985). Sucrose synthase of soybean nodules. Plant Physiology 78, 149-154. Morett, E., Moreno, S. and Espen, G. (1985). Impaired nitrogen fixation and glutamine synthesis in methionine sulfoximine sensitive (MY) mutants of Rhizobium phaseoli. Molecular and General Generics 200. 229-234. Nautiyal, C. S. and Modi, V. V. (1987). Malate dehydrogenase and isocitrate dehydrogenase in root nodules of Trigonella. Phytochemistry 26, 1863-1865. Nautiyal, C. S., vanBerkum, P., Sadowsky, M. J . and Keister, D. L. (1989). Cytochrome mutants of Bradyrhizobiuni induced by transposon Tn5. Plant Physiology 90, 553-559. Newcomb, E. H., Kaneko, Y. and VandenBosch, K. A. (1989). Specializationofthe inner cortex for ureide production in soybean root nodules. Protoplasma 150, 15&159. Newcomb. E . H., Tandon, S. H. and Kowal, R. R. (1985). Ultrastructural specialization for ureide production in uninfected cells of soybean root nodules. Protoplasma 125, 1-12. Newcomb, W. (1976). A correlated light and electron microscopic study of symbiotic growth and differentiation in Pisutn sativum root nodules. Canadian Journal of Botany 54. 2163-2186. Newcomb, W . (1981). Nodule morphogenesis and differentiation. fnternational Review of Cytology 13,247-298. Newcomb, W. and McIntyre, L. (1981). Development of root nodules of mung bean (Vigna radiata): a reinvestigation of endocytosis. Canadian Journal of Botany 59,2478-2499. Newcomb, W. and Wood, S. M. (1986). Fine structure of nitrogen-fixing leguminous root nodules from the Canadian arctic. Nordic Journa! of Botany 6, 609-626. Newcomb, W . , Sippell, D. and Peterson, R. L. (1979). The early morphogenesis of Glycine max and Pisum sativum root nodules. Canadian Journal of Botany 57, 2603-26 16. Nguyen, J., Machal, L., Vidal, J., Perrot-Rechenmannt C. and Gadal, P. (1986). Immunochemical studies on xanthine dehydrogenase of soybean root nodules. Planta 167, 19S195. Nikaido, H. and Vaara, M. (1985). Molecular basis of bacterial outer membrane permeability. Microbiological Reviews 49, 1-32. Nur, I., Okon, Y . and Henis, Y. (1982). Effect of dissolved oxygen tension of production of carotenoids, poly-13-hydroxybutyrate, succinate oxidase and superoxide dismutase by Azospirillum brasilense Cd grown in continuous culture. Journal of General Microbiology 128, 2937-2943. O’Brian, M. R. and Maier, R. J. (1989). Molecular aspects of the energetics of nitrogen fixation in Rhizobium-legume symbiosis. Biochimica et Biophysica Acta 974, 229-246. O’Gara, F., Birkenhead, K., Noonan, B. and Manian, S. S. (1988). Dicarboxylic acid utilisation and regulation of nitrogen fixation in Rhizobium species. Journal of Plant Physiology 132,439441. O’Hara, G. W . . Riley, I. T., Glenn, A. R. and Dilworth, M. J. (1985). The ammonium permease of Rhizobium leguminosarum MNF 3841. Journal of General Microbiology 131, 757-764. Ohyama, T. and Kumazawa, K. (1980a). Nitrogen assimilation in soybean nodules I. The role of GSGOGAT system in the assimilation of ammonia produced by N2-fixation. Soil Science and Plant Nutrition 26, 109-115. Ohyama. T. and Kumazawa, K. (1980b). Nitrogen assimilation in soybean nodules

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

177

11. "Nz assimilation in bacteroid and cytosol fractions of soybean nodules. Soil Science and Plant Nutrition 26, 205-21 3. Orme-Johnson, W. H. (1985). Molecular basis of biological nitrogen fixation. Annual Review of Biophysics and Biophysical Chemistry 14, 419-459. Pankhurst, C. E. and Sprent. J. I. (1975). Surface features of soybean root nodules. Protoplasrna 85, 85-98. Pankhurst. C. E . , Craig, A. S. and Jones, W. T. (1979). Effectiveness of Lotus root nodules. I . Morphology and flavolan content of nodules formed on Lotus pedunculatiis by fast-growing Lotus rhizobia. Journal of Experimental Botany 30, 1085-1093. Pate. J . S . . Gunning, B. E. S. and Briarty, L. G. (1969). Ultrastructure and functioning of the transport system of the leguminous root nodule. Planta 85. 11-34. Patel, J . J. and Yang. A. F. (1981). Light and electron microscopic studiesof nodule structure of alfalfa. Canadian Journal of Microbiology 27, 3643. Peoples, M. B.. Pate. J. S . , Atkins, C. A. and Bergersen. F. J. (1986). Nitrogen nutrition and xylem sap composition of peanut (Arachis hypogaea L. cv Virginia Bunch). Plant Physiology 82, 946951. Peterson. J. B . and Evans, H. J. (1978). Properties of pyruvate kinase from soybean nodule cytosol. Plant Physiology 61, 909-914. Peterson, J. B . and Evans, H. J. (1979). Phosphoenolpyruvate carboxylase from soybean nodule cystol. Evidence for isoenzymes and kinetics of the most active component. Biochimica et Biophysica Acta 567,445-452. Peterson, J. B. and LaRue, T. A. (1981). Utilization of aldehydes and alcohols by soybean bacteroids. Plant Physiology 68, 489493. Peterson, J . B. and LaRue, T. A. (1982). Soluble aldehyde dehydrogenase and metabolism of aldehydes by soybean bacteroids. Journal of Bacteriology 151, 1473- 1484. Pfeiffer, N . E., Torres, C. M. and Wagner, F. W. (1983). Proteolytic activity in soybean root nodules. Activity in host cell cytosol and bacteroids throughout physiological development and senescence. Plant Physiology 71. 797-802. Phillips. D. V., Wilson, D. 0. and Dougherty, D. E. (1984). Soluble carbohydrates in legumes and nodulated nonlegumes. Journal of Agricultural and Food Chemistry 32, 1289-1291. Pladys, D. and Rigaud, J. (1988). Lysis of bacteroids in vitro and during the senescence in Phaseolus vulgaris nodules. Plant Physiology and Biochemistry 26, 179-186. Pladys. D., Trinchant, J.-C. and Rigaud. J. (1986). Proteases from French-bean nodule host-cells: in vitro effects on bacteroids. Physiologie VPgttale 24, 697-705. Preston, G. G.. Zeiher, C., Wall, J. D. and Emerich. D. W. (1989). Acetateactivating enzymes of Bradyrhyizobium japonicum bacteroids. Applied and Environmental Microbiology 55. 165-170. Price, G. D., Day, D. A . and Gresshoff. P. (1987). Rapid isolation of intact peribacteroid envelopes from soybean nodules and demonstration of selective permeability to metabolites. Journal of Plant Physiology 130, 157-164. Puppo, A . , Dimitrijevic, L. and Rigaud, J. (1987). O2consumption and superoxide disrnutase content in purified mitochondria from soybean root nodules. Plant Science 50, 3-1 1. Quiocho, F. A. (1990). Atomic structures of periplasmic binding proteins and the high-affinity active transport systems in bacteria. Philosophical Transactions of the Royal Society of London, Series B 326, 341-351.

178

JOHN G . STREETER

Rainbird, R. M. and Atkins, C. A . (1981). Purification and some properties of urate oxidase from nitrogen-fixing nodules of cowpea. Biochimica et Biophysica Acta 659, 132-140. Rainbird, R. M . , Hitz, W. D . and Hardy, R. W. F. (1984). Experimental determination of the respiration associated with soybeanlRhizobium nitrogenase function, nodule maintenance, and total nodule nitrogen fixation. Plant Physiology 1 5 , 49-53, Rao, N. V., Reddy, R. S. and Sastry, K. S. (1988). Allantoinases of nodulated Arachis hypogaea. Phytochemistry 21, 693-695. Raven, J. A . , Sprent, J. I . , McInroy, S. G. and Hay, G. T. (1989). Water balance of N2-fixing root nodules: Can phloem and xylem transport explain it? Plant, Cell and Environment 12, 683-688. Rawsthorne, S. and LaRue, T . A . (1986a). Preparation and properties of mitochondria from cowpea nodules. Plant Physiology 81, 1092-1096. Rawsthorne, S. and LaRue, T. A . (1986b). Metabolism under microaerobic conditions of mitochondria from cowpea nodules. Plant Physiology 81, 10971102. Rawsthorne, S., Minchin. F. R., Summerfield, R. J., Cookson, C. and Coombs, J . (1980). Carbon and nitrogen metabolism in legume root nodules. Phytochemistry 19. 341-355. Reding, H. K. and Lepo. J . E. (1989). Physiological characterization of dicarboxylate-induced pleomorphic forms of Bradyrhizobium japonicum. Applied and Environmental Microbiology 55, 666-671. Reibach, P. H. and Streeter, J. G. (1983). Metabolism of ''C-labeled photosynthate and distribution of enzymes of glucose metabolism in soybean nodules. Plant Physiology 72,634-640. Reibach, P. H . and Streeter, J. G. (1984). Evaluation of active versus passive uptake of metabolites by Rhizobium japonicum bacteroids. Journal of Bacteriology 159,47-52. Reibach, P. J., Mask, P. L. and Streeter, J. G . (1981). A rapid one-step method for the isolation of bacteroids from root nodules of soybean plants, utilizing self-generating Percoll gradients. Canadian Journal of Microbiology 2 7 491-495. Reynolds, P. H . S. and Farnden, K . J . F. (1979). The involvement of aspartate aminotransferase in ammonium assimilation in lupin nodules. Phytochemistry 18, 1623-1630. Reynolds, P. H . S . , Boland. M. J. and Farnden, K. J. F. (1981). Enzymesof nitrogen metabolism in legume nodules: Partial purification and properties of the aspartate aminotransferases from lupine nodules. Archives of Biochemistry and Biophysics 209, 524-533. Reynolds, P. H. S . , Boland, M. J., Blevins, D. G., Schubert, K. R. and Randall, D. D . (1982). Enzymes of amide and ureide biogenesis in developing soybean nodules. Plant Physiology 69, 1334-1338. Reynolds, P. H. S . , Hine, A . and Rodber, K. (1988). Serine metabolism in legume nodules: Purification and properties of phosphoserine aminotransferase. Physiologia Planfarum 14, 194-199. Robertson, J. G. and Lyttleton, P. (1984). Division of peribacteroid membranes in root nodules of white clover. Journal of Cell Science 69, 147-157. Robertson, J . G . and Taylor, M. P. (1973). Acid and alkaline invertases in roots and nodules of Lupinus angustifolius infected with Rhizobium lupini. Planta 112, 1-6. Robertson, J . G . , Farnden, K. J . F., Warburton, M. P. and Banks. J . M . (1975).

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

179

Induction of glutamine synthetase during nodule development in lupin. Australian Journal of Plant Physiology 2. 265-272. Robertson, J. G.. Lyttleton, P., Bullivant, S. and Grayston, G . F. (1978a). Membranes in lupin root nodules. I. The role of Golgi bodies in the biogenesis of infection threads and peribacteroid membranes. Journal of Cell Science 30, 129-149. Robertson, J. G., Warburton, M. P., Lyttleton, P.. Fordyce. A . M. and Bullivant, S. (1978b). Membranes in lupin root nodules. 11. Preparation and properties of peribacteroid membranes and bacteroid envelope inner membranes from developing lupin nodules. Journal of Cell Science 30, 151-174. Roland, J. C., Lembi. C. A. andMorre, D. J. (1972). Phosphotungstic acidchromic acid as a selective electron-dense stain for plasma membranes of plant cells. Stain Technology 47, 195-200. Romanov, V. I . , Fedulova, N. G., Tchermenskaya, I. E., Shramko, V. I., Molchanov, M. I . and Kretovich. W. L. (1980a). Metabolism of polyB-hydroxyhutyric acid in bacteroids of Rhizobium lupini in connection with nitrogen fixation and photosynthesis. Plant and Soil 56,379-390. Romanov. V. I . . Ivanov, B. F., Fedulova, N . G., Raikhinshtein, M. V., Chermenskaya. I. E., Zemlyanukhin, A. A . and Kretovich, V. L. (1980b). Glucose metabolism in isolated bacteroids of lupine nodules. Biokhimiya 45, 213% 2145. Romanov, V. I . , Hajy-zadeh, B. R.. Ivanov, B. F., Shaposhnikov, G . L. and Kretovich, W. L. (1985). Labelling of lupine nodule metabolites with I4CO2 assimilated from the leaves. Phytochemistry 24. 2157-2160. Ronson, C. W. and Primrose, S. B. (1979). Carbohydrate metabolismin Rhizobium trifolii: Identification and symbiotic properties of mutants. Journal of General Microbiology 112, 77-88. Ronson, C. W., Lyttleton. P. and Robertson, J. G. (1981). C.+-dicarboxylatetransport mutants of Rhizobium trifolii from ineffective nodules on Trifolium repens. Proceedings of the National Academy of Science USA 78, 4284-4288. Ronson, C. W., Astwood, P. M. and Downie, J. A. (1984). Molecular cloning and genetic organization of Cj-dicarboxylate transport genes from Rhizobium leguminosarum. Journal of Bacteriology 160, 903-909. Ronson, C. W., Astwood, P. M. and Nixon, T. B. (1987). Deduced products of C4-dicarboxylic transport regulatory genes of Rhizobium leguminosarum are homologous to nitrogen regulatory gene products. Nucleic Acids Research 15, 7921-7934. Rosendahl, L.. Vance, C. P. and Pedersen, W. B. (1990). Products of dark COz fixation in pea root nodules support bacteroid metabolism. Plant Physiology 93, 12-19. Roth, E. L. and Stacey, G. (1989a). Bacterium release into host cells of nitrogenfixing soybean nodules: The symbiosome membrane comes from three sources. European Journal of Cell Biology 49, 13-23. Roth. E. L. and Stacey. G . (1989b). Cytoplasmic membrane systems involved in bacterium release into soybean nodule cells as studied with two Bradyrhizobium japonicum mutant strains. European Journal of Cell Biology 49, 24-32. Roth, E. L., Jeon, K. and Stacey, G. (1988). Homology in endosymbiotic systems: The term “symbiosome”. In “Molecular Genetics of Plant-Microbe Interactions” (R. Palacios and D. P. s. Verma, eds), pp. 220-225. APS Press, St Paul, Minnesota. Ryan, E . , Bodley. F. and Fottrell, P. F. (1972). Purification and characterization of

180

JOHN G . STREETER

aspartate aminotransferases from soybean root nodules and Rhizobium japonicum. Phytochemistry 11, 957-963. Salminen, S. 0. and Streeter, J. G. (1986). Enzymes of a,a-trehalose metabolism in soybean nodules. Plant Physiology 81, 538-541. Salminen, S. 0. and Streeter, J. G. (1987a). Uptake and metabolism of carbohydrates by Bradyrhizobium japonicum bacteroids. Plant Physiology 83. 535-540. Salminen, S. 0. and Streeter. J. G. (1987b). Involvement of glutamate in the respiratory metabolism of Bradyrhizobium japonicum bacteroids. Journal of Bacteriology 169, 495-499. Salminen, S. 0. and Streeter, J. G. (1990). Factorscontributing to the accumulation of glutamate in Bradyrhizobium japonicum bacteroids under microaerophilic conditions. Journal of General Microbiology 136,2119-2126. SanFrancisco, M. J . D. and Jacobson, G. R. (1986). Glucose uptake and phosphorylating activities in two species of slow-growing Rhizobium. FEMS Microbiology Letters 35, 71-74. Saroso, S., Glenn, A. R. and Dilworth, M . J . (1984). Carbon utilization by freeliving and bacteroid forms of cowpea Rhizobium strain NGR234. Journal of General Microbiology 130. 1809-1814. Saroso. S . , Dilworth, M. J. and Glenn, A. R. (1986). The use of activities of carbon catabolic enzymes as a probe for the carbon nutrition of snakebean nodule bacteroids. Journal of General Microbiology 132. 243-249. Sawhney, V., Saharan, M. R. and Singh, R. (1987). Nitrogen fixing efficiency and enzymes of CO2 assimilation in nodules of ureide and amide producing legumes. Journal of Plant Physiology 129, 201-210. Schubert, K. R. (1981). Enzymes of purine biosynthesis and catabolism in Glycine max. I. Comparison of activities with N2 fixation and composition of xylem exudate during nodule development. Plant Physiology 68, 1115-1 122. Schubert, K. R. (1986). Products of biological nitrogen fixation in higher plants: Synthesis, transport, and metabolism. Annual Review of Plant Physiology 37, 539-574. Scott, D. B . , Farnden, K. J . F. and Robertson, J. G. (1976). Ammonia assimilation in lupin nodules. Nature 263, 703-705. Selker, J. M. L. (1988). Three dimensional organization of uninfected tissue in soybean root nodules and its relation to cell specialization in the central region. Protoplasma 147, 178-190. Selker, J. M. L. and Newcomb. E. H. (1985). Spatial relationships between uninfected and infected cells in root nodules of soybean. Planta 165, 446-454. Sen, D. and Weaver, R. W. (1980). Nitrogen fixing activity of rhizobial strain 32H1 in peanut and cowpea nodules. Plan! Science Letters 18, 315-318. Sen, D., Weaver, R. W. and Bal, A. K. (1986). Structure and organization of effective peanut and cowpea root nodules induced by rhizobial strain 32H1. Journal of Experimental Botany 37, 356-363. Sengupta-Gopalan, C. and Pitas, J. W. (1986). Expression of nodule specific glutamine synthetase genes during nodule development in soybeans. Plant Molecular Biology 7, 189-199. Shantharam, S., Engwall, K. S. and Atherly, A. G. (1988). Symbioticphenotypesof soybean root nodules associated with deletions and rearrangements in the symbiotic plasmid of R. fredii USDA 191. Journal of Plant Physiology 132, 43 1-438. Shearer, G. and Kohl, D. H. (1989). Natural lSN enrichment of amide exporting legume nodules. Physiologia Plantarum 76, 586-590.

TRANSPORT AND METABOLISM OF CARBON AND NITROGEN

181

Shearer, G., Kohl, D . H . and Harper, J. E. (1980). Distribution of "N among plant parts of nodulating and nonnodulating isolines of soybeans. Plant Physiology 66, 57-60. Sheehy, J. E., Minchin, F. R. and Witty, J. F. (1983). Biological control of the resistance to oxygen flux in nodules. Annals of Botany 52, 565-571. Shelp, B. J . and Atkins, C. A. (1983). Role of inosine monophosphate oxidoreductase in the formation of ureides in nitrogen-fixing nodules of Cowpea (Vigna unguiculata L. Walp.). Plant Physiology 72, 1029-1034. Shelp, B. J. and Atkins. C. A . (1984). Subcellular location of enzymes of ammonia assimilation and asparagine synthesis in root nodules of Lupinus albus L. Plant Science Letters 36, 225-230. Shelp, B . J., Atkins, C. A., Storer, P. J. and Canvin, D. T. (1983). Cellular and subcellular organization of pathways of ammonia assimilation and ureide synthesis in nodules of cowpea (Vigna unguiculata L. Walp.). Archives of Biochemistry and Biophysics 224, 429-441. Sinclair, T. R. and Goudriaan, J . (1981). Physical and morphological constraints on transport in nodules. Plant Physiology 67, 143-145. Smith, A. M. (1985). Capacity for fermentation in roots and Rhizobium nodules of Pisum sativum L. Planta 166, 264-270. Smith, S. E. and Smith, F. A . (1990). Structure and funciion of the interfaces in biotrophic symbiosis as they relate to nutrient transport. New Phytologist 114, 1-38. Snapp, S. S. and Vance, C. P. (1986). Asparagine biosynthesis in alfalfa (Medicago sativa L.) root nodules. Plant Physiology 82. 39G395. Somerville. J. E., Shatters, R. G. and Kahn, M. L. (1989). Isolation characterization, and complementation of Rhizobium meiiloti 104A14 mutants that lack glutamine synthetase I1 activity. Journal of Bacteriology 171, 5079-5086. Sprent, J. 1. (1980). Root nodule anatomy, type of export product and evolutionary origin in some Leguminosae. Plant, Cell and Environment 3, 35-43. Sprent, J. I. (1989). Which steps are essential for the formation of functional legume nodules? New Phytologist 111, 129-153. Stone, S. R.,Copeland, L. and Kennedy, I. R. (1979). Glutamate dehydrogenase of lupin nodules: Purification and properties. Phytochemistry 18, 1273-1278. Stovall, I. and Cole, M. (1978). Organic acid metabolism by isolated Rhizobium japonicum bacteroids. Plant Physiology 61, 787-790. Stowers, M. D . (1985). Carbon metabolism in Rhizobium species. Annual Review of Microbiology 39, 89-108. Streeter, J . G. (1979). Allantoin and allantoic acid in tissues and stem exudate from field-grown soybean plants. Plant Physiology 63,478-480. Streeter, J . G. (1980). Carbohydrates in soybean nodules. 11. Distribution of compounds in seedlings during the onset of nitrogen fixation. Plant Physiology 66, 471-476. Streeter, J . G. (1982). Enzymesof sucrose, maltose, and a,a-trehalosecatabolism in soybean root nodules. Planta 155, 112-115. Streeter, J . G. (1985). Accumulation of a , a-trehalose by Rhizobium bacteria and bacteroids. Journal of Bacteriology 164, 78-84. Streeter. J. G. (1986). Effect of nitrate on acetylene reduction activity and carbohydrate composition of Phaseolus vulgaris nodules. Physiologia Plantarum 68, 294-300. Streeter, J. G. (1987). Carbohydrate, organic acid, and amino acid composition of bacteroids and cytosol from soybean nodules. Plant Physiology 85,768-773. Streeter, J . G. (1989a). Analysis of periplasmic enzymes in intact cultured bacteria

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and bacteroids of Bradyrhizobium japonicum and Rhizobium leguminosarum biovar phaseoli. Journal of General Microbiology 135, 3477-3484. Streeter, J. G. (1989b). Estimation of ammonium concentration in the cytosol of soybean nodules. Plant Physiology 90, 779-782. Streeter, J . G . and Morre D. J . (1990). Release of bacteroid proteins during the preparation of peribacteroid membrane and peribacteroid space fractions from soybean nodules (Glycine max (L). Merr.). Symbiosis 8,161-173. Streeter, J . G. and Salminen, S. 0. (1990) Periplasmic metabolism of glutamate and aspartate by intact Bradyrhizobium japonicum bacteroids. Biochimica et Biophysics Acta 1035. 257-265. Stripf, R. and Werner, D. (1978). Differentiation of Rhizobium japonicum. 11. Enzymatic activities in bacteroids and plant cytoplasm during the development of nodules of Glycine max. Zeitschrift fur Naturforschung, Teil C 33, 373-381. Stumpf, D. K. and Burris, R. H. (1981a). Biosynthesis of malonate in roots of soybean seedlings. Plant Physiology 68,992-995. Stumpf, D. K. and Burris, R. H. (1981b). Organic acid contents of soybean: Age and source of nitrogen. Plant Physiology 68, 989-991. Suganuma, N. and Yamamoto, Y. (1987). Respiratory metabolism of mitochondria in soybean root nodules. Soil Science and Plant Nutrition 33, 93-101. Suganuma, N., Kitou, M. and Yamamoto, Y. (1987). Carbon metabolismin relation to cellular organization of soybean root nodules and respiration of mitochondria aided by leghemoglobin. Plant Cell Physiology 28, 113-122. Suzuki, A., Vidal, J., Nguyen, J. and Gidal, P. (1984). Occurrence of ferredoxindependent glutamate synthase in plant cell fraction of soybean root nodules (Glycine max). FEBS Letters 173,204-208. Ta, T.-C., Faris, M. A. and MacDowall, F. D. H. (1986). Pathways of nitrogen metabolism in nodules of alfalfa (Medicago sativa L.). Plant Physiology 80, 1002-1005. Ta, T.-C., MacDowall, F. D. H., Fans, M. A. and Joy, K. W. (1988a). Metabolism of nitrogen fixed by root nodulesof alfalfa (Medicagosativa L.): I. Utilization of [14C, '5N]glutamate and [14C,"N]glutamine in the synthesis oPy-aminobutyrate. Biochemistry and Cell Biology 66,1342-1348. Ta, T.-C., MacDowall, F. D. H . , Faris, M. A. and Joy, K. W. (1988b). Metabolism of nitrogen fixed by nodules of alfalfa (Medicago sativa L.): 11. Asparagine synthesis. Biochemistry and Cell Biology 66, 1349-1354. Ta, T.-C., MacDowall, F. D. H. and Faris, M. A . (1989). Asparagine synthetase from root nodules of alfalfa. Biochemistry and Cell Biology 67,455-459. Tajima, S. and Kouzai, K. (1989). Nucleotide pools in soybean nodule tissues, a survey of NAD(P)/NAD(P)H ratios and energy charge. Plant Cell Physiology 30,589-593. Tajima, S. and LaRue, T. A. (1982). Enzymes for acetaldehyde and ethanol formation in legume nodules. Plant Physiology 70, 388-392. Tajima, S . and Yamamoto, Y. (1975). Enzymes of purine catabolism in soybean nodules. Plant Cell Physiology 16, 271-282. Tajima, S . and Yamamoto Y, (1984). Fluctuation of enzyme activities related to nitrogen fixation, C6/C1 ratios, and nicotinamide nucleotide contents during soybean plant development. Soil Science and Plant Nutrition 30, 85-94. Tajima, S., Sasahara, H., Kouchi, H., Yoneyama,T. andkhizuka, J . (1985). Effects of oxygen concentration and leghemoglobin on organic acid degradation by isolated soybean nodule bacteroids. Agricultural and Biological Chemistry 49, 3473-3479.

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Tajima, S., Kimura, I. and Sasahara, H. (1986). Succinate metabolism of isolated soybean nodule bacteroids at low oxygen concentration. Agricultural and Biological Chemistry 50, 1009-1014. Thony-Meyer, L., Stax, D. and Hennecke, H. (1989). An unusual gene cluster for the cytochrome bcl , complex in Bradyrhizobium japonicum and its requirement for effective root nodule symbiosis. Cell 57, 683497. Thorne, D. W. and Burris, R. H. (1940). Respiratory enzyme systems in symbiotic nitrogen fixation. 11. The respiration of Rhizobium from legume nodules and laboratory cultures. Journal of Bacteriology 39, 187-196. Thummler, F. and Verma, D. P. S. (1987). Nodulin-100 of soybean is the subunit of sucrose synthase regulated by the availability of free heme in nodules. The Journal of Biological Chemistry 262, 14730-14736. Tjepkema, J. D. and Yocum, C. S. (1974). Measurement of oxygen partial pressure within soybean nodules by oxygen electrodes. Planta 119,351-360. Trinchant, J . C. and Rigaud, J . (1987). Acetylene reduction by bacteroids isolated from stem nodules of Sesbania rostrata. Specific role of lactate as an energyyielding substrate. Journal of General Microbiology 133, 37-43. Trinchant, J. C., Birot, A. M. and Rigaud, J. (1981). Oxygen supply and energyyielding substrates for nitrogen fixation (acetylene reduction) by bacteroid preparations. Journal of General Microbiology 125, 159-165. Triplett, E. W., Blevins, D. G. and Randall, D. D. (1980). Allantoic acid synthesis in soybean root nodule cytosol via xanthine dehydrogenase. Plant Physiology 65, 1203-1206. Triplett, E. W., Blevins, D. G . and Randall, D. D. (1982). Purification and properties of soybean nodule xanthine dehydrogenase. Archives of Biochemistry and Biophysics 219, 39-46. Truchet, G.. Camut, S. deBilly, F . , Odorico, R. and Vasse, J. (1989). The Rhizobium-legume symbiosis. Two methods to discriminate between nodules and other root-derived structures. Protoplasma 149, 82-88. Tsien, H. C., Dreyfus, B. L. and Schmidt, E. L. (1983). Initial stages in the morphogenesis of nitrogen-fixing stem nodules of Sesbania rostrata. Journal of Bacteriology 156, 888-897. Tuzimura, K. and Meguro, H. (1960). Respiration substrate of Rhizobium in the nodules. Journal of Biochemistry 47, 391-397. Udvardi, M. K., Price, G. D., Gresshoff, P. M. and Day, D. A. (1988a). A dicarboxylate transporter on the peribacteroid membrane of soybean nodules. FEBS Letters 231, 36-40. Udvardi, M. K., Salom, C. L. and Day, D. A. (1988b). Transport of L-glutamate across the bacteroid membrane but not the peribacteroid membrane from soybean nodules. Molecular Plant-Microbe Interactions 1, 250-254. Upchurch, R. G. and Elkan, G. H. (1978). Ammonia assimilation in Rhizobium japonicum colonial derivatives differing in nitrogen-fixing efficiency. Journal of General Microbiology 104, 219-225. Urban, J. E. and Dazzo, F. B. (1982). Succinate-induced morphology of Rhizobium trifolii 0403 resembles that of bacteroids in clover nodules. Applied and Environmental Microbiology 44,219-226. Urban, J. E. and Nelke, M. (1988). Succinate-induced swelling in a variety of rhizobia. Canadian Journal of Microbiology 34, 910-913. vanBerkum, P., Sloger, C., Weber, D. F., Cregan, P. B . andKeyser, H. H. (1985). Relationship between ureide N and N2 fixation, aboveground N accumulation, acetylene reduction, and nodule mass in greenhouse and field studies with Glycine max L. (Merr). Plant Physiotogy 77, 53-58.

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vanBrussel, A. A. N., Planque, K. and Quispel, A. (1977). The wall of Rhizobium leguniinosarum in bacteroid and free-living forms. Journal of General Microbiology 101, 51-56. Vance, C. P. and Stade, S. (1984). Alfalfa root nodule carbon dioxide fixation. 11. Partial purification and characterization of root nodule phosphoenolpyruvate carboxylase. Plant Physiology 75, 261-264. Vance, C. P.. Johnson, L. E. B., Halvorsen, A. M., Heichel, G. H. and Barnes, D. K. (1980). Histological and ultrastructural observations of Medicago sativa root nodule senescence after foliage removal. Canadian Journal of Botany 58, 295-309. Vance, C. P., Stade, S. and Maxwell, C. A. (1983). Alfalfa root nodule carbon dioxide fixation. I. Association with nitrogen fixation and incorporation into amino acids. Plant Physiology 72, 469473. Vance, C. P., Boylan, K. L. M., Maxwell, C. A., Heichel, G . H. and Hardman, L. L. (1985). Transport and partitioning of COz fixed by root nodules of ureide and amide producing legumes. Plant Physiology 78, 774-778. Vance, C. P., Reibach, P. H. and Pankhurst, C. E. (1987). Symbiotic properties of Lotus pedunculatus root nodules induced by Rhizobium loti and Bradyrhizobium sp. (Lotus).Physiologica Plantarum 69, 435-442. VandenBosch, K. A. and Newcomb, E. H. (1986). Immunogold localization of nodule-specific uricase in developing soybean root nodules. Planta 167, 425-436. VandenBosch, K. A., Noel, K. D., Kaneko, Y. and Newcomb, E. H. (1985). Nodule initiation elicited by noninfective mutants of Rhizobium phuseoli. Journal of Bacteriology 162, 950-959. Vanlerberghe, G. C., Horsey, A. K., Weger, H. G. and Turpin, D. H. (1989). Anaerobic carbon metabolism by the tricarboxylic acid cycle. Evidence for partial oxidative and reductive pathways during dark ammonium assimilation. Plant Physiology 91, 1551-1557. Vasse, J . , deBilly, F., Camut, S. and Truchet, G. (1990). Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules. Journal of Bacteriology 172,4295-4306. Vaughn, K. C. (1985). Structural and cytochemical characterization of three specialized peroxisome types in soybean. Physiologia Plantarurn 64, 1-12. Vella, J. and Copeland, L. (1990). UDP-glucose pyrophosphorylase from the plant fraction of nitrogen-fixing soybean nodules. Physiologia Plantarum 78, 140-146. Verma, D. P. S. and Bal, A. K. (1976). Intracellular site of synthesis and localization of leghemoglobin in root nodules. Proceedings of the National Academy of Science USA 73,3843-3847. Verma, D. P. S. and Fortin, M. G. (1989). Nodule development and formation of the endosymbiotic compartment. Cell Culture and Somatic Cell Genetics of Plants 6,329-353. Verma, D. P. S., Kazazian, V., Zogbi, V. and Bal, A. K. (1978). Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules. Journal of Cell Biology 78, 919-936. Vezina, L.-P. and Langlois, J. R. (1989). Tissue and cellular distribution of glutamine synthetase in roots of pea (Pisum sativum) seedlings. Plant Physiology 90, 1129-1133. Vidal, J . , Nguyen, J., Perrot-Rechenmann, C. and Gadal, P. (1986). Phosphoenolpyruvate carboxylase in soybean root nodules. An immunochemical study. Planta 169, 198-201.

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Walsh, K. B., McCully. M.E. and Canny, M. J . (1989a). Vascular transport and soybean nodule function: Nodule xylem is a blind alley, not a throughway. Plant, Cell and Environment 12, 395-405. Walsh. K. B., Canny, M. J. and Layzell, D . B. (1989b). Vascular transport and soybean nodule function: 11. A role for phloem supply in product export. Plant, CeN and Environment 12. 713-723. Ward, A. C., Rowley, B. I. and Dawes, E. A . (1977). Effect of oxygen and nitrogen limitation of poly-R-hydroxybutyrate biosynthesis in ammonium-grown Azotobacter beijerinckii. Journal of General Microbiology 102, 61-68. Waters, J. K., Karr, D . B . and Emerich, D . W. (1985). Malate dehydrogenase from Rhizobium japonicum 3Ilb-143 bacteroids and Glycine max root-nodule mitochondria. Biochemistry 24. 6479-6486. Watson, R. J. (1990). Analysis of the C4-dicarboxylatetransport genes of Rhizobium meliloti: Nucleotide sequence and deduced products of dctA, dctB, and dctD. Molecular Plant-Microbe Interactions 3, 174-181. Watson, R . J., Chan, Y.-K., Wheatcroft, R., Yang, A.-F. and Han, S. (1988). Rhizobium meliloti genes required for C4-dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid. Journal of Bacteriology 170,927-934. Webb, M. A. and Newcomb, E. H. (1987). Cellular compartmentation of ureide biogenesis in root nodules of cowpea (Vigna unguiculata (L.) Walp.). Pfanta 172, 162-175. Werner, D . and Morschel, E. (1978). Differentiation of nodules of Glycine m u . Ultrastructural studies of plant cells and bacteroids. Planta 141, 169-177. Werner, D. Dittrich, W. and Thierfelder, H. (1982). Malonate and Krebs cycle intermediates utilization in the presence of other carbon sources by Rhizobium japonicum and soybean bacteroids. Zeitschrift fur Naturforschung, Teil C 37, 921-926. Werner, D . , Morschel, E . , Kort, R., Mellor, R. B. and Bassarab, S. (1984). Lysis of bacteroids in the vicinity of the host cell nucleus in an ineffective (fix-) root nodule of soybean (Glycine max). Planta 162, 8-16. Werner, D., Morschel, E., Garbers, C., Bassarab, S. and Mellor, R. B. (1988). Particle density and protein composition of the peribacteroyd membrane from soybean root nodules is affected by mutation in the microsymbiont Bradyrhizobium japonicum. Planta 174,263-270. Wittenberg, J. B., Bergersen, F. J., Appleby, C . A. and Turner, G. L. (1974). Facilitated oxygen diffusion. The role of leghemoglobin in nitrogen fixation by bacteroids isolated from soybean root nodules. The Journal of Biological Chemistry 249,4057-4066. Witty, J. F., Minchin, F. R. and Sheey, J. E. (1983). Carbon costs of nitrogenase activity in legume root nodules determined using acetylene and oxygen. Journal of Experimental Botany 34, 951-963. Witty, J. F., S k ~ tL. , and Revsbech, N. P. (1987). Direct evidence for changes in the resistance of legume root nodules to O2 diffusion. Journal of Experimental Botany 38, 1129-1 140. Wong, P. P. and Evans, H . J. (1971). Poly-R-hydroxybutyrate utilization by soybean (Glycine max Merr.) nodules and assessment of its role in maintenance of nitrogenase activity. Plant Physiology 47, 750-755. Yarosh, 0. K., Charles, T. C. andFinan, T . M. (1989). Analysisof C4-dicarboxylate transport genes in Rhizobium meliloti. Molecular Microbiology 3, 813-823. Yates, M . G. (1980). “Biochemistry of nitrogen fixation”. In “The Biochemistry of Plants” (B. J. Miflin, ed.), pp. 1-64. Academic Press, New York.

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Yoneyama, T., Karasuyama, M., Kouchi, H. and Ishizuka, J. (1985). Occurrence of ureide accumulation in soybean plants. Effect of nitrogen fertilization and Nz fixation. Soil Science and Plant Nutrition 31, 133-140. Yoneyama, T., Fujita, K., Yoshida, T., Matsumoto, T., Kambayashi, I. and Yazaki, J. (1986). Variation in natural abundance of I5N among plant parts and in lsN/I4N fractionation during NZ fixation in the legume-rhizobia symbiotic system. Plant Cell Physiology 27, 791-799. Zlotnikov, K. M., Marunov, S. K. and Khmel’nitskii, M. I. (1984). Disturbance in assimilation of fixed nitrogen by soybean plants in symbiosis with the ASPbacterium Rhizobium japonicum. Doklady Akademii Nauk SSSR 275, 189-192.

NOTES ADDED IN PROOF (1) Closer examination of the literature on poly-p-hydroxybutyrate, including one paper which was overlooked (Gerson et al., 1978), indicates that substantial variation in content of the polymer in Lupinus nodules occurs in response to growth stage and to treatments which affect plant carbohydrate status-such as darkness. Typically, there is a close negative relationship between nitrogenase activity and poly-p-hydroxybutyrate concentration suggesting a potential role in carbon metabolism for the polymer in these nodules (Kretovich et al., 1977; Gerson et al., 1978; Romanov et al., 1980a). (2) Two very recent papers suggest the possible operation of a malate/ aspartate shuttle mechanism in some nodules. A mutant of R. meliloti lacking aspartate aminotransferase activity was found to be Fix- (Rastogi and Watson, 1991); unlike the results for B. japonicum (Zlotnikov et al., 1984). In the second paper (Appels and Haaker, 1991), analysis of the plant cytoplasmic aspartate aminotransferase activity and analysis of substrate consumption by R. leguminosarum bacteroids in response to various substrate combinations led to the suggestion for the existence of an operative shuttle in Pisum sativum nodules. Thus, although a malate/aspartate shuttle mechanism seems unlikely for B. japonicum (Streeter and Salminen, 1990), it cannot be dismissed as a mechanism in fast-growing rhizobia. A more general conclusion from points 1 and 2 is that it may be too early to develop detailed general descriptions of carbon metabolism in bacteroids. (3) Unpublished results from Dr. S. A. Tchetkova (personal communication), have shown that nitrogenase isolated anaerobically from dctA mutant bacteroids of R . meliloti had no enzyme activity, (nitrogenase isolated from wild type bacteroids had typical activity). dctB and dctD regulatory mutants which had low but measurable dicarboxylate transport also had low but measurable nitrogenase activity in vitro. Curiously, nitrogenase protein could be detected in extracts of dctA mutant bacteroids even though there is no detectable enzyme activity. Thus, there is apparently some previously undescribed relationship between the development of

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a functional discarboxylate transport system in bacteroids and the formation of active nitrogenase, and the results summarized in Table I11 should be interpreted with caution. ADDED REFERENCES

Appels, M. A. and Haaker, H. (1991). Glutamate oxaloacetate transaminase in pea root nodules. Participation in a malate/aspartate shuttle between plant and bacteroid. Plant Physiology 95,740-747. Gerson, T., Patel, J. J. and Wong, M . N. (1978). The effects of age, darkness and nitrate on poly-13-hydroxybutyratelevels and nitrogen-fixing ability of Rhizobium in Lupinus angustifolius. Physiologia Plantarum 42, 42Q-424. Rastogi, V. K . and Watson, R. J. (1991). Aspartate aminotransferase activity is required for aspartate catabolism and symbiotic nitrogen fixation in Rhizobium meliloti. Journal of Bacteriology 173, 2879-2887.

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Plants and Wind

P . VAN GARDINGEN and J . GRACE University of Edinburgh. tnstitute of Ecology and Resource Management. Darwin Building. The Kings Buildings. Mayfield Road. Edinburgh. EH9 3JU. U K

I . Introduction

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Wind Regimes Around Plants and their Role in Transport . . . . A . TheClassicalMicrometeorologicalApproach . . . . . . . B . What Classical Micrometeorology is Unable to Do . . . . .

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Wind and Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Energy Balance Equation B . Boundary Layer Conductance . . . . . . . . . . . . . C . Convective Energy Flux . . . . . . . . . . . . . . . . D . Temperature . . . . . . . . . . . . . . . . . . . . . E . Transpiration . . . . . . . . . . . . . . . . . . . . . F . Stomata1 Conductance . . . . . . . . . . . . . . . . . G . Cuticular Conductance . . . . . . . . . . . . . . . . . H . Solving the Energy Balance Equation for Transpiration and Surface Temperature . . . . . . . . . . . . . . . .

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IV . Facts, Fallacies and Mysteries A . Thigmomorphogenesis . B . Abrasion . . . . . . C . Ecological Phenomena . V.

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

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Conclusions

Appendix I . . . . . . . . . . . . . . . . . . . . . . . . 246 Appendix I1 . . . . . . . . . . . . . . . . . . . . . . . . . 248 Copyright @ 1991Academic Press Limited Advances in Botanical Research Vol . 18 ISBN &12-005918-5

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SYMBOLS AND ABBREVIATIONS Leaf area Areas of a set of plant parts Coefficient of expansion of air The drag force on a single leaf The sum of all values of B Heat flux by convection Drag coefficient Specific heat capacity of air at constant pressure Rate of change of wind speed u with respect to height z Rate of change of temperature with respect to height Vertical concentration gradient Deflection of the free end of a stem Diameter Zero plane displacement Characteristic dimension of object parallel to flow for heat and mass transfer Molecular diffusion coefficient Diffusion coefficient for water vapour in air Rate of evapotranspiration Flux (molar); in units of mol m-* s-’ Flux (mass units): in units of g m-* s-’ Acceleration due to gravity Conductance Boundary-layer conductance Cuticular conductance Leaf conductance Stomata1 conductance Rate of heat conduction to and from the plant part Grashof number Height of vegetation Intensity of turbulence (a coefficient of variation) Second moment of area Thermal diffusivity Von Karman’s constant Coefficient of eddy diffusion (a constant of proportionality) Turbulent transfer coefficient for heat Turbulent transfer coefficient for water Turbulent transfer coefficient for momentum Latent heat of vaporization Length Length (in dimensional analysis) Mass d.

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Ta TL Ts T' U

U* V VH V'

P W W'

W WUE Xa

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Relative molecular mass Nusself number (heat transfer) Correction factor, as outlined in Dyer (1974) Energy flux converted by photosynthesis and respiration Atmospheric pressure Density of air Resistance Aerodynamic or boundary layer resistance Aerodynamic resistance to heat transfer Aerodynamic resistance to momentum transfer Aerodynamic resistance to gas transfer Cuticular resistance Mesophyll resistance Stomata1 resistance Universal gas constant Reynolds number Net radiation flux Energy flux to and from storage Sherwood number Saturation vapour pressure deficit Standard deviation of the u component of wind Downward flux of horizontal momentum Time a fluid particle has been travelling once it moves away from a leaf Temperature of air Characteristic time a fluid particle moves in one direction before changing course Temperature of plant surface Fluctuating component of temperature (undefined) Horizontal streamwise component of the vector wind speed, o r simply the wind speed. Mean wind speed at height z Fluctuating component of uw Mean of sum of squares of all the u values Friction velocity Kinematic viscosity of air Horizontal lateral component of vector windspeed Fluctuating component of V H Mean of sum of squares of all v' values Vertical component of vector wind speed Fluctuating component of w Applied force Water use efficiency Mole fraction of water vapour of external atmosphere

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xevap xs

Y Z

zo

P. VAN GARDINGEN AND J . GRACE

Mole fraction of water vapour at sites of evaporation in a leaf Mole fraction of water vapour at leaf surface Young’s modulus Height Roughness length of vegetation

I. INTRODUCTION Advances in the study of plants and wind have been made on several fronts during the last 25 years or so. In this period, the role of air movement in the transport of heat and gases between leaves and the atmosphere has become widely appreciated and micrometeorological theory has been applied by agronomists and foresters in the analysis of crop response to the weather. Profiles of mean wind speed over vegetation reveal how the roughness of the vegetational surface and the wind above the canopy determine the vertical mixing of air. This work has wide-reaching implications and provides a theoretical framework for understanding how microclimates develop, how water and CO2 are exchanged and the role of vegetation as a sink for atmospheric pollutants. In recent years, new and more flexible approaches have been proposed for measuring air movement and heat and mass transfer in the “awkward” cases of forests, agroforests and patchy vegetation. These involve the use of rapidly responding sensors to examine the fine structure of air movement in relation to equally rapid sensing of temperature or gas concentration. At the smaller scale of the leaf, the role of wind in determining tissue temperature and plant water use is much better understood than previously, although problems remain in adequately describing natural flows and in defining the response of leaves to turbulence. Moreover, there are still formidable problems in “scaling-up” from a knowledge of the characteristics of leaves to the vegetation in the context of landscape. Finally, much is now known about the physiological responses of plants to wind, including the influence of abrasion on the epidermis which produces the characteristic “scorching” known to gardeners, and also the anatomical response to mechanical stimulation which causes the well-known “stunted” appearance of plants in windy places. In this chapter, we have tried to outline these advances, emphasizing the most important conclusions.

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11. WIND REGIMES AROUND PLANTS AND THEIR ROLE IN TRANSPORT A. THE CLASSICAL MICROMETEOROLOGICAL APPROACH

This approach was developed in the 1960s and concerns the analysis of micrometeorological measurements made with a vertical array of sensors mounted on a mast over an extensive area of crop, land or sea. It yields a set of techniques for evaluating the vertical flux of several important entities; notably heat, water vapour, C 0 2 and pollutant gases, Thus, diurnal and seasonal fluctuations in the rate of photosynthesis and transpiration may be measured and related to the stage of the crop, the physiological state of the plants or to the weather. The basic conditions and assumptions required for this approach are well known. These conditions are: (i) that the crop is uniform and extensive so that the sensors can be placed in a turbulent air flow which has totally adjusted to the roughness of the crop and the strength of its physiological sinks or sources; and (ii) that transfer within this turbulent boundary layer occurs by a process known as turbulent diffusion, where the vertical flux of entities such as heat, water and COz between the vegetation and the atmosphere is proportional to their concentration gradient F = - K.6XI62

(1)

where F is the flux, 6xI6z is the vertical concentration gradient and K is the constant of proportionality known as the coefficient of eddy diffusion, its value varying over orders of magnitude depending on the wind regime. This equation has the same form as that for molecular diffusion from a large flat plate, except that the constant of proportionality is then the molecular diffusion coefficient (usually denoted D ) , is much smaller than K , and depends on the characteristics of the diffusing molecule and the medium in which diffusion occurs.

1. The wind profile above the vegetation The mean wind speed is often measured with light-weight cup anemometers. It has been observed that, in many conditions, the wind speed increases with height above the ground in a logarithmic manner

where u(z) is the mean wind speed at height z , u, is the friction velocity, k is von Karman’s constant, which has been found to have a value of about 0.41, zo is the roughness length of the vegetation and d is the zero-plane displacement.

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To fit the equation to a measured wind profile is not straightforward as there are three unknowns ( u * , d and zo). The most common method is to iterate d around its suspected value, plotting In(z-d) against u(z) at each iteration, until the value of d is found at which the graph becomes a straight line, viz.

k ln(z-d) = -(u(z)) u,

+ In zo

(3)

Then, the slope provides an estimation of (klu,) and hence of u, and the intercept (In 20) yields the roughness length 20. An example from Thom (1971) illustrates this with data from the bean crop (Fig. 1). Practical difficulties arise if the meteorological and site conditions required to obtain a

0

1.0

0.5

Leaf area

Wind speed ( m s-'1

5

-

4

-0 I

'u Y

= 3

2

0

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2

3

Wind speed ( m s-I)

Fig. 1 . Profiles of mean wind speed in and above a bean crop, with logarithmic plot of the five uppermost data points (Thom, 1971). The four data sets are from days with different wind speed.

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logarithmic profile do not obtain, or if one or more anemometers is miscalibrated. In such cases a straight line may never occur, or, if it does, it may yield a spurious value of d . The physical meaning of the parameters u,, d and zo have been discussed by Thom (1975). The friction velocity u, (ms-l) can be regarded as the velocity at which turbulent eddies at the top of the canopy rotate. The zero plane displacement length d is a measure of the height at which the sink for momentum, comprising the roughness elements (leaves etc.), is elevated from the ground by the stalks. The roughness length zo is a measure of the effectiveness of the canopy in absorbing momentum from the wind. The determination of d and zo is very susceptible to measurement error: as zo is obtained as an intercept in a regression equation (eqn (3)) it is very sensitive to quite small errors (ca. 1%)in the determination of wind speed. For this reason, anemometers must be frequently calibrated and interchanged. Other limitations are as follows. (1) Mean wind speeds must be determined over a sufficiently long period to overcome the temporal variation inherent in the flow. In practice, 20 minutes is usually sufficient. (2) Sensors must be placed within the boundary layer which has developed over that particular vegetation. For this reason, a large field is recommended, to supply sufficient fetch. According to one estimate, a logarithmic profile of depth 1m requires 100 m of fetch. As it is difficult to fit a set of six anemometers, even if they are small, into a vertical range of less than 1m, the 100m is a minimum. (3) Logarithmic profiles are found only in meteorologi/cal conditions of near-neutral stability. If the air temperature increases with height (stable conditions) then the upward movement of the turbulent eddies is suppressed and higher wind speeds than otherwise are recorded at any particular height. If the air temperature decreases very rapidly with height, buoyancy occurs (instability) and vertical mixing is enhanced. Lower wind speeds than those calculated in with eqn (2) would then obtain. When the lapse rate of temperature is known, correction terms can be calculated and so zo, d and u, can still be found. (Monteith and Unsworth, 1990). 2. Typical values of d and zo A very large number of determinations have now been made of d and 20, and empirical relationships have been suggested to relate these to the height of the vegetation h. For many crops, d is in the range 0.6-0.7 h and 20 is about 0.1 h (see Legg etal., 1981). For very sparse vegetation (e.g. desert scrub) or crowded and smoothed vegetation, these relationships do not hold as the vegetation is considerably less effective ,at absorbing momentum from the wind than expected from its height. Data on the range of zolh for roughness

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elements arranged with variable packing suggest that zo can be estimated for almost any vegetation if the silhouette area of the vegetation, measured normal to the wind is known in relation to the land area occupied by the plant (Garratt, 1977; Raupach et al., 1980). Others have proposed relationships based on leaf area index (Jacobs and van Boxel, 1988). Thus, estimates of wind profiles can be made on the basis of the structural organization of plant communities. Wind profiles are usually not particularly useful or interesting in their own right, their main value comes when attempting to measure fluxes of heat and gases to and from the crop, as described in the next section.

3. Use of wind profiles in estimating heat and mass transfer The rate of change of wind speed u with respect to height z may be used to define the drag force 7 that the vegetation exerts on the air

+K M . ( ~ u / ~ z )

(4) 7 may also be regarded as the downward flux of horizontal momentum, a concept explained by Thom (1975) by dimensional analysis. The dimensions of 7 are (mass x velocity) per area of ground per unit of time, i.e. M L T 2 / (L2T).The constant of proportionality K M is the turbulent transfer coefficient for momentum and can be calculated from u, in eqn (3) using the expression 7

=

K M = ku, (2-d) (5) The flux of gases Fand heat C between vegetation and atmosphere follows a similar relationship, where p is the density of air and cpis the heat capacity of air at contant pressure.

As momentum, heat and gases are all transported by turbulent eddies, the corresponding transfer coefficients K M , KV and K H might be expected to have the same numerical value (but see Thom (1975)). Thus, KM found from eqn (5) may be inserted into eqns (6) and (7) so that, if the concentration gradients (6x/Sz) and (6T/6z) are known, then F and C can be calculated. This really is very useful, as uptake rates of COz and pollutant gases, and the loss of water are matters of great concern. The calculation of KM from u, has to be modified when dealing with non-neutral stability.

K M = ku, ( Z - d ) / $ M (8) where $, is a correction factor calculated as outlined in Dyer (1974). Stability correction factors have been found to be inaccurate when used over

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forests, where observed diffusivities for heat and mass exceed those calculated (Thom et af., 1975; Raupach, 1979; Denmead and Bradley, 1985). Thus, determination of heat and mass transfer over forest canopies is best made using the technique of eddy correlation (see section on Statistics of Turbulence), or perhaps by the Bowen ratio method which assumes only that diffusivities for heat and mass are equal (see Denmead and Bradley, 1985). The general approach outlined here is usually called “flux gradient analysis”. Examples of the successful use of this technique include the studies reported by Biscoe et al. (1975) and Garland (1977).

4. Diffusion resistances Plant physiologists have for a long time modelled the diffusion of CO;?and water through stomata1 pores as an electrical analogue. This is possible because diffusion is proportional to the concentration gradients just as current is proportional to a gradient of electrical potential. The concept of aerodynamic resistance r, was advanced by Gaastra (1959) to model diffusion in air above individual leaves. In an analogous fashion, an entire crop can be regarded as a big leaf, having an aerodynamic resistance which can be found from KM:

More specifically, it is related mathematically to the parameters of the wind profile as

where ramis the resistance to momentum transfer between the vegetation and the reference height z. In non-neutral conditions, an additional term is required to make the resistance smaller in unstable conditions and larger in stable conditions (Monteith and Unsworth, 1990). An interesting conclusion from eqn (10) is that tall vegetation displays much lower aerodynamic resistances than short vegetation. Some calculations on the magnitude of this, and its likely consequences for evaporation and transpiration are given by Grace (1981) and Jarvis (1981). In practice, the corresponding resistances for heat and gases TaH and rav may be somewhat higher than ram because surfaces at right angles to the wind (bluff surfaces) are more efficient at exchanging momentum than heat and gas (Thom, 1975). This leads to the use of an additional resistance, the excess resistance, estimated as 4/u,.

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P. VAN GARDINGEN AND J . GRACE

5. Profiles within the canopy The drag exerted by the canopy on the air flow, manifest as the wind profile observed above the canopy, arises from the sum of the individual drag forces of the leaves. The drag force B on a single leaf is given by

where p is the air density, c d is the drag coefficient and A is the area of the leaf. The drag coefficient c d depends on the roughness of the leaf and can be determined on a force balance in a wind tunnel (Thom, 1968; Holland et al., 1991). For large broad leaves, c d is in the range of 0.8-1.0 when leaves are placed normal to the air flow, and as low as 0.05 when the leaf is held parallel to the lines of flow (Thom, 1968). The sum of all values of B , integrated as follows from z = 0 to z = h , might be expected to be equal to the drag on the canopy T measured from wind profiles above the canopy h

where A l , represents the areas of a set of plant parts (leaves, stems, fruits, etc.). In practice, B’ has been found to exceed T by a factor of 3.5, presumably because many of the plant parts within the canopy are “sheltering” immediately behind and in the wake of other plant parts. For this reason, it is not possible to forecast an exact description of the within-canopy profile from a knowledge of the distribution of plant parts, and most authors have used an empirical relationship (Cionco, 1965; Thom,’1971; Landsberg and James, 1971; Oliver, 1975; Baldocchi e t a l . , 1983). In all cases the mean horizontal wind speed declines towards the ground, though in stands of trees a “bulge” has been noted in the region of the trunks. Some selected examples from the literature are given elsewhere (Grace, 1977). There are great difficulties in making useful comments about profiles of mean wind speed inside canopies for several reasons. The flow is spatially very variable and the mean profile is dependent on the location of the measurement mast in relation to the stems and shoots of plants. Moreover, the flow is turbulent and contains a considerable vertical component of velocity that has been completely ignored until recently (see section B.l on Measured Turbulence). For similar reasons, it is also difficult to apply the techniques of flux-gradient analysis in this region, although several people have attempted to do so.

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B. WHAT CLASSICAL MICROMETEOROLOGY IS UNABLE TO DO

As we have seen, classical micrometeorology provides a tool for understanding mean transfer rates of momentum, water and other gases, to and from extensive areas of vegetation, and also enables mean wind speeds above and within crops to be estimated. It has been used with great success for the case of short crops of cereal, and occasionally for semi-natural vegetation from tundra to tropical forest (see other examples in Monteith (1976)). The main limitations of the classical approach as outlined above appear to be as follows. (1) It deals with mean values and says nothing about the fluctuating nature of wind or of other variables. Many natural phenomena such as the release and dispersal of spores and seeds, catastrophic gale damage and the morphogenetic response of plants depend less on the mean velocity and more on the intermittent and turbulent nature of the wind. Micrometeorology has developed in the way that it has because mean values of the main variables were easier to collect and handle. For example, the most common type of anemometer was originally the cup anemometer which measures the mean horizontal wind speed. It is only relatively recently that field sensors able to resolve the fine detail of natural flows have been available, and that data loggers and computers to handle such information have been in widespread use. (2) Most non-cultivated vegetation does not conform to the basic requirements: it is patchy rather than uniform and, consequently, it is impossible to define a region above the vegetation where the boundary layer can be said to be characteristic of that vegetation type. (3) Difficulties arise because sources and sinks of momentum, heat and water vapour are not the same. Thus, the path length for respective diffusion resistances are different, and somewhat arbitrary corrections must be made. (4) The discrepancy between observed and calculated stability corrections over tall vegetation is not understood, but does severely restrict the utility of flux-gradient relationships over forests. ( 5 ) Turbulent diffusion is a useful concept only when the turbulent eddies are small in relation to the scale of the gradient or the distance over which transport is being considered. Eddies in plant communities are, however, often very large, as shown by spectral analysis of data from forests (Allen, 1968; McBean, 1968; Crowther and Hutchings, 1985) and by the release of smoke (Oliver, 1975). Denmead and Bradley (1985) demonstrated a clear example of the failure of flux-gradient analysis within a 16m tall pine forest. Independent measurements of heat and water vapour transport were made with eddy correlation equipment. Upward flux of sensible heat and water vapour

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were accompanied by negative (downward) gradients of temperature and humidity. For sensible heat, 70% of the observations were in this category. Inspection of the recordings in one 15-min run suggested that most of the heat transport occurred in five short events, four of which were gusts that penetrated the canopy from above, carrying down warmer drier, GO;! depleted air (Fig. 2). The counter-gradient position is merely the obvious extreme of a situation that is presumed to commonly exist. An important question, therefore, is: When can flux-gradient analysis be usefully applied? This question was addressed in a wind-tunnel study done by Raupach and Legg (1984) who used gravel to simulate vegetation and heating wires to provide a planar heat source dissipating energy at a known rate. Wind

co,

H,O

flux

flux

Heot flux

CO, concentration

Water vopour mixing ratio

Temperature

W

U

f

12.5 min

-

/

Fig. 2. Fluxes of COz,water and heat over a forest, with correspondingrecords of temperature, T , vertical wind, wand horizontal wind, u . Peaks of flux are seen to correspond to peaks of w. Redrawn from Denmead and Bradley (1985).

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profiles and turbulence characteristics were taken with hot wire anemometers and temperature was sensed with a fine (1.2pm) resistance thermometer. Heat transport was greater than that calculated (from fluxgradient theory with stability corrections) over a surprisingly large distance above the ground. The discrepancy was x 2 near the gravel surface and declined with height z until z = 100 20 was reached. If this were also true in the natural environment, it would pose very serious difficulties in finding suitable sites. The authors warn about the use of measurements made too close to the vegetation. A theoretical basis for modelling heat and mass transport has not yet been achieved (see Finnigan, 1985). Current approaches use the Lagrangian specification to model the dispersal of entities in the turbulence field (Raupach, 1989). In Lagrangian fluid dynamics, the positions and velocities of wandering fluid particles are considered. The leaves, etc., in the canopy are considered as sources and sinks for scalars. Transport is modelled in much the same way as they would be if we were dealing with a population of chimneys emitting plumes of smoke. It is necessary to know the profile of the standard deviations of the vertical component of the wind, u,,,, and the characteristic time a fluid particle moves in one direction before changing course ( TL). Once a fluid particle moves away from the leaf it is characterized by the time it has been travelling t . In the period when tccTL,flow is dispersive rather than diffusive, and this is used to define a nearfield. Later on, when b TL, a far field is defined in which flux gradient relationships do apply. It is too early to say whether this approach will eventually be more successful than the classical one. It seems likely that the latter will continue to be used for some time, especially where surface resistances are required for models of regional and global transfers of water, CO;,and pollutants.

I . The nature of flows near plants The true nature of flows near plants is revealed effectively by the release of smoke or other small particles than by the use of cup anemometers. The routine recording of mean horizontal flows may have led to considerable misunderstanding of flows in the past. For example, much of what is commonly said regarding the shelter effect of wind-breaks may be wrong. It used to be thought that a hedge of height h creates useful shelter to a distance of 20 h. It now appears that hedges create a highly turbulent flow in a zone of around 8-12 h and a reverse flow in the region 0-8 h (Heisler and DeWalle, 1988; McNaughton, 1989). Rapid fluctuations may be sensed in the field by light-weight propeller anemometers and sonic anemometers (Grace, 1985). Both require associated signal-processing equipment or fast data loggers, and are able to resolve the vector wind into u , v and w components corresponding to horizontal streamwise, horizontal lateral and vertical components.

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Moreover, they enable fluctuations of the order of 1 H z or better to be resolved. Working with models in wind tunnels, it is common to use hot-wire anemometers which resolve fluctuations of 1kHz or better. These instruments are not so robust and have several features which make them less suitable for field work, although they are sometimes used. Statistics of turbulence. The wind speed at any instant ( u , v or w )may be considered as a mean (a, P, or W ) and a fluctuating component ( u ’ , v f or w f )

+ u f (horizontal streamwise) v = ri + v f (horizontal lateral) w = W + w ’ (vertical) u = li

(13) The standard deviation of the fluctuating part measures the spread of values: 12 0.5 uu=(u )

(14)

where (ur2) is the mean of the sum of squares of all u f values. The coefficient of variation is termed the intensity of turbulence

i, = u U h (15) where readings are taken with a cup anemometer, u‘ and V ’ are not resolved, in which case the intensity of turbulence, i, may be given the following meaning.

.

lU =

(u’2

+ v’2)0,5 l i

The ability to measure instantaneous values of the wind components leads to the development of the eddy correlation technique for the measurement of fluxes of sensible heat. This is a particularly direct method, in which vertical heat-laden gusts are averaged thus:

-

C = pc, w fT’

(17)

where T is the fluctuating part of the temperature signal. Similar relationships can be written for other entities such as water vapour and C 0 2 but with the latter the technique has so far been limited by the relatively slow response time of the available COz analyses. A common manipulation is the calculation of the power spectrum. This involves the presentation of the data in the frequency domain, such that the total variance (power) in the data is partitioned according to frequency. For a further discussion of this technique the reader is referred to Chatfield (1984). Measured turbulence. The turbulence intensities measured inside canopies often range between 0.2 and 0.8 for the horizontal velocity components

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and 0.1 and 0.5 for the vertical components (Cionco, 1972; Shaw e t a f . ,1974; Finnigan, 1979a). The profile of intensity within the canopy can be related to the distribution of roughness elements and perhaps their flexibility (Finnigan, 1979a). Some of the turbulence results from the passage of gusts over the canopy, with penetration of air into the canopy. Other contributions include the turbulence produced in the wakes of leaves and stems, and the waving of plant parts.

E

.+ a

$ 0.8 c

.-In U

x .-+ In

0.6 0.4

0.8 0.6

-

-3

( C )

-2

-1

0 (Y -

1

2

3

r 1/U"

Fig. 3. Probability density distribution of the differences from the mean of (a) vertical, (b) streamwise and (c) lateral components of the wind velocity within an almond orchard. Data were collected at two heights within the canopy: 0.14h (---)and0.51 h (-). Reproduced with permission from Baldocchi and Hutchinson (1987).

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P. VAN GARDINGEN AND J. GRACE

Information on the variation in u‘, v‘ and w ’may also be presented as a probability density distribution (Baldocchi and Hutchinson, 1987). In this case the deviations from the mean are presented as a fraction of the standard deviation (Fig. 3). Note that this technnique enables an estimate to be made of the probability of recording extreme gusts. “Rare” gusts may be very important in effecting dispersal of pollen or spores, and the breakage of plant parts. Recent work has been concerned with establishing the extent of skewness in such distributions. An early example of power spectra of turbulence is provided by Allen (1968) who investigated the flow at various heights in a 10m tall Japanese larch plantation (Fig. 4). He recognized a predominance of low-frequency peaks at about 0.04 Hz (i.e about 25 s) corresponding to gusts of 100 m wavelength. These large gusts were found even at the forest floor. Further up the canopy there were other peaks in the spectrum, corresponding to the size expected to be produced by the eddy shedding of individual trees. Although such flows have mainly been seen in forests (see Section IV) there is no reason to believe they are absent in shorter vegetation. Arkin and Perrier (1974) showed very pronounced vorticular air movement by using smoke as a tracer in the rows between crop plants.

Wakes of individualplants. Smooth solid objects interact with flows in a fairly predictable manner: they shed regular eddies at a frequency which can be calculated if the dimensions of the object and the velocity of the flow are

Frequency (Hz)

Fig. 4. Power spectra of turbulence at various heights in a larch plantation. Reproduced with permission from Allen (1968).

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205

known. Photographs of this are given in many textbooks, such as Tritton (1977). Downstream, these eddies break into smaller ones and the flow becomes completely chaotic. Ultimately the eddies become so small as to be non-detectable. Plants are rather rough objects with gaps through which the air flows. Studies with smoke tracers show that they behave as porous bluff bodies (Wilson and Crowther, 1985). The lines of flow diverge around the shoots, but some of the air flows straight through the gaps. The wakes of individual trees have been investigated by hot-wire anemometry in a wind tunnel (Ruck and Schmitt, 1986), and by computational means (Gross, 1987). In both cases, a relatively “quiet” zone is formed immediately downstream of the canopy containing reverse flow (Fig. 5 ) . Ruck and Schmitt (1986) include a line drawing as an interpretation of the flow field. Similar regions of reverse flow occur behind wind breaks (Plate, 1971; McNaughton, 1989). 2. Wind and waving plants This subject has received attention recently because of its practical importance in forestry and agriculture. It is particularly important in oceanic climates because a substantial part of the crop occurs in areas so windy that the size to which the trees may be safely grown is limited. The topic illustrates the importance of understanding the fluctuating nature of the wind, rather than the mean speed. In gales, trees may be uprooted or the stems may break. The mean drag forces on the canopy may be calculated from the distribution of leaf area and the within-canopy profile of wind speed (eqn 11). The drag coefficients (Cd) of entire trees have been determined by Mayhead (1973) using a large wind tunnel. The drag coefficient decreases as the wind speed increases because the branches and leaves are swept back (“streamlining”). The turning moments tending to topple the tree or break the roots are shown in Fig. 6. They include components of: (1) the wind; and (2) the mass of the canopy once it has been displaced from the vertical. This second component may be very large if the crown is loaded with snow or rime ice (Petty and Worrell, 1981). Rain is an additional loading. The extent to which the stem is displaced from the vertical in response to an applied force may be found by experiment or be estimated from the geometry of the stem and its mechanical properties. To calculate the deflection it is usual to assume that the plant is a perfectly elastic cantilever, and to apply standard equations obtained from engineering texts. It is instructive to examine these equations in their simplest form. For a cantilever, the deflection of the free end, 8, is given by

w 13 o+ 3 YI,

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P. VAN GARDINGEN AND J . GRACE

-

0.5 0.4

-i

-

0.3

V

-g 0.2I

--.

0.1 -

0-

B

.0.35

-0.12

0.11 0.20 0.270.350 42 0.50 Distance ( m )

Fig. 5 . Mean flow around an individual small tree in a wind tunnel. The lines with arrows represent the wind vector (mean speed and direction). Reproduced with permission from Ruck and Schmitt (1986).

where W is the applied force, 1, is the length, Y is Young’s modulus and I , is the second moment of the area, which for a solid stem is IT d4/64 where d is the diameter. Young’s modulus is a measure of the stiffness of a material and I , is a cross-sectional shape factor, such that YIg describes how easily a sample undergoes elastic deformation. For a tree, the force attributable to the wind, W, will depend mainly on the area of leaf in the canopy A arid the square of the wind speed (W = 1/2 pu2CdA,see eqn (11)). In the early life of a tree, the cross-sectional area of the stem is proportionate to the leaf area, and the length is proportional to the diameter (Hamilton and Christie, 1971). If these allometric relationships are inserted into eqn (18), we conclude that the deflection caused by a given wind speed will grow as the tree develops, in proportion to the square root of the leaf area. However, this simple approach may not apply. Morgan and Cannell (1988) and Cannell and Morgan (1988) point out some of the difficulties. It must be appreciated that the tree is not perfectly elastic, that the deflection may be so large as to invalidate the use of the formula, and that Young’s modulus for tree stems is very variable and depends not only on the species but also on the water content. Fraser and Gardiner (1967) measured the actual turning moment needed to uproot trees by pulling them with a winch. The results were used to compare the susceptibility of trees to wind-throw when growing on different soil types. O n surface water gleys the roots were shallow and the trees were more easily uprooted. On brown earths the root system developed “sinker” roots which conferred additional stability on the tree. Subsequently, these observations have led to further work on the root system in relation to anchorage in the soil (Waldron and Dakessian, 1981; Coutts, 1983, 1986).

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The process of wind-throw is likely to depend on the dynamic properties of bending trees. In a gale, trees sway violently to and fro and their root systems “work loose”. This seems to involve breakage of minor roots and disturbance to the soil. During this process water may become mixed with soil particles to form a slurry, thus reducing the shear resistance of the soil. Attention has thus been directed towards the swaying of trees. The maximum deflections are likely to be caused by resonance phenomena, where the gust ‘frequency is the same as the natural period of sway of the stem. Swaying of trees can be measured using spring-loaded displacement transducers or accelerometers. Wind speed measurements taken at the same time require an anemometer system that responds rapidly so that the important fluctuations are detected, such as lightweight propeller anemometers or sonic anemometers (Grace, 1985). It is possible to derive an exact physical equation for wind-induced sways (Finnigan and Mulhearn, 1978; Milne, 1991). However, the approach taken with trees has so far been a statistical one, using the techniques of time-series analysis (Holbo et al., 1980; Mayer, 1987). A long run of data on wind velocity and acceleration (or displacement) of the tree may be expressed in the frequency domain by using Fourier transformation. Typically, such data are collected at a rate of 5-10Hz over a period of tens of minutes. The resulting power spectra of velocity or acceleration may be compared and inferences drawn about the response of the tree (Figs 7 and 8). In this scheme, note that the transfer of energy from

Fig. 6 . Forces acting on a tree crown attributable to the wind and gravity, w , and w2. with corresponding turning moments ml and m2.

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P. VAN GARDINGEN AND J . GRACE

Power spectrum

Power spectrum

Power spectrum of tree response

M U Aerodynamic transfer function

Mechanical transfer function

Fig. 7. The spectral method of analysis of tree vibrations used by Mayer (1987). The power spectrum of wind speed and of tree response are measured, the rest are derived.

the spectrum of wind speed to that of the tree response occurs selectively, as in a band-pass filter. The characteristics of these “filters” furnish the essential information we require on the ability of the tree to “extract” energy from the wind and may be described as “transfer functions”. The selectivity between the power spectrum of wind speed and that of wind loading furnishes information about the frequency dependence of Cd. The second transfer function yields information about the mechanical response of the tree, and this is of great theoretical and practical interest, as it may alter according to the dimensions of the stem and the degree of contact between crowns of trees. Thus, its variation might be used to assess the influence of silvicultural treatment on the tendency of the tree to sway in the wind. The mechanical transfer functions so far obtained show rather narrow peaks at the primary period of sway of the tree (Fig. 8) (Mayer, 1987); for a mature tree, this is often in the range 0.3-0.6 Hz. Outside this range, rather little energy is extracted from the wind, despite early suggestions that secondary and tertiary modes of vibration may be important, and rotational modes also. Sway periods of trees can easily be found by artificially displacing the stem and recording the subsequent movement (Mayhead, 1973; White et al., 1976). The natural sway periods of trees may also be calculated from the formulae given by Blevins (1979) using information about Y ,I,, length, density and degree of taper.

111. WIND AND ENERGY TRANSFER A . ENERGY BALANCE EQUATION

The energy transfer between a plant part and the external environment can be expressed as an energy balance equation with the algebraic sum of the separate energy fluxes equal to zero.

R,

+ AEt + C + S + C + P = 0

(19) where R, is the net heat flux through radiation, AE, is the rate of evapotranspiration multiplied by the latent heat of vaporization (A) in order to express

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PLANTS A N D WIND

transpiration as an energy flux, C i s convective heat flux, S is the energy flux to and from storage, G is the rate of heat conduction to and from the plant part and P represents the rate at which energy is converted into chemical bonds in photosynthesis or released by respiration. When written in this way, the term would be positive to denote fluxes of energy to the plant and

1.0-

Streamwise ( u )

L I

I

1

1

0001

002

004

1

1

007 01

I

I

I

1

02

04

07

1

Frequency (Hz)

10'

-10-

-EB

N

B

10-

1o

-~

10-1

10-2

1

Frequency(Hz1

Fig. 8. (a) The power spectra of the u , v and w components of the wind, measured just above the forest, and (b) the power spectrum of longitudinal tree acceleration. Data were recorded in a spruce forest (Mayer, 1987). Reproduced with permission from Mayer (1987).

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P. VAN GARDINGEN AND J. GRACE

negative to denote flux away from the plant. In practice, the terms S, G and P make only minor contributions to the total energy balance of leaves and can be omitted to form a simplified form of the energy balance equation (eqn 20) which shows that the total energy available from radiation during the day is partitioned into two components: convection and evaporation. Both C and hEt can be defined in terms of a driving force or gradient multiplied by a conductance term by analogy to Ohms' law. R,+XE,+C=O + cp( Ts-

Rn + X(Xs-Xa)gleaf

Ta)gca = 0

(20)

This equation differs from those which are found in most current textbooks in that the fluxes are defined in molar units, a practice which has recently become widespread in plant physiology but not in micrometeorology . The reader is referred to Appendix I for a discussion of the common units used to describe flux estimates and conversion factors. Equation (20) shows that the transpiration term is proportional to the difference in the mole fraction of water vapour between the leaf surface (xs) and the external atmosphere (x,) multiplied by the leaf conductance (g&. The leaf conductance is calculated from three conductances, the boundary layer (gca),stomata1 (gcs), and cuticular conductances. The convective energy flux is proportional to the temperature difference between the plant surface (T,) and the air (T,) multiplied by the boundary layer conductance. This molar flux is converted into energy units using cp, the molar specific heat capacity of air at constant pressure. Fluxes of energy and gas molecules can be accurately modelled by analogy to an appropriate electrical circuit. Very complicated systems can be simplified using Kirchoff's law for electrical circuits. The flux is then represented by a driving force or potential difference divided by a resistance term (Monteith, 1973). Conductance is simply the reciprocal of the resistance and is preferred since flux rates are linearly related to conductance. The transpiration term in eqn (20) is driven by the water vapour mole fraction difference (xs-xa) and convection by the temperature difference ( T,- T,) between the leaf surface and the air. Both fluxes are proportional to the appropriate conductance term gleaffor transpiration and g,, for convection. The nature of these interactions is examined in the following sections.

B. BOUNDARY LAYER CONDUCTANCE

In this section, the nature of the boundary layer, and how wind influences the magnitude of the boundary layer or aerodynamic conductance is examined. Boundary layer conductance describes the influence of the boundary layer on heat and mass transfers, and depends on the size and shape of the plant part, as well as wind speed. It is the single physical parameter which

PLANTS A N D WIND

21 1

best describes the direct effect of wind on heat and mass transfers between a plant part and the atmosphere. The boundary layer is the transition zone from immediately above the plant surface to the external or ambient atmosphere away from the influence of the plant part. The nature of the boundary layer is therefore determined by the characteristics of both plants and atmosphere, and is the interface zone between them. This point introduces an important concept in that, whilst the rates of energy and mass transfer from a plant are determined in part by the atmospheric environment, the atmospheric environment is itself modified by the presence of vegetation, at levels ranging from microclimate, to the regional climate. The boundary layer conductance can be considered to be a measure of the coupling between the plant and atmosphere. A plant part with low boundary layer conductance would have an atmospheric environment near its surface which is determined by the characteristics of the plant and would be considered to be uncoupled from the atmosphere. High boundary layer conductance results in conditions near the plant’s surface that are more like those of the ambient atmosphere. 1. Convection Heat transfer by convection occurs by two distinct processes: natural and forced convection. Natural convection occurs when a vertical flow of air is induced as a result of density gradients created by local warming or cooling of air in contact with the plant surface, and at low wind speeds it predominates the convection process. In most environments there is some degree of air movement and free convection tends to be less important than forced convection induced by movement of air over the plant surface. Free convection is, however, a process of great importance for plants growing in sheltered environments, especially in glasshouses where protection from the wind may be complete. In contrast, forced convection is driven by an external influence, that of air flow. The rate of heat transfer from the plant surface depends on the transfer of heat to the airstream moving over the plant surface. 2. Boundary layer structure The nature of air flow over a plant part such as a leaf is determined by the turbulence of the impinging air stream and characteristics of the leaf such as size, shape and surface roughness. Air flow may be laminar, turbulent, or a complex mixture of both in the transition zone between laminar and turbulent flow (Fig. 9). The flow of air over a leaf increases the rate of heat and mass transfer as the boundary layer conductance increases. A laminar boundary layer is defined as the situation where lines of air flow are parallel to each other and the leaf surface. Under certain circumstances, the lines of flow tend to become disturbed into random motion as a turbulent boundary

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+ Wind direction

I3

d

_+

Free stream

Turbulent boundary layer

Laminar sublayer

Fig. 9. Air Row over a smooth flat plate, showing the transition from laminar to turbulent flow.

layer develops. The tendency for the turbulence to develop is measured by a non-dimensional number, the Reynolds number: Re=-

UdC V

where u is the wind speed, dc is the dimension or length of the surface over which the air has passed, and v is the kinematic viscosity of air. Typical values for Re calculated for leaves are in the range O-104. In engineering studies, it is generally accepted that the critical Re required to initiate turbulence in smooth airflows is about lo5 (Monteith, 1973). Turbulent boundary layers may develop over leaves since, in nature, the airflow incident on a leaf will always have a degree of turbulence. Leaf surface details such as veins and serrations also have the effect of disturbing the airflow and causing turbulence at very low Reynolds numbers (Grace and Wilson, 1976). A leaf in a natural environment will move with the wind, and this flapping motion tends to create a turbulent boundary layer (Parlange et al., 1971; Schuepp, 1972; Grace, 1978). The structure of the boundary layer is determined by wind speed, leaf size and, to a lesser extent, surface detail. Small leaves have relatively thin boundary layers with high conductance, and hence their temperatures are closely coupled to the external environment. Large leaves form thick boundary layers with lower conductance and their temperature may differ significantly from that of the surrounding air. As has been indicated previously, the thickness of the boundary layer is reduced with increasing wind speed and, consequently, the boundary layer conductance increases with wind speed. If the wind speed exceeds a critical value air flow over the leaf changes from laminar to turbulent. The resulting vertical component of transfer increases the boundary layer conductance, and hence rates of transfer. The magnitude of the boundary layer conductance can be

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computed in two ways. Firstly, g,, may be calculated from theoretical considerations of heat transfer, developed principally for the engineering sciences. The second method is to calculate g,, from carefully measured rates of heat or mass transfer from real or model leaves. 3. Calculating g,, Heat and mass transfers between an object and any fluid are increasingly expressed as entities which have no units, known as non-dimensional numbers. Heat transfer is expressed as the Nusselt number, Nu, which has a simple relationship to the boundary layer conductance, gca:

Nu = gcadcR Ta KP,

where d, is the characteristic dimension of the object parallel to the flow (Monteith, 1973; Grace et al., 1980; Dixon and Grace, 1983), K is the thermal diffusivity of the fluid, Pa is the atmospheric pressure,and R is the universal gas constant. The corresponding non-dimensional number for mass transfer is the Sherwood number, Sh,

where D is the diffusion coefficient of the gas in air. It is obvious that if the values of Nu and Sh can be determined these equations can be solved for gca. For forced convection with laminar boundary layers the value of Nu can be calculated using the Polhausen relationship, which is derived from theoretical considerations, and gives a good representation of the large body of experimental data in the engineering sciences (Welty et af., 1969; Monteith, 1973). Nu = 0.66 ReO.s vo.33 (24) By rearranging these equations, and substituting (ud,/v) for R e an estimate of g,, can be derived:

The corresponding equation for turbulent flow is entirely empirical (Monteith, 1973): Nu = 0.03 Re".' and gives a similar equation for g,,

(26)

The boundary layer conductance for mass transfer can be calculated by replacing the value of K with the appropriate diffusion coefficient D , or by

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P. VAN GARDINGEN A N D J . GRACE

multiplying to the boundary layer conductance for heat by a conversion ( ) For water vapour the value of ( D H z 0 l ~ ) " is 1.09. These factor of ( D / K )67. equations show that g,, increases more rapidly with wind speed if the boundary layer is turbulent, and that in this case the conductance is relatively insensitive to the leaf dimension d,. Heat transfer by laminar free convection can be related to another nondimensional number, Gr, the Grashof number, which measures the tendency for free convection to occur. Gr = agd: (T,- T,)lv2 (28)

''

This relationship suggests that the tendency for free convection to occur depends on two properties of the plant, the characteristic leaf dimension ( d , ) and the temperature difference between leaf and air (T,- TJ, where a is the coefficient of expansion of air, and g is the acceleration due to gravity. An estimate of the Nusselt number can be obtained from Gr using standard relationships which are documented for a variety of geometric shapes (Monteith, 1973; Grace and Dixon, 1985). For a flat plate the value of Nu is defined as Nu = 0.54/(Grv/~)"25 (29) and consequently the boundary layer conductance is defined as gc'l

8000

=

0.54Ko7ig0 2Sa" 25 ( T, - T,)" 25 Pa d! 2 i V 0 IiRT,

(30)

-

0

1

2

3

4

5

0

2

4

6

8 1 0 1 2 1 4

< - T , ("C) Fig. 10. Boundary layer conductances for heat flux from leaves with dimensions d = 1 mm to 1 m.(a) The conductance for forced convection as a function of windspeed (eqn 25). (b) The conductance for free convection as a function of the temperature difference between the leaf and air (T,- T.) (eqn 30). Care must be taken when calculatingg,, for leaves. Heat transfer will occur from both sides of the leaf, and hence g,, will be computed as the parallel sum of the conductances on both sides. For most leaves this is equivalent to halving the conductance calculated for one side from the projected area of the leaf. The situation becomes more complex, however, when the two sides of the leaf have radically different surface features, such as the dense hairs present on the underside of many leaves. The boundary layer conductance for water vapour will depend on g,, for heat, and on whether the leaf is hypostomatous or basistomatous. u (rn s-'1

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Using the above relationships it is possible to calculate values of go for a range of leaf sizes, wind speeds, and temperature differences (Fig. 10). The figures show that the boundary layer conductance for forced convection is usually much larger than the values obtained for free convection even when large temperature differences exist between the leaf and the external atmosphere. In addition to free and forced convection, a mixture of both, termed hybrid or mixed convection, may occur particularly at low wind speeds. It is difficult to decide how to treat hybrid convection in energy balance models. Alternative approaches exist, both calculate the boundary layer conductances for free and forced convection. One method then uses the higher of the two conductances (McAdams, 1954), the alternative being to assume that both pathways operate in parallel and to add the conductances (Grace, 1983). In this review, we have used the second approach, calculating the free and forced convection using eqns (27) and (30), respectively. Experimental studies using leaf models, cylinders and spheres have been used to estimate the effect of wind on convection andg,, for simple shapes of leaves, stems and fruits (Gates and Papian, 1971; Kreith, 1973; Grace and Wilson, 1976; Mitchell, 1976; Grace et al., 1980; Dixon and Grace, 1983). Such studies also present the opportunity to be able to compare measured values ofg,, with those calculated from theory as detailed above. The results obtained from experimental studies have illustrated the problems associated with applying the theory to plant structures, since rates of transfer may sometimes be up to two times greater than predicted. The differences may be attributed to the formulae having been developed for use with large smooth surfaces in laminar flows, and higher temperature differences such as might occur with power station heat exchangers. The results obtained with real leaves may reflect the development of some degree of turbulence over the leaf surface because of leaf minutiae. These studies have served to illustrate that the formulae from engineering studies must be used with caution since the actual boundary layer conductance of a leaf is likely to be higher than the calculated value. C. CONVECTIVE ENERGY FLUX

The convective energy flux is a function of the temperature difference ( Ts- T,) and the boundary layer conductance gca(eqn 20). In the previous section, it was shown that the nature of the boundary layer depends on the mode of convection. Forced convection occurs when heat is transferred from the plant surface to a stream of air moving over the plant part. Free convection occurs by the formation of convection plumes during the local heating and cooling of air by the plant surface. It is thus important to recognize that the temperature difference term occurs twice in the calculation of free convection as both the driving force, and in determining the boundary layer conductance.

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P. VAN GARDINGEN AND J . GRACE

A number of experiments have been conducted to estimate rates of heat and mass transfer and boundary layer conductance for model leaves (reviewed by Grace (1977,1981)). In these experiments, the measured rates of transfer often exceeded the calculated values by up to a factor of 2. Dixon and Grace (1983) showed that transfer of heat by natural convection was twice that predicted by theory. They noted that the difference was greatest with microphyllous leaves which suggests some kind of edge effect. This conclusion is supported by the work of Schuepp (1973) who used visualization techniques to demonstrate the presence of convection plumes on a microscale, and showed that the edges of his model leaves were particularly active. Forced convection presents an additional problem in that there is a question of whether the boundary layer is laminar or turbulent. Studies of leaves in both laminar and turbulent airflows show that these objects have an aerodynamically rough surface and that some degree of turbulence exists in the boundary layer in virtually all conditions (Fig. 11) (Grace and Wilson, 1976). This observation may explain why many experimental data exceed the calculated values. Another possible reason for any

Wind direction cm

L

al

w

U

0 lcm Horizontal scale Fig. 11. Air flow over a Populus leaf. The thick black line is a representation of the leaf, showing roughness because of the topographyand veins. Onlypart of the leaf behaved like a flat plate; the diagram shows that the boundary layer was turbulent in places. Reproduced with permission from Grace and Wilson (1976).

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discrepancy may be related to poor experimental technique, especially associated with measurements of mass transfer of water vapour. An especially accurate method for measuring g,, involves carefully measuring the cooling rates of model leaves which have been heated to a few degrees above ambient temperature. This technique was utilized by Grace et al. (1980) in a series of experiments on large leaved tropical trees, and good agreement was found between calculated and observed heat transfer rates. There were, however, indications that leaf minutiae could influence transfer rates, especially with high wind speeds. Experiments have been conducted to investigate the effects of a turbulent airstream and variations of leaf form on heat flux (Dixon, 1982). This study concluded that turbulence in the impinging airstream, and serrated or pleated margins increased the rate of heat transfer by up to 20%. D . TEMPERATURE

The temperature of a plant part is a vitally important biological characteristic. Temperature determines the metabolic rates of all living tissues, which in turn influences growth and development. Temperature extremes can either severely reduce plant growth, or kill the plant outright. Temperature may explain the distribution of organisms, and processes such as timberline formation (Tranquillini, 1979; Berry and Raison, 1981; Kappen, 1981; Larcher and Bauer, 1981; Grace, 1989). 1 . Calculating surface temperature The temperature of a plant part is dependent on that of the ambient environment (T,) and the energy flux within the system. The simplified energy balance equation (eqn 20) may be solved for (T,-T,) using an iterative procedure (Gates and Papian, 1971; Grace, 1983). This procedure has been used in the section on transpiration to illustrate the effect of wind on the energy balance of a range of leaf sizes in several environments. Alternative approaches to solving this problem are available. One method uses the Penman-Monteith equation (Monteith and Unsworth, 1990) and requires measurements of the boundary layer conductance, surface conductance, air humidity and net radiation absorbed by the leaf. Other methods are described by Campbell (1981), Monteith (1981) and Nobel (1983). An energy balance approach can be extended to include photosynthetic data and then relationships between leaf temperature and water use efficiency can be described (Ball et al., 1988).

2. Calculating surface temperature The importance of the temperature difference between a plant part and the free atmosphere ( Ts-T,) has been alluded to in previous sections. Temperature difference is the driving force for heat transfer by convection, and

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indirectly for transpiration via its influence on water vapour partial pressure. The boundary layer conductance for free convection is also affected by the temperature difference. In a system with a low rate of evaporation, such as overwintering buds, or leaves with their stomata shut, small changes in wind speed have a large effect on the surface temperature. In such cases as wind speed increases the temperature tends to approach that of the ambient environment. The result is more complex when the rate of evaporation is larger, such as with a transpiring leaf, since the system is also sensitive to stomata1 conductance, and the saturation vapour pressure deficit (SVPD) of the atmosphere.

3. Shelter Shelter is used in agricultural systems as a simple method of modifying wind. In shelter trials, the temperature of plant surfaces are seldom measured, but measurements of air temperature immediately above the crop can be used to infer that of the plant. Typically the air above a sheltered crop is one or two degrees warmer than above a nearby unsheltered area (Aslyng, 1958; Brown and Rosenberg, 1972; Carr, 1985). The temperature increase associated with shelter is undoubtedly one of the main influences underlying the shelter effect in cold or temperate parts of the world. In other climates where temperatures exceed the growth optima, shelter may only exacerbate the problem. 4. Meristem temperature The temperature of plant meristems plays an important role in determining the rate of plant growth and development. Meristem temperature determines the rates of cell division, and leaf extension, and in many species plays an important role in the release of winter dormancy of buds. The temperature of apical meristems or buds which are exposed to direct sunlight may be several degrees warmer than the ambient air. In contrast, the meristem may be only slightly warmer than the ambient atmosphere in vegetation with sheltered meristems, such as grasses, which have the meristem within a leaf sheath at the base of the plant. E. TRANSPIRATION

The loss of water from plant parts is a complex process controlled by a number of interacting factors. The discussion of the movement of water from the plant surface will be limited to leaves in this review, but the basic principles can be applied to any structure. I . Transpirational fluxes Transpiration from a leaf can be considered in two ways, as an energy flux (e.g. eqn 20) or as the diffusion of water vapour molecules (mass transfer). These two processes are simply related as shown in eqn 20 since the energy

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flux equals the mass flux multiplied by the latent heat of vaporization (A). It then follows that the rates of evaporative transfers of energy and mass will be limited by the diffusion of water vapour molecules from the plant surface. Mass transfer by molecular diffusion is analogous to heat transfer by thermal agitation in still air. In moving air, both entities are carried in the moving airstream. It would then be expected that heat and mass transfer rates through boundary layers would be simply related. In a previous section it was shown that the boundary layer conductance for molecular diffusion of water vapour (gca)is simply related to the value of g,, for heat by the ratio (%)llh7

where D H ,is~the diffusion coefficient of water vapour in air.

K

2 . Calculating transpiration rates In eqn 20 the rate of evaporation or transpiration was expressed as an energy flux but more often we are interested in the amount of water used by a plant. The transpiration rate therefore tends to be expressed as either a mass or molar quantity. Molar units are to be preferred since they permit the direct comparison of the fluxes of different gas molecules (Cowan, 1977). The simplest method to calculate molar transpiration rates expresses the water vapour gradient as a mole fraction, and the flux then equals the mole fraction gradient multiplied by the leaf conductance.

3. Factors influencing the transpiration flux The mole fraction gradient (xs-xa) is a function of three parameters, the temperature and SVPD of the external atmosphere, and the temperature difference between the leaf surface and the air ( Ts-T,). Air temperature and SVPD are not influenced by wind speed but interact with the temperature difference term which is dependent on the energy balance of the leaf, and hence on wind speed. The change in (xs-xa) with increasing (TS-Ta) increases with temperature (Fig. 12) and thus the effect of wind on transpirational fluxes will increase with ambient temperature. The terms which are grouped into the leaf conductance parameter can be determined by considering the possible pathways for the movement of water vapour from the substomatal cavities, to the external atmosphere (Fig. 13). In Fig. 13 the pathways for water vapour are compared with those for heat and CO2. In the case of water vapour the leaf conductance comprises three terms; those associated with stomata, the cuticle (gee), and the boundary layer. The surface conductance equals the sum of two parallel conductances g,, and g,, and the leaf conductance equals the sum of the boundary layer conductance for water vapour (grca)and the surface conductance in series:

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P. VAN GARDINGEN AND J . GRACE 140

--120-

80 (a

1

60 70

I

-

(b)

T. = 30°C

L

1

I? I?

50-

- 40E

X

0-

0

-10.-

I

10

20

30 ("C)

40

50

-5

0

5

10

15

(&-&) ("C)

Fig. 12. Dependence of vapour pressure on temperature. (a) Saturated water vapour pressure in air above liquid water at a temperature of 0-50°C. (b) Vapour pressure difference between leaf and air as determined by the temperature difference between leaf and air.

The boundary layer conductance was discussed earlier in relation to heat transfer, and since there is a simple relationship between the conductances for heat and water vapour, the factors influencing g,, do not need to be reiterated. The two conductance considered here are those of stomatal and cuticular conductance. Of these parameters, under normal conditions g,, is much larger than g,, often by a factor of up to 100-1000, and hence most of the transfers occur via the stomatal pores. Stomata1 conductance is a function of the size, number and distribution of stomatal pores over the leaf surface, and several interacting environriiental parameters. Cuticular conductance is a function of the thickness, permeability and integrity of the leaf cuticle. Heat

5 Boundary layer Cuticle Epidermis

Mesophyll

Fig. 13. Resistances for gas and heat exchange at the surface of a single leaf ra, boundary layer; rs, stomatal; r,,, cuticular; r,, mesophyll or residual. The drawing on the right-hand side shows the pathways in relation to leaf structure. To convert resistance to conductance g, = l/r; for most leaves ra < rswcu,or g,, > g,pg,.

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22 1

F. STOMATAL CONDUCTANCE

Stomata represent the principal pathway for the diffusion of water and other gases between the leaf mesophyll and the external environment. Stomata have evolved a structure which can control the rates of gaseous mass transfer between plants and the atmosphere. Stomata1 conductance is an estimate of the limitation of mass transfer, with low conductances resulting in low rates of transfer. I . Variation in stornatal conductance Observations made over many years have shown that the aperture size of stomata vary in response to several environmental parameters. Change in the width of aperture is the principal cause of short-term changes in stomatal conductance. During the ontogeny of a leaf, stomatal conductance changes according to the number and size of pores which are capable of opening. In a fully expanded leaf, the number of pores which respond to the stimulus, and the maximum aperture which is obtained, determines the maximum value of gcs. Recent work suggests that stomata respond to stimuli as a population of individual apertures (Laisk et al., 1980; Terashima et al., 1988; Spence, 1987; van Gardingen et al., 1989). This hypothesis implies that the conductance is a function of the proportion of responsive stomata, the sensitivity and maximum aperture of each individual pore, as well as the magnitude of the stimulus.

2. Water use eficiency When considering the role of stomata in regulating transpirational water loss, it is important to recognize that any reduction in transpiration will also reduce the rate of photosynthesis. This is often called the “Paradox” of stomata and has led to the development of theories which propose that stomata have adaptations that will maximize the water use efficiency (WUE), the carbon gain per unit water loss, It has become fashionable to consider that plants have evolved mechanisms whereby stomata can sense environmental parameters and act to minimize WUE. These concepts are derived from the ‘feed-forward’’ hypothesis proposed by Farquhar (1978), who suggested that responses of stomata to atmospheric humidity are the result of the apparatus sensing the saturation vapour pressure deficit (SVPD) and closing to prevent water loss. This contrasts with previous explanations which suggested that stomatal closure with increasing SVPD was because of water loss and a subsequent reduction in turgor pressure in the guard cells (“feed-back” hypothesis). There is now much experimental data which can be interpreted to show the feed-forward response to SVPD, in a range of plant types (Losch and Tenhunen, 1981; Schulze and Hall, 1982). Feed-back mechanisms are, however, important for long-term

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responses to water status such as soil drying. Changes in stomatal conductance due to responses to SVPD or plant and soil water potential, depend on both the type of plant and the prevailing environmental conditions.

3. Responses to environmental parameters The response of stomata to light is important in maximizing the WUE of plants. Stomata tend to open with increasing light, which limits transpiration during periods of low light intensity and hence photosynthetic activity. The light response of stomata is dependent on plant type and environment. Stomata respond to a range of environmental parameters in a complex manner and the reader is referred to several recent reviews for further information (Jarvis and Mansfield, 1981; Hall, 1982; Schulze and Hall, 1982; Jones, 1983; Nobel, 1983; Schulze, 1986). 4. Wind and stomatal conductance The processes by which wind influences stomatal conductance is not well documented. In the few studies which have been conducted, stomatal conductance was found to decrease with increasing wind speed (reviewed by Dixon and Grace (1984)). It is now apparent that there is no direct effect of wind on stomatal conductance. Experimental results suggesting such a relationship can instead be explained by changes in the temperature or VPD

10

20

10

x , ~ . ,-~ X ,

20

10

20

( mPa pa-' )

Fig. 14. Leaf conductance to water vapour as a function of the mole fraction water vapour difference between the sites of evaporation in the leaf (xevap)and at the leaf surface (x5). Wind speed: (0) 3.0ms-I; (0)OSms-'. (a) Glycine mux; (b) Abutilon theophrusti; (c) Duturu strumonium. Reproduced with permission from Bunce (1985).

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at the leaf surface associated with changes in the boundary layer conductance. Grace et al. (1975) suggested that stomata might respond to changes in the water vapour pressure of the air at the leaf surface, which is a function of the ambient water vapour pressure, the boundary layer conductance, and the surface conductance of the leaf. This hypothesis was recently tested by Bunce (1985), who was able to demonstrate that changes in stornatal conductance with increasing wind speed (Fig. 14) could be totally explained by the increase in the vapour pressure gradient between the evaporation site and that calculated for the leaf surface.

G.

CUTICULAR CONDUCTANCE

The permeability of the cuticle to water vapour is described by the cuticular conductance. The cuticular conductance of most plant leaves is low in comparison to stornatal conductance and hence most transpiration occurs via the stornatal pathway. There are occasions, however, when the cuticular pathway increases in importance, either because of high cuticular conductance or, conversely, low stornatal conductance. Cuticular transpiration may represent the major pathway of water loss when stomata are closed, such as during the night or after a long period of drought.

I . The structure of the cuticle A continuous cuticular layer covers the epidermis of the aerial organs of most higher plants and ferns. The cuticle is tightly attached to the tangential walls of the epidermal cell and may extend partially onto the anticlinal walls and into part of the substomal cavities. Cuticles are heterogeneous lipid membranes with regions of different structure and properties. The bulk of the membrane consists of a polymer matrix with high water permeability. The permeability of cuticles to water vapour, and hence the cuticular conductance, is determined by a chloroform soluble fraction, which contains long hydrophobic chains that are unbranched and highly orientated. This fraction may only constitute a small fraction by weight (2-30%) of the total membrane mass. For more information on the structure of cuticle the reader is referred to the reviews by Juniper and Jeffree (1983), Pitcairn and Grace (1985), Schonherr (1982). 2 . Development of the cuticle The cuticle develops on the tangential walls of the epidermal cells as the young leaf grows and expands. The nature of the cuticle is species dependent, and is influenced by the environmental conditions during development. A leaf with poorly developed cuticle would have high cuticular conductance, and may have difficulty in controlling water loss. Inadequate development of the cuticle has been used to explain the formation of

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timberlines by Michaelis (1934). Michaelis suggested that new shoots of trees above a critical altitude did not mature sufficiently during the short alpine growing season. The hypothesis of Michaelis then postulated that cuticle development was inadequate to protect the new tissue from desiccation damage. The Michaelis hypothesis was restated by Wardle (1971) and other workers (e.g. Tranquillini, 1979) as part of a theory for the causes of timberline. The experimental evidence for this is by no means conclusive, as desiccation may be caused by cuticular abrasion as well as inadequate cuticular thickness (Grace, 1989). 3. Wind damage of the cuticle The cuticle may be damaged by abrasive particles carried by the wind, or by rubbing against other plant parts as the plant moves with the wind. Initially, damage is limited to disruption of the epicuticular waxes, but even this may have a major effect on the transpiration rate (Pitcairn et al., 1986). With more severe damage the epidermis may itself be breached, with a very large increase in the “cuticular” conductance (van Gardingen et al., 1991). H. SOLVING THE ENERGY BALANCE EQUATION FOR TRANSPIRATION AND SURFACE TEMPERATURE

The energy balance equation (eqn 20) is subject to complex interactions between plants and the physical environment. In order to illustrate the effect of these factors on surface leaf temperature and transpiration, eqn 20 has been solved using an iterative procedure for a range of environmental conditions. A simple Pascal computer program for this purpose is contained in Appendix 11. The results show the leaf temperature and transpiration rates predicted from the energy balance model, which in this simple simulation assumes that stornatal conductance is constant and does not respond to either light or SVPD. This is obviously an unrealistic constraint, but it is required so that the effect of wind speed can be shown. The effect of wind speed is mediated via the boundary layer conductance and increases with leaf size as would be predicted from Figure 10. The leaf model has been applied to four generalized environments contrasting from montane to arid. In all environments, four model leaves with characteristic dimensions (d,) ranging between 1 and 100 mm having a stornatal conductance of 500 mmol m-2 $,-1 are subjected to a range of wind speeds and radiant fluxes.

1. Montane environment A leaf in a montane environment experiences low ambient air temperatures, and often low values for the atmosheric saturation vapour pressure deficit (SVPD). The montane environment was characterized in this study as 5°C and 1.5 mPa Pa-’ SVPD. The leaf temperature of a small (1 mm) leaf was

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predicted to be close to that of the ambient air for all windspeed and radiation values (Fig. 15). Such a leaf can be said to be closely coupled to the atmosphere. As would be expected, the transpiration of this leaf (Fig. 16) also shows a minimal response to radiation and wind speed, but a slight increase is observed with radiation, and a decrease with increasing wind speed is observed with high net radiation. The boundary layer conductance decreases with increasing leaf size (Fig. 10) and the temperature difference (Fig. 15) and transpiration rate (Fig. 16) show a more pronounced effect of wind speed and radiation. It is apparent that with increasing wind speed the temperature difference tends towards zero, that is the leaf becomes more closely coupled to the atmosphere. Figure 15 shows that large leaves in low wind conditions may be several degrees colder than ambient during the night (R, negative), and nearly 10°C warmer on a sunny day. Figure 16 shows the effect of the temperature difference on transpiration, with the highest rates occurring with high leaf temperatures on large leaves in low wind speeds under high net radiation. 2. Temperate environment The temperate environment was defined as 15°C and 8mPa Pa-' SVPD. The model again shows that the 1mm leaf would be closely coupled to the atmosphere, but responds to wind and radiation more than in the montane environment because of the higher ambient temperature and SVPD (Fig. 17). Leaf temperature tends towards ambient with increasing windspeed, for all leaf dimensions, with the temperature differences becoming more pronounced with larger leaves, as g,, decreases. Transpiration rates increase with net radiation, and with wind speed at low values of R, (Fig. 18). With higher values of R,, transpiration decreases with increasing wind speed because of a leaf temperature effect. The explanation for the different wind response at high and low values of R, is related to the balance between convective and transpirational energy fluxes. When the leaf has a transpiration rate such that the transpiration energy flux exceeds the value of R,, the leaf temperature will be lower than ambient (from eqn 20). In a temperate environment, this occurs with low values of R,. In the situation when AE, is greater than R,, (T,-T,) is negative, and increases in wind speed results in (T,-T,) becoming less negative. The convective term C, however, becomes more negative with the increase in the value of g,,. The energy balance is maintained with an increase in the transpiration rate. A similar situation is observed when R, is greater than AE,. Increases in wind speed result in a reduction of ( T S - T a ) , that is it becomes less positive, but the reduction in gcaresults in an increase in C. Transpiration then decreases with wind speed, because of the reduction in the water vapour gradient between the leaf surface and the external atmosphere. These observations can be summarised in that the rate of convective transfers (Figs. 23 and 24) tends to increase with increasing wind

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P. VAN GARDINGEN AND J . GRACE d-lmm

d=lOmm

d = 50mm

d=100mm

Fig. 15. Temperature difference between the leaf surface and air for a range of leaf dimensions ( d ) in a montane environment. The figures show the effects of wind speed ( u ) and net radiation (R,) on ('T-TJ as calculated using an energy balance model (eqn 20). The environmental conditions were: air temperature 5°C. and SVPD 1.5 mPa Pa-'. The leaf stomatal conductance was 500mmol m-'s-'.

speed since the magnitude of the increase in gcais more important than the reduction in the absolute value of (T,- T a ) . 3. Tropical environment The response surfaces produced by the model are very similar to those for the temperate environment, but the effect of leaf temperature difference (Ts-Ta) is amplified by the,increase in ambient temperature (see Fig. 12). For this reason the rates of transpiration are higher than for the temperate environment, with appropriate changes in convective transfer depending on whether (Ts- Ta)is positive or negative (Figs. 19 and 20). In actual tropical environments, a SVPD of 8 mPa Pa-' might occur often in the rainy seasons. In the dry season, SVPDs of 40mPa Pa-' are likely. 4. Arid environment The arid environment (Figs. 21 and 22) is characterized by high temperature (40°C) and very high SVPD values (60 mPa Pa-'). The model predicts that

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Fig. 16. Transpiration rates for a range of leaf dimensions ( d ) in a montane environment. Other details are as in Fig. 15.

for all leaf sizes and combinations of windspeed and R, the leaf temperature will be less than ambient. For the 1and 10 mm leaves increases in wind speed resulted in an increase in leaf temperature as the boundary layer conductance increases. Leaf temperature increases with R, for all leaf dimensions. The 50 and 100mm leaves showed a decrease in leaf temperature with increasing wind speed which can be attributed to the convective transfer term becoming less negative. This shows that the two largest leaves were poorly coupled to the ambient atmosphere.

5. General trends The solution of the heat balance model for ( T s - T a ) , transpiration and convective transfer, has shown complex interactions between leaf temperature, boundary layer conductance and SVPD. It is, however, possible to summarize some of the predictions for the leaf model. Leaf temperature is closely coupled to ambient temperature in small leaves, and the temperature difference tends towards zero with increasing wind speed (Fig. 23). Large leaves (Fig. 24) are less well coupled to the atmosphere and may show an increase in the absolute magnitude of the temperature difference with

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P. VAN GARDINGEN AND J. GRACE d=lmm

d=10mm

d=bOmm

d=100mm

Fig. 17. Temperature differences between the leaf surface and air for a range of leaf dimensions ( d ) in a temperate environment. The figure shows the effects of windspeed ( u ) and net radiation ( R , ) on (Ts-Ta) as calculated using an energy balance model (eqn 20). The environmental conditions were: air temperature 15°C. and SVPD 8 mPaPa-'. The leaf stornatal conductance was 500 mmol m-2s-1.

increasing wind speed when the transpiration rate and R, are both high (e.g. Fig. 21). Transpiration rate increases with R , in all examples and tends to decrease with wind speed if ( Ts- T J is positive, and increase when ( Ts- T.,) is negative. 6. Effect of changing stornatal conductance A major deficiency of the model as presented in this current review, is that stomatal conductance is treated as a constant. In most plants, stomata respond to both light and VPD. Stomatal opening with increasing light would reduce the transpiration rate in the dark, amplifying the effect of R, on transpiration. The leaf temperature at low R, values would tend to be less negative, because of the reduced transpiration flux. Stomatal closure with increasing VPD (or leaf temperature difference via its effect on VPD) would reduce transpiration and increase leaf temperature. The effects of a range of values for stomatal conductance are shown for the temperate environment in Figs. 25 and 26. The principal effect of g,, is to

229

PLANTS A N D W I N D d=lmm

d = 10mm

d =50mm

d=100mm

Fig. 18. Transpiration rates for a range of leaf dimensions ( d ) in a temperate environment. Other details are in Fig. 17.

increase transpiration as the stomatal conductance increases (Fig. 20). The change in the rate of transpiration will then influence both leaf temperature and the convective heat flux. Increases in transpiration tend to decrease leaf temperature because of evaporative cooling. The energy balance is then maintained by the convective heat flux decreasing, but the absolute value of the flux increases if (T,- T,) is negative. The computer program contained in Appendix I1 can easily be altered to support a submodel for stomatal conductance. In the listing of the program a subroutine (PROCEDURE CALC-GS;) is provided which calculates the actual stomatal conductance from the maximum value for the leaf. The subroutine could be modified to include light and VPD responses which would then be calculated after each iteration to allow for changes in the leaf temperature. This approach has been used to illustrate the interactions between the physical process of heat and mass transfer and physiological adaptations of leaves (van Gardingen and Grace, 1989). 7. Problems of scaling up Difficulties arise when the results of such calculations are to be “scaled up” from the leaf level to the level of vegetation as a whole. Consider, for

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P. VAN GARDINGEN AND J. GRACE d=lrnrn

d = lOmm

d =50mm

d=100mm

Fig. 19. Temperature difference between the leaf surface and air for a range of leaf dimensions ( d ) in a tropical environment. The figure shows the effects of wind speed ( u ) and net radiation (R,) on (T,-T,) as calculated by an energy balance model (eqn 20). The environmental conditions were: air temperature 30”C, and SVPD 8mPaPa-I. The leaf stomata1 conductance was 500mmol m-’s-’.

example, the case of a glasshouse crop in which it is required to forecast the likely effect on transpiration or leaf temperature of increasing the air movement in the environment by turning on a fan. The calcuations are likely to predict adequately the initial effect of the increase in wind speed. Soon, however, the environment around the leaf will change as the water and heat are transferred between the leaf and the air. Quite soon the air will be more humid than at first and so the calculation based on the initial conditions will not apply. In short, feedback will occur. In the outdoor environment a similar process will apply, but because of mixing of the air near the plant with that in the vegetation as a whole, the effect is likely to be smaller. In short vegetation, where this mixing is not very thorough, a local microclimate will soon develop, so that a “prediction” made from consideration of a single leaf will not apply. To cope with this, an entire micrometeorological model would be needed, to “mix” the air near the plants with the air above the canopy and to recalculate the temperature and humidity of the air. This “mixing” is dependent on the wind speed and

231

PLANTS AND WIND d=lmm

d=lOmm

d=50mm

d=100mm

Fig. 20. Transpiration rates for a range of leaf dimensions ( d ) in a tropical environment. Other details are as in Fig. 17.

on structural parameters of the crop, particularly height (Monteith and Unsworth, 1990). On the landscape scale, an analogous problem will arise. Here, the change of state of the planetary boundary layer must be calculated. For a quantitative treatment of this problem the reader is referred to Jarvis and McNaughton (1986).

IV.

FACTS, FALLACIES AND MYSTERIES

The discussion in the preceding sections was concerned mainly with the conceptual and physical aspects of plants and wind. The biological responses to patterns of air flow (Section 11) are well documented but not well understood. Certainly, most biological responses to wind are insufficiently characterized to enable quantitative predictions to be made about the influence of shelter on plant growth or agronomic yield. This is not surprising, partly because of the many ways wind may influence biological processes (Table I) and, secondly, because a change in wind speed is necessarily accompanied by other microclimatological variations which may have a profound effect on the plant.

232

P. VAN GARDINGEN AND J . GRACE d=lmrn

d=lGrnm

d=bGmrn

d=100rnm

5a

Fig. 21. Temperature differences between the leaf surface and air for a range of leaf dimensions ( d ) in an arid environment. The figure shows the effects of windspeed ( u ) and net radiation (R,) on (T,- Ta) as calculated using an energy balance model (eqn 20). The environmental conditions were: air temperature 40°C. and SVPD 60mPaPac'. The leaf stornatal conductance was 500mmol m-2sc'.

A . THIGMOMORPHOGENESIS

In this field, more than in most fields of inquiry, physical and biological ideas are closely tied together. This is illustrated in the earliest paper we have found, published in the English Mechanic and World of Science in 1873. The author describes a not very well controlled experiment on an unnamed species:

. . . I selected two young shoots of a creeper belonging to the grape tribe which grow on a wall, apparently under the same condition for they were both ten and a-quarter inches long. I fastened one on the wall in three different places, and left the other swinging in the air. The sun never shone on the wall or ever near it all the year round, and it was always colder than the air; but when I measured them on the twelfth day, I found the one fastened to be three-quarters of an inch longer than the one swinging free. After that, I tried others, and found that they grew several feet longer in the course of the summer, and quite evident to the human observer (Schucht, 1873). The author (Schucht, 1873) had clearly discovered that wind exerted a

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Fig. 22. Transpiration rates for a range of leaf dimensions ( d ) in an arid environment. Other details are as in Fig. 21.

strong effect on morphogenesis, preceding by exactly 100 years the seminal paper o n thigmomorphogenesis published in Pluntu (Jaffe, 1973). An imaginative interpretation of the experiment was couched in strict biophysical terms (Schucht, 1873): The reason for the extra vigour in the one fastened on the wall, I think we may find in the following. By creating a higher pitch, we create in fact a higher temperature in the plant. The waves of a higher pitch have a more acute angle, and in consequence, have a greater power to forward the juice through the cells. (Schucht, 1873). Schucht may not have had a thermocouple to hand, or a means of measuring sap flow. Moreover, the explanation seems quite improbable, but are we any nearer now to explaining how plants detect mechanical perturbations and grow less tall when they are mechanically excited? The phenomenon is apparently ubiquitous, having been shown in over 50 species for more than 20 families. The response to shaking, flexure and rubbing, includes the production of the growth regulator ethylene, callose, and several of the so-called elicitors. This is very similar to the response to wounding (Erner et al., 1980). What is remarkable is that such a small

234

-

P. VAN GARDINGEN AND J. GRACE Temperate

Manlone

Arid

Tropical

550

250

'E

-s

o -250

-500 15

; .

-300

I

-600

c,

-900

-1200

5

Fig. 23. Sensible heat transfer by convection ( C )from a leaf with characteristic dimension ( d ) of 1 mm. The figure shows the effects of windspeed ( u ) and net radiation (R,) on C as calculated by an energy balance model (eqn 20). The environmental conditions were: montane, air temperature 5 ° C SVPD 1.5 mPaPa-'; temperate, air temperature W C , SVPD 8mPa Pa-'; tropical, air temperature 30"C, SVPD 8mPa Pa-'; arid, air temperature 40"C, SVPD 60 mPa Pa I . The leaf stomata1 conductance was 500 mmol m-? s- I . ~

stimulus can have a very large effect, as Nee1 and Harris (1971) demonstrated. Their Liquidamber plants were shaken for only 30s daily and extension growth was reduced to 20-30% of the unshaken controls. It is in woody plants that responses to mechanical perturbations are most highly developed, with the development of flexure wood and compression wood as described by Time11 (1986) and Telewski (1989). In Arabidopsis touch stimulates specific RNA transcription, and in this species we may be very near to elucidating the genetic control of a sequence of events which lead to the development of shorter petioles in plants which are touched, sprayed with water, or stimulated by wind. So far, five genes have been identified (Braam and Davis, 1990). Response to touch is well developed in a few species, but in their case not linked to morphogenesis. In Mimosa the leaves fold up when touched. The cells of the leaf transmit a message to pulvini at the base of each leaflet. When the message is received there is a flux of Kf which changes the osmotic

235

PLANTS AND WIND Temperate

Montane

Arid

Tropical

Fig. 24. Sensible heat transfer by convection ( C ) from a leaf with characteristic dimension ( d ) of 100mm.Other details are as in Fig. 23.

potential in such a way as to increase turgor and cause the leaf to fold (Allen, 1969). Clearly, there are several ways in which wind action could conceivably be sensed, and Mimosa type sensing is only one. Another would be to sense acceleration. Biological accelerometers do occur, in the ear for example. Something akin to them is believed to occur in the nodes of grasses, where the direction of the gravitational field is detected, and this may also occur in both shoots and roots, enabling negative and positive tropisms in relation to gravity. Perhaps this is also how plants detect wind, by sensing accelerations and/or displacement from the vertical. B . ABRASION

There is no doubt that abrasive damage commonly causes an increase in the surface conductance of leaves. Scanning electron microscopy reveals polishing of cuticular waxes and ruptured epidermal cells. The phenomenon is well documented in the case of the grass Festuca arundinacea (Grace,

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Fig. 25. Temperature difference between the leaf surface and air for a range of leaf stomata1 conductance (gcs)in a temperate environment. The figure shows the effect of wind speed ( u ) and net radiation (R,) on (T,- Ta) as calculated by an energy balance model (eqn 20). The environmental conditions were: air temperature 1 5 T , and SVPD 8 mPa Pa- *. The leaf dimension ( d ) was SO mm.

1974; Thompson, 1974; Pitcairn et al., 1986) and a general review is provided by Pitcairn and Grace (1985). There does appear to be a fundamental difference between species in the extent to which cuticle is affected by abrasion, apparently arising from differences in cuticular structure. In Trifolium leaves and Mulus fruits, gentle polishing increases the transpiration rate (Hall and Jones, 1961; Hall, 1966). It would seem that in these species the cuticular resistance largely resides in the epicuticular wax. In contrast, tough-leafed species such as Picea sitchensis are less affected by severe treatments in the wind tunnel, their epicuticular wax being apparently a small part of the total resistance to diffusio of gases (Grace et al., 1975; van Gardingen et al., 1991). More work is needed before we understand how the structure of the cuticle is related to the susceptibility of the plant to abrasive damage. Abrasive damage may disrupt the normal functioning of stomata by upsetting the turgor balance between the guard cells and the surrounding epidermal cells. Thus, stomata may fail to close at night, or open more

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Fig. 26. Transpiration rate of a leaf in a temperate environment with a range of leaf stornatal conductances. Other details are as in Fig. 25.

widely than is normal during the day. This is likely to cause water stress, and it may predispose the leaf to pollution stress. However, so far it has proved very difficult to know whether any increase in conductance is simply water loss from damaged epidermal cells or whether there really is an increased stornatal opening. Techniques which enable stornatal apertures to be precisely and unequivocally measured without having to peel the epidermis are required for this task and one possible approach was recently described by van Gardingen et al. (1989). C . ECOLOGICAL PHENOMENA

Wind patterns are often imprinted on vegetation as a whole and, although these have been described in some detail, the processes which cause them are largely obscure. Pehaps the most intriguing is the wave-regeneration pattern seen in natural forests. These are spatially repeating patterns of stand development and destruction, in which over a wavelength of 100m or so, a gradient of tree age is seen to culminate in a dieback zone of standing dead trees. These waves move as the trees age. Two mysteries surround

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TABLE I Processes or phenomena which ure wind-speed dependent Process or phenomenon

Key reference

Transpiration Swaying plants Deposition of rime ice Cloud water deposition Spore release Dispersal in the atmosphere Abrasion of epidermis Macroscopic damage to leaves Sand blast Interactions with nutrition Thigmomorphogenesis Strengthening of trees Flexure wood in trees Growth and partitioning Wind shaping of trees Wave regeneration patterns in vegetation Agronomic benefit of shelterbelts Tree lines Abrasion of tree crowns Catastrophic damage to natural forest Salt spray communities

Dixon and Grace (1984) Mayer (1987) Foster (1988a,b) Lovett and Reiners (1986) Grace and Collins (1976) Hanna (1982) Pitcairn and Grace (1985) Wilson (1980) Woodruff (1956) Jaffe (1973) Jacobs (1954), Larson (1965) Telewski (1989) Russell and Grace (1978) Holroyd (1970) Robertson (1987) Brandle et al. (1988) Grace (1989) Putz, Parker and Archibald (1984) Foster (1988a,b) Boyce (1954)

these waves. Firstly, their direction of travel with respect to the direction of the wind is in dispute. Most authors report that the wave moves in the same direction as the prevailing wind (e.g. Foster, 1988a,b). Robertson (1987), reporting on a case in Newfoundland found the waves moved perpendicular to the direction of the wind. He postulated the existence of large vortices with a diameter similar to those of the wavelength of the regeneration cycle (Fig. 27). Secondly, the mechanisms responsible for the forest dieback are obscure. Foster (1988a,b) considers that damage is done to the canopies by deposition of rime ice, and presents some data on photosynthetic capacity of the foliage, but these are not conclusive. Another suggestion is that sea-spray is the prime cause of the decline. The possibility of large roller-type vortices does, however, provide a clue to the development and maintenance of forest waves. The vortices might have originally occurred at irregularities in the forest. The downward sweeps of cold air might have created an unfavourable microclimate or could have brought salt-laden air to the canopy, so that the trees were retarded. At a distance of one-half wavelength, a sheltered zone between the rollers might occur, with a corresponding favourable microclimate and a more rapid tree growth. Another phenomenon which has been widely discussed in the popular press and attributed to vortices by some authors, is the appearance of

239

PLANTS A N D WIND Helical roll vortices

/-7

100rn

Fig. 27. Hypothetical helical roll vortices which, according to Robertson (1987). may account for wave-regeneration forests. Reproduced with permission from Robertson (1987).

circular areas of damage in cereal fields (Meadon, 1989). A sceptical view is that these areas are the result of a hoax intended to promote belief in the supernatural!

V.

CONCLUSIONS

The interaction between air flow and exchange processes in the canopy is currently being re-examined. This has been prompted by a general disillusionment with the classical technique for evaluating heat and mass transfer from micrometeorological profiles (the one-dimensional flux-gradient approach). Not only is the technique inapplicable to non-homogeneous vegetation and uneven terrains, but complete failure to yield correct results has been demonstrated for forests (where heat seems to flow against the temperature gradient) and in model stands in a wind tunnel. More recently, instruments with a very rapid response time enable heat and mass transfer to

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be measured using eddy-correlation, and furnish turbulence statistics of the u, v and w components of wind at different places in the canopy. Many problems involving wind in the canopy are more amenable to analysis by a theory which rests on the dispersal of materials in parcels of air rather than quasi-diffusion in one dimension. The behaviour of propagules in air, the mechanical excitation of tall plants and the optimal design of shelter are all examples of such problems. At the level of individual leaves, heat and mass transfer as well as temperatures, may be estimated from a description of the environment. A model to do this is presented to examine the effect of wind and speed on water use and surface temperatures under a range of conditions. The result is very sensitive to structural parameters of the leaf, its size and the integrity of the cuticle. Difficulties arise in “scaling up” the results from the leaf to the canopy, and from the canopy to the landscape. Physiological and ecological responses to wind are characterized. The stunting response of plant material to mechanical excitation was discovered over 100 years ago. Although documented in over 50 species, the basis of perception and response are not well understood. The abrasion of plant surfaces is also a common component in plant response to wind, and can result in excessive water loss.

REFERENCES Allen, L. H. (1968). Turbulence and wind speed spectra within a Japanese larch plantation. Journal of Applied Meteorology 7, 73-78. Allen, R. D. (1969). Mechanism of the seismonastic reaction in Mimosa pudica. Plunt Physiology 44, 1101-1107. Arkin, G. F. and Perrier, E. R. (1974). Vorticular air flow within an open row crop. Agricultural Meteorology 13, 359-374. Aslyng, H. C. (1958). Shelter and its effect on climate and water balance. Oikos 9, 282-310. Baldocchi, D. D. and Hutchinson, B. A. (1987). Turbulence in an almond orchard: vertical variations in turbulent statistics. Boundary-layer Meteorology 40, 127-146. Baldocchi, D. D., Verma, S. B. and Rosenberg, N. J. (1983). Characteristics of air flow above and within soybean canopies. Boundary-layer Meteorology 25, 43-52. Ball, M. C., Cowan, I. R. and Farquhar, G. D. (1988). Maintenance of leaf temperature and the optimisation of carbon gain in relation to water loss in a tropical mangrove forest. Australian Journal Plant Physiology 15, 263-276. Berry, J. A. and Raison, 5. K. (1981). In “Physiological Plant Ecology I , Encyclopedia of Plant Physiology” (0.L. Lange, P. S. Nobel, C. B . Osmund and H. Ziegler, eds), Vol. 12A, pp. 277-338. Springer-Verlag, Berlin. Biscoe, P. U., Clark, S. A., Gregson, K., McGowan, M . Montieth, J . L. and Scott, R. K. (1975). Barley and its environment. 1. Theory and practice. Journal of Applied Ecology 12, 227-257.

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Blevins. R. D. (1979). “Formulas for Natural Frequency and Mode Shape”. Van Nostraad. New York. Boyce, S. G. (1954). Salt-spray communities. Ecological Monographs 24, 29-67. Braam, J . and Davis. R. W. (1990). Rain. wind and touch-induced expression of calmodulin and calmodulin related genes. Cell 60, 352-364. Brandle, J. R.. Hinze. D. L. and Sturrock, J . W. (1988). “Windbreaks”. Elsevier, Amsterdam. Brown. H. W. and Rosenberg, N. J . (1972). Shelter effects on microclimate, growth and water use by irrigated sugar beets in the Great Plains. Agricultural Meteorology 9. 241-263. Buck. A . L. (1981). New equations for computing vapour pressure and enhancement factor. Journal of Applied Meteorology 20, 1527-1532. Bunce. J . A. (1985). Effect of boundary layer conductance on the response of stomata to humidity. Plant Cell Environment 8, 55-57. Campbell, G. S. (1981). Fundamentals of radiation and temperature relations. In “Physiological Plant Ecology I. Encyclopedia of Plant Physiology” (0. L. Lange, P. S. Nobel, C. B. Osmund and H. Ziegler, eds), Vol. 12A, pp. 11-40. Springer-Verlag. Berlin. Cannell. M . G. R. and Morgan, J. (1988). Youngs modulus of sections of living branches and tree trunks. Tree Physiology 3. 355-364. Carr, M. K. V. (1985). Some effects of shelter on the yield and water use of tea. Progress in Biometeorology 2, 127-144. Chatfield, C. (1984). “The Analysis of Time Series-An Introduction”, 3rd edn. Chapman and Hall, London. Cionco. R. M. (1965). A mathematical model for air flow in a vegetative canopy. Journal of Applied Meteorology 4, 517-522. Cionco. R. M. (1972). Intensity of turbulence within canopies with simple and complex roughness elements. Boundary-layer Meteorology 2, 456465. Coutts. M. P. (1983). Root architecture and tree stability. Plant Soil 71, 171-188. Coutts. M. P. (1986). Components of tree stability in Sitka spruce on peaty gley soil. Forestry 59. 173-197. Cowan. I . R. (1977). Stomata1 behaviour and environment. Advances in Botanical Research 4. 117-228. Crowther, J . M. and Hutchings, N . J . (1985). Correlated vertical wind speeds in a spruce canopy. In “The Forest-Atmosphere Interaction” (B. A. Hutchison and B. B. Hicks, eds), pp. 534-561. Reidel. Dordrecht. Denmead, 0. T. and Bradley, E. F. (1985). Flux gradient relationships in a forest canopy. In “The Forest-Atmosphere Interaction” (B. A. Hutchison and B. B. Hicks, eds). pp. 421-442. Reidel, Dordrecht. Dixon. M. (1982). ‘*Effectof Wind on the Transpiration of Young Trees”. Ph.D. Thesis. University of Edinburgh, Edinburgh, UK. Dixon. M . and Grace. J. (1983). Natural convection from leaves at realistic Grashof numbers. Plant Cell Environment 6. 665-670. Dixon. M. and Grace, J . (1984). The Effect of wind on the transpiration of young trees. Annals of Botany 53.81 1-819. Dyer. A . J. (1974). A review of flux-profile relationships. Boundary-layer Meteorology 7,363-372. Erner, Y.. Biro, R. and Jaffe, M. F. (1980). Thigmomorphogenesis: evidence for a translocatable thigmomorphogenetic factor induced by mechanical perturbation of beans (Phuseolus vulgaris). Physiology of Plants 50,21-25. Farquhar, G . D. (1978). Feed forward responses of stomata to humidity. Australian Journal of Plant Physiology 5 , 787-800.

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Finnigan, J. J. (1979a). Turbulence in waving wheat. I . Mean statistics and Honami. Boundary-layer Meteorology 16, 213-236. Finnigan, J. J. (1985). Turbulent transport in flexible plant communities. I n “The Forest-Atmosphere Interaction’’ (B. A. Hutchison and B. B. Hicks, eds), pp. 443480. Reidel, Dordrecht. Finnigan, J. J. and Mulhearn. P. J. (1978). Modelling waving crops in a wind tunnel. Boundary-layer Meteorology 14,253-277. Foster, D. R. (1988a). Species and stand response to catastrophic wind in central New England, USA. Journal of Ecology 76, 135-151. Foster, D. R. (1988b). The potential role of rime ice defoliation in tree mortality in wave-regenerated balsam fir forests. Journal of Ecology 76, 172-180. Fraser, A. I . and Gardiner, J . B. H. (1967). Rooting and stability in Sitka spruce. Bulletin of the Forestry Commission 40. Gaastra, P. (1959). Photosynthesis of crop plants as influenced by light, carbon dioxide, temperature and stomatal diffusion resistance. Mededelingen van de Landbou whoogeschool te Wageningen 59, 1-68. Garland, J. A . (1977). The dry deposition of sulphur dioxide to land and water surfaces. Proceedings of the Royal Society 354, 245-268. Garrett, J. R. (1977). Division of Atmospheric Physics, Technical Paper 29, 1-19. Gates, D. M. and Papian, L. E. (1971). “Atlas of Energy Budgets of Plant Leaves”. Academic Press, New York. Grace, J. (1974). The effect of wind on grasses I. Cuticular and stomatal transpiration. Journal of Experimental Botany 25, 542-555. Grace, J. (1977). “Plant Response to Wind”. Academic Press, London. Grace, J. (1978). The turbulent boundary layer over a flapping Populus leaf. Plant Cell and Environment 1, 35-38. Grace, J. (1981). Some effects of wind on plants. I n “Plants and Their Atmospheric Environment” (J. Grace, E. D. Ford and P. G. Jarvis, eds), pp. 31-56. Blackwell Scientific Publications, Oxford. Grace, J. (1983). “Plant-Atmosphere Relationships”. Chapman and Hall, London. Grace, J. (1985). The measurement of wind speed. I n “Instrumentation for Environmental Physiology” (B. Marshall and F. I. Woodward, eds), pp. 101-121. Cambridge University Press, Cambridge. Grace, J. (1989). Tree lines. Philosophical Transactions of the Royal Society of London Series B 324,233-245. Grace, J. and Collins, M. A. (1976). Spore liberation from leaves by wind. I n “Microbiology of Aerial Plant Surfaces” (C. H. Dickinson and T. F. Preece, eds), pp. 185-198. Academic Press, London. Grace, J. and Dixon, M. (1985). Convective heat transfer from leaves. Progress in Biorneteorology 2. 1-16. Grace, J. and Wilson, J . (1976). The boundary layer over a Populus leaf. Journal of Experimental Botany 27. 231-241. Grace, J . , Malcolm, D. C. and Bradbury, I. (1975). The effect of wind and humidity on leaf diffusive resistance in Sitka spruce seedlings. Journal of Applied Ecology 12. 931-940. Grace, J., Fasehun, F. E. and Dixon, M. (1980). Boundary layer conductance of some tropical trees. Plant Cell and Environment 3. 443-450. Gross, G. (1987). A numerical study of the air flow within and around a single tree. Boundary-layer Meteorology 40, 31 1-327. Hall, A. E . (1982). Mathematical models of plant water loss and plant water relations. I n “Physiological Plant Ecology 11, Encyclopedia of Plant Physio-

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243

logy“ (0.L. Lange, P. S. Nobel, C. B. Osmund and H. Ziegler, eds), Vol. 12B, pp. 231-262. Springer-Verlag. Berlin. Hall, D. M. (1966). A study of the surface wax deposits on apple fruit. Austrian Journal of Biologicul Science 19. 1017-1 102. Hall, D. M. and Jones, R. L. (1961). Physiological significance of surface wax on leaves. Nature 191, 95-96. Hamilton, G . J . and Christie, J. M. (1971). Forest management tables (metric). Forestry Commission Booklet 34. Hanna, S. R. (1982). Turbulent diffusion: chimneys and cooling towers. In “Engineering Meteorology” (E. Plate, ed.), pp. 429479. Elsevier, Amsterdam. Heisler. G. M. and DeWalle, D. R . (1988). Effect of windbreak structure on wind flow. I n “Windbreak Technology” (J. Brandle, M. Hinze and H. Sturrock, eds), pp. 41-70. Elsevier, Amsterdam. Holbo, H. R.. Corbett, T. C. and Horton. P. J. (1980). Acromechanical behaviour of selected Douglas-fir. Agricultural Meteorology 21, 81-9 1. Holland, M. R., Grace, J . and Hedley, C. L. (1991). Momentum absorption by dried-pea crops 11. Wind tunnel measurements of drag on isolated leaves and pods. Agricultural and Forest Meteorology 54. 81-93. Holroyd, E. W. (1970). Prevailing winds on Whiteface Mountain as indicated by flag trees. Forest Science 16, 222-229, Jacobs. A. F. G. and van Boxel, J . H. (1988). Computational parameter estimation for a maize crop. Boundary-layer Meteorologv 42, 265-279. Jacobs, M. R. (1954). The effect of wind sway on the form and development of Pinus radiata. Australian Journal of Botany 2. 35-5 1. Jaffe, M. J. (1973). Thigmomorphogenesis: the response of plant growth and development to mechanical stimulation. Planta 114, 143-157. Jarvis, P. G . (1981). Stornatal conductance, gaseous exchange and transpiration. In “Plants and Their Atmospheric Environment” (J. Grace, E. D. Ford and P. G. Jarvis, eds). pp. 175-204. Blackwell Scientific Publications, Oxford. Jarvis, P. G. and Mansfield, T. A. (1981). “Stornatal Physiology. Society for Experimental Biology Seminar Series No. 8”. Cambridge University Press, Cambridge. Jarvis, P. G. and McNaughton, K . G. (1986). Stornatal control of transpiration: scaling up from leaf to region. Advances in Ecological Research 15, 1 4 9 . Jones, H. G. (1583). “Plants and Microclimate“. Cambridge University Press, Cambridge. Juniper, B. E. and Jeffree. C. E. (1983). “Plant Surfaces”. Edward Arnold, London. Kappen, L. (1981). Ecological significance of resistance to high temperature. In “Physiological Plant Ecology I , Encyclopedia of Plant Physiology” (0. L. Lange, P. S. Nobel, C. B. Osmund and H. Ziegler, eds), Vol. 12A, pp. 439-474. Springer-Verlag, Berlin. Kreith, F. (1973). “Principles of Heat Transfer,” 3rd edn. Harper and Row, New York. Laisk, A . , Oja, V. and Kull. K. (1980). Statistical distribution of stomatal apertures of Vicia f a h a and Hordeum vulgare and the Spunnungsphase of stomatal opening. Journal of Experimental Botany 31, 45-58. Landsberg. J. J. and James, G. B. (1971). Wind profiles in plant communities: studies on an analytical model. Journal of Applied Ecology 8, 729-741. Larcher, W. and Bauer, H. (1981). Ecological significance of resistance to low temperature. In “Physiological Plant Ecology I, Encyclopedia of Plant Physiology” (0.L. Lange, P. S. Nobel, C. B. Osmund and H. Ziegler, eds), Vol. 12A, pp. 403438. Springer-Verlag, Berlin.

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Larson, P. R. (1965). Stem form of young Lariv as influenced by wind and pruning. Forest Science 11. 412-424. Legg. B. J., Long, I. F. and Zemroch. P. J. (1981). Aerodynamic properties of field beans and potato crops. Agricultural Meteorology 23, 21-43. Losch, R. and Tenhunen, J. D. (1981). Stomatal responses to humidity - phenomenon and mechanism. In “Stomatal Physiology” (P. G. Jarvis and T. A. Mansfield, eds), pp. 137-161. Cambridge University Press, Cambridge. Lovett. G. M. and Reiners. W. A. (1986). Canopy structure and cloud water deposition in subalpine forests. Tellus 38B, 319-327. Mayer, H. (1987). Wind-induced tree sways. Trees 1, 195-206. Mayhead, G. J. (1973). Sway periods of forest trees. Scottish Forests 27. 19-23. McAdams, W. H. (1954). “Heat Transmission”, 3rd edn. McGraw Hill. New York. McBean, G. M. (1968). An investigation of turbulence within the forest. Journalof Applied Meteorology 7,41&416. McNaughton, K. G. (1989). Micrometeorology of farm forests and shelter. Proceedings of the Royal Society of London B324, 351-368. Meaden, G. T. (1989). Weather 44, 2-10. Michaelis, P. (1934). Okologishe studien an der alpinen Baumgrenze V. Osmotischer wert und wassergehalt wahrend des winters in der verschiedenen Hohenlager. Jahrbuch fur Wissenschaftliche Botanik 80. 337-62. Milne. R. (1991). Tree Physiology, in press. Mitchell, J. W. (1976). Heat transfers from spheres and other animal forms. Biophysical Journal 16. 561-569. Monteith, J. L. (1965). Evaporation and environment. In “The State and Movement of Water in Living Organisms” (G. E. Fogg, ed.), pp. 205-234. Cambridge University Press. Cambridge. Montieth, J. L. (1973). “Principles of Environmental Physics”. Edward Arnold, London. Montieth,J. L. (1975). “VegetationandtheAtmosphere”,Vol. 1,Academicpress,London. Montieth,J. L. (1976). “Vegetationandthe Atmosphere”,Vol. 2. Academicpress,London. Montieth, J. L. (1981). Evaporation and surface temperature. Quarterly Journal of the Royal Meteorological Society 107, 1-27. Monteith. J. L. and Unsworth, M. H. (1990). “Principles of Environmental Physics”. Edward Arnold, London. Morgan, J. and Cannell, M. G . R. (1988). Structural analysis of tree trunks and branches: tapered cantilever beams subject to large deflections under complex loading. Tree Physiology 3, 365-374. Neel. P. L. and Harris, R. W. (1971). Motion-induced inhibition of elongation and induction of dormancy in Liquidamber. Science 173, 58-59, Nobel, P. S. (1983). “Biophysical Plant Physiology and Ecology”. W. H. Freeman, San Francisco. Oliver, H. R. (1975). Ventilation in a forest. Agricultural Meteorology 14, 347-355. Parlange, J. Y . , Waggoner, P. E . and Heichel, G. H. (1971). Boundary layer resistance and temperature distribution on still and flapping leaves. Plant Physiology 48, 437-442. Petty, J. A. and Worrell, R. (1981). Stability of coniferous tree stems in relation to damage by snow. Forestry 54, 115-128. Pitcairn, C. E. R. and Grace, J. (1985). Wind and surface damage. Progress in Biometeorology 2, 115-126. Pitcairn, C. E. R., Jeffree, C. E. and Grace, J. (1986). The influence of polishing and abrasion on the diffusive conductance of the leaf surface of Festuca arundinacea Schreb. Plant Cell and Environment 9, 191-196.

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Plate, E. J. (1971). The aerodynamics of shelter belts. Agricultural Meteorology 8. 202-222. Putz, F. E., Parker, G . G. and Archibald. R. M. (1984). Mechanical abrasion and intercrown spacing. American Midland Naturalist 112, 24-28. Raupach, M. R. (1979). Anomolies in flux gradient relationships over forests. Boundary-layer Meteorology 16, 467-486. Raupach, M. R. (1989). In “Plant Canopies: their Growth Form and Function” (G. Russell, B. Marshall and P. G. Jarvis, eds), pp. 41-61. Cambridge University Press, Cambridge. Raupach,M. R. and Legg,B. J. (1984). Theusesandlimitationsoffluxgradient relationships in micrometeorology. Agricultural Water Management 8, 119-131. Raupach. M. R., Thom, A. S. and Edward, I. (1980). A wind tunnel study of turbulent flow close to regularly arrayed rough surfaces. Boundary-layer Meteorology 18, 373-397. Robertson. A . (1987). The centroid of tree crowns as an indicator of abiotic processes in a balsam fir wave forest. Canadian Journal of Forestry Research 17, 746755. Ruck, B. and Schmitt, F. (1986). Das Stromungsfeld der Einzelbaumumstromung. Forstwissenschaftliches Centralblutt Hamburg 105, 178-196. Russell, G . and Grace, J. (1978). The effect of windspeed on the growth of grasses. Journal of Applied Ecology 16,507-514. Schonherr, J. (1982). Resistance of plant surfaces to water loss: transport properties of Cutin, Suberin and associated lipids. I n “Physiological Plant Ecology 11, Encyclopedia of Plant Physiology” (0.L. Lange. P. S. Nobel, C. B. Osmund and H . Ziegler, eds), Vol. 12B, pp. 153-180. Springer-Verlag, Berlin. Schucht, J . H. (1873). Vibration and its artificial effect on animals and vegetation. English Mechanic and World of Science 457, 7082. Schuepp, P. H. (1972). Studies of forced-convections, heat and mass transfer of fluttering realistic leaf models. Boundary-layer Meteorology 2, 263-274. Schuepp, P. H. (1973). Model experimentson free convection heat and mass transfer of leaves and plant elements. Boundary-layer Meteorology 3, 454-467. Schulze. E. D. (1986). Carbon dioxide and water vapour exchange in response to drought in the atmosphere and in the soil. Annual Reviews in Plant Physiology 37,247-274. Schulze, E. D. and Hall, A. E. (1982). Stomata1 responses, water loss and COz assimilation rates of plants in contrasting environments. In “Physiological Plant Ecology 11. Encyclopedia of Plant Physiology” (0.L. Lange, P. S. Nobel, C. B. Osmund and H. Ziegler, eds), Vol. 12B, pp. 181-230. SpringerVerlag, Berlin. Shaw. R. H., den Hartog, G., King, K. M. and Thurtell. G . W. (1974). Measurements of mean wind flow and three-dimensional turbulence within a mature corn canopy. Agricultural Meteorology 13. 419-425. Spence. R. D. (1987). The problem of variability in stomata1 responses, particularly aperture variance, to environmental and experimental conditions. New Phytologist 107, 303-315. Telewski, F. W. (1989). Thigmomorphogenesis: changes in the morphology and chemical composition induced by mechanical perturbation in 6-month-old Pinus taeda seedlings. Tree Physiology 5, 113-121. Terashima, I . , Wong, S. C., Osmond, B. and Farquhar, G. D. (1988). Characterisation of non-uniform photosynthesis induced by abscisic acid in leaves having different mesophyll anatomies. Plant Cell Physiology 29,385-394. Thom. A. S. (1968). The exchange of momentum, mass and heat between an

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artificial leaf and the air flow in a wind tunnel. Quarterly Journal of the Royal Meteorological Society 94, 44-55. Thorn, A . S. (1971). Momentum absorption by vegetation. Quarterly Journal of the Royal Meteorological Society 97, 414-428. Thorn, A. S. (1975). Momentum, mass and heat exchange of plant communities. In “Vegetation and the Atmosphere“ (J. L. Montieth. ed.). Vol. 1. pp. 57-105. Academic Press. London. Thorn. A . S . , Stewart. J . B., Oliver, H. R . and Gash, J. H. C. (1975). Comparison of aerodynamic and energy budget estimates of fluxes over a pine forest. Quarterly Journal of the Royal Meteorological Society 101, 93-105. Thompson, J. R. (1974). The effect of wind on grasses 11. Mechanical damage in Festuca arundinacea Schreb. Journal of Experimental Botany 25,965-972. Timell, T. W. (1986). “Compression Wood in Gymnosperms”, Vol. 2. SpringerVerlag. Berlin. Tranquillini. W. (1979). “Physiological Ecology of the Alpine Timberline”. Springer-Verlag. Berlin. Tritton, D. J. (1977). “Physical Fluid Dynamics”. van Nostrand Reinhold, Wokingham. Van Gardingen, P. R. and Grace, J. (1989). Surface temperature and transpiration rates of leaves. In “Proceedings of the 4th Australian Conference on Heat and Mass Transfer” (D. L. Evans, ed.). pp. 199-207. University of Canterbury, Christchurch, New Zealand. Van Gardingen, P. R., Jeffree, C. E. and Grace, J. (1989). Variation in stomatal aperture in leaves of Avena fatua L. observed by low-temperature scanning electron microscopy. Plant Cell and Environment 12, 887-897. Van Gardingen, P. R., Grace, J. and Jeffree, C. E. (1991). Abrasive damage by wind to the needle surfaces of Picea sitchensis (Bong.) Carr. and Pinus sylvestris L. Plant Cell and Environment 14, 185-193. Waldron. L. J. and Dakessian, S. (1981). Soil reinforcement by roots: calculations of increased soil shear resistance from root properties. Soil Science 132,427-435. Wardle, P. (1971). An explanation for alpine timberline. New Zealand Journal of Botany 9, 371-402. Welty, J. R., Wicks, C. F. and Wilson, R. E. (1969). “Fundamentals of Momentum, Heat and Mass Transfer”. Wiley, New York. White, R. G., White, M. F. and Mayhead. G. J. (1976). “Institute of Sound and Vibration Research Report 86”. University of Southampton, Southampton, UK. Wilson, C. E. and Crowther, J. M. (1985). Flow visualisation and the study of shelter effects for vegetation at the microscale. Progress in Biometeorology 2, 17-36. Wilson, J. (1980). Macroscopic features of wind damage to leaves of Acerpseudoplatanus L. and its relationship with season, leaf age and wind speed. Annuals of Botany 46. 303-31 1. Woodruff, N. P. (1956). Wind-blown soil abrasive injuries to winter wheat plants. Agronomy Journal 48,499-504.

APPENDIX I CONVERSION O F UNITS FOR MASS TRANSFERS

1. Resistance and conductance The use of resistances in both micrometeorology and stomatal physiology follow from the resistance analogies used to describe heat and mass transfers

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247

according to Ohm's and Fick's laws. Conductances are, however, becoming increasingly used since flux is directly proportional to conductance, whilst being inversely proportional to resistance. 1

S=r

When there are a number of resistances or conductances the algebraic sum of the components will depend on whether the fluxes are in series or parallel. The equation for parallel conductance or resistance is (1.2) and that for as series is (1.3). g \ u m = 61+ S?,

2. Conductance units The units for conductance in both micrometeorology and plant physiology until recently have been expressed as centimetres per second (cm s-I) (seconds per centimetre (s cm-') for resistance) or similar. Plant physiologists, however. increasingly tend to use molar units for conductance, commonly millimoles per square metre per second (mmol m-* s-') or moles per square metre per second (mol m-2 s-I). The main advantage of molar units is that flux and conductance can be expressed in the same units, a feature which simplifies the computation and understanding of gas exchange problems. If g' is a conductance expressed in centimetres per second (cm s-I) it can be computed from a molar conductance using eqn (1.4). It is worth noting that the conversion includes temperature and pressure terms, indicating that g' is dependent on these parameters.

where g' has units of centimetres per second (cm s-I), g has units of millimoles per square metre per second (mmol m-2 s-'), R is the universal gas constant, P, is the atmospheric pressure and Tis the temperature in Kelvin. For conditions of 20°C and Pa = 101.3kP, the conversion factor is 0.24.

3. Units ofpux There is only one unit which is routinely used for energy flux: that of Watts per square metre (W mP2). Unfortunately, a plethora of units have been used to describe mass flux. Plant physiologists have standardized on molar units for the reasons detailed above, whilst micrometeorologists base the

248

P. VAN GARDINGEN AND J. GRACE

units on the mass of gas. The most common unit for water vapour flux is that of grams of H 2 0 per square metre per second (g H20m-2s-'), however, many variations exist, the main difference being in the area and time terms. Occasionally one may encounter units which have been expressed on the basis of leaf dry weight, and as such are not strictly flux measurements. These can be converted to a unit-area basis by multiplying by the specific leaf area (the area per unit weight). To convert from molar to mass units of flux one simply multiplies by the relative molecular mass ( M , ) at the diffusing entity.

F* = M,F

(1.5)

where F' is the flux in grams per square metre per second (g m-2s-') and Fis the flux in moles per square metre per second (mol m-2 s-'). For water flux the conversion unit is thus 18.

APPENDIX I1 PASCAL COMPUTER PROGRAM FOR SOLVING THE ENERGY BALANCE EQUATION

This program was written for the Microsoft Pascal compiler. It should run under all versions of Pascal, but the line starting {$ real: 8)should be modified to select double precision reals numbers for other compilers. PROGRAM HEAT-BALANCE(INPUT,OLITPUT):

[sreal:8]

( Use Double precision Reals

1

const CP

= 29.3: ( Molal heat capacity of air ( J mol-1 K-1) = 44154.0: ( Molal heat of vaporisation of water ( J mol-1) d if €-const = 0.22: ( Diffusion coefficient of heat in air(cm2 5-11 } viscos = 0.15; ( Kinematic viscosity of dry air (cm2 s-1) gravity = 981; ( Acceleration due to gravity (cm s - 1 ) 1 therm-exp = 3.41297E-03: [ Coefficient of thermal expansion (K-1) 1 atmosqressure = 1013: ( Atmospheric pressure (Wa) 1

lamda

var t-amb, t-leaf, dt I start-d t , new-dt , old-dt, delta-dt, VPD, dw I

( ( ( ( ( ( [

Ambient temperature 1 Leaf temperature I Temperature difference (T-AMB - T-LEAF) First estimate of dt 1 New estimate of dt 1 Last estimate of dt 1 Change in dt (NEW-DT - OLD-DT)

1

[ Atmospheric saturation vapour pressure deficit] ( Mole fraction difference between leaf and air

1

249

PLANTS AND WIND [ [ ( [ [

9s-max I gs,

:

rs, ra-water , ra-heat, transp-heat, transp-moles, convect , wind-speed, dimension, Ne t-r ad, comp-cn, rn-error, last-rn-error Real;

( [ [

[ [ { ( [ [

Maximum Stomata1 conductance ) Actual stomata1 conductance ] Stomata1 resistance I Boundary layer resistance for water vapour 1 Boundary layer resistance € o r heat transfers 1 Transpiration rate expressed in W/m2 1 Transpiration rate expressed in mmol/m2/s Convective heat flux (W/m2) ) Wind speed ( m / s ) Characteristic leaf dimension (cm) 1 Net radiation (W/m2) 1 Sum of heat balance (TRANSP-HEAT + CONVECT) 1 Abs (NET-RAD COMP-RN) 1 Last value for RN-ERROR 1

-

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

PROCEDURE INITIALISE: ( INITIALISE 1 ( Start with the constants

BEGIN

1

wr i teln; writeln('P1ease enter environmental parameters'): )'=======:=====.=..=....=.............'(nletirw wc iteln: write('Atm0spheric VPD (mPa/Pa) >> ');readln(vpd): write('Ambient temp (C) >> ');readln(t-amb); >> '):readln(gs-max); write('qs (mmol/mZ/s) qs-max : = qs-max / 1000.0; [ Convert into mol m-1 5-1 1

.

write('Initia1 (Ts-ta) (C) >> '):readln(start-dt); >> '):readln(net-rad): write('Rn ( W / m Z ) write('Wind Speed ( m / s ) >> '):readln(wind_speed): write('Leaf Dimension ( m m ) >> ');readln(dimension); dimension : = dimension / 1 0 ; [ Convert into cm 1 dt := start-dt: END ;

[ INITIALISE

1

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

FUNCTION COMP-X(TEMP,VPD:REAL):REAL; [

Computes the mole fraction of air at temperature TEMP and saturation vapour pressure deEicit VPD. This Function uses Eormulae derived by Buck (1981) to calculate the vapour pressure of water in mBar and then converts this number into a mole fraction, by subtracting the VPD and then dividing by the atmospheric pressure (which is set a s a constant) 1 CONST A B C

= 6.1121; = 17.502; = 240.97:

VAK SVP

beg in svp : = a

: REAL:

[ COMP-X ) exp( b temp /(temp

comp-x := (svp end;

-

[ Saturation vapour pressure

c)); [ mbar 1 vpd) / atmos-pressure; [ Mole fraction [ COMP-X 1

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

t

1

I

250

P. VAN GARDINGEN A N D J. GRACE

FUNCTION CALC-GA(W1ND-SPEED,D:REAL):REAL; VAR free-ga, forced-ga. sum-ga

[ Free convection boundary layer conductance 1 [ Forced convection boundary layer conductance :

Sum of free and forced convection terms real;

FUNCTION PWR(A,B:REAL):REAL: Function t o return Ab

1

1

begin [ PWR I if (a = 0.0 ) then pwr := 0 else ~ W K:= exp(b*ln(a)); ( PWR I end; begin [ CALC-GA 1 wind-speed := wind-speed 100;

[ cm s - 1

1

( Compute forced and free convection terms for ga (cm s-1) 1 forced-ga := 0 . 6 6 * pwr(diEf-const,0.67) sqrt(wind-speed)/ (sqrt(d) * pwr(viscos.0.17) 1 ;

pwr(gravity,0.25) free-ga := 0 . 5 4 * pwr(diEE-const.0.75) pwr(Therm-exp,0.25) * pwr(abs(dt),0.25) / ( pwr(d.0.25) * pw~(viscos,O.25)); sum-ga := free_ga*2 + forced-ga; ( parallel sum of resistances ( Convert to m m o l / m ~ / s

1

CalC-ga := sum-ga atmosqressure / ( 8 . 3 1 4 end; [ CALC-GA 1

FUNCTION COMP-DT:REAL; VAR new-d t

:

real: New estimate for dt

[----------I FUNCTION SIGN-OF(X

:

REAL):INTEGER;

( SIGN-OF begin if ( x > = O ) then sign-of : = 1 else sign-of := -1; end; [ SIGN-OF [----------)

1

1

1

(t-amb

t 273

)):

25 1

PLANTS AND WIND ( COMP-DT 1 ( solve energy balance for DT

begin

new-dt := ( Net-cad - (lamda delta-dt := new-dt-old-dt: (

1

dw) / (Ra-water t r s ) )

Ra-heat / cp;

If the change in DT is greater than 0.5 C find out the direction of change and add 0 . 2 5 times the sign of DELTA-DT 1 if (abs(delta-dt) > 0.5 ) then SIGN-OF(delta-dt); new-dt : = old-dt t 0.25

(

Compute the new value of DELTA-DT and assign the value of NEW-DT to the function delta-dt := new-dt-old-dt: camp-dt := new-dt: end; ( COMP-DT

1

(===...?========....=I-...=:.=======..==============================~=======)

PROCEDURE NEW-PARAM: Const convert-ra (

= 0.93:

Constant used to convert the value of the boundary layer resistance f o r heat to that E O K water 1

{ Boundary layer conductance ) ( Boundary layer resistance ) : real;

begin t-leaf := t-amb [

( NEX-PARAM t

)

dt:

Compute the mole fraction diEference ( O W ) between the leaf and air ) dw :=

comp-x(t-leaf,O)

calc-gs; KS :=

1

/ 9s:

- comp_x(t-amb,vpd);

( New estimate of stomata1 conductance ( Stomata1 resistance J

ga : = calc-ga(wind-speed,dimension); r a : = 1 / ga: [ Boundary layer resistance (

1

Compute estimates for the energy transfers by convection and mass transfer of water vapour 1 convect :=cp * dt/(ra-heat): transp-heat : = lamda * dw/(ra-water+rs):

(

1

Compute the boundary layer resistances for heat and water vapour transfers. RA-HEAT is divided by two since transfers can OCCUK €rom b@th sides of the leaf. For water vapour transfers i t is assumed that the Leaf i s basistomatous 1 ra-heat := ra/2: ( allows f o r two sides ra-water : = ra convert-ra:

(

1

Compute the transpiration rate in moles

1

252

P. VAN GARDINGEN AND J . GRACE

transp-moles := transp-heat / lamda (

1000: (mmol/m2/s)

+

Compute the sum of the energy balance and work out the errnr between the computed estimate of net radiation with the actual value 1 comp-rn : = convect + transp-heat; rn-error := abs( comp-rn - Net-rad end: ( NEW-PARAM )

)

;

(.=.======:=========--=-.=-I--:----;===========================================]

PROCEDURE GET-TEMP: CONST max-outer

= 5000: ( Maximum number of iterations )

VAR outer-count fail-converge

(

: :

integer: boolean:

Main procedure which i s used to determine the temperature diEference at which energy balance is obtained. An estimate for DT is obtained by solving the energy balance equation for DT using the function COMP-DT. The new estimate oE DT is then used for the next ireration if i t reduces the size of RN-ERROR which is computed b y the procedure NEW-PARAM. The procedure then checks if the maximum number of iterations (MAX-OUTER) has been exceeded. If the new estimate o f DT doesn't reduce the error term then the procedure SPLIT-TEMP tries using a smaller change in DT. The procedure GET-TEMP will finish with one oE two conditions. If RN-ERROR is less than 0.05 W/m2, the procedure is considered to have converged. If the maximum number of iterarions is exceeded the flag FAIL-CONVERGE is set to true, and an error message is printed 1 (----------)

PROCEDURE SPLIT-TEMP; const max-inner

= 10;

var inner-count : integer; exceed-inner : boolean: ( SPLIT-TEMP 1 begin inner-count := 0; repeat

( ~ r ay smaller change in DT

1

delta-dt := delta-dt / 2 : dt := old-dt t delta-dt; [

Use NEW-PARAM to see calculate the new values oE the energy balance equation. newgaram; inner-count := inner-count + 1 ;

1

253

PLANTS AND WIND exceed-inner : = (inner-count>lO): until ((rn-error < last-rn-error) or exceed-inner end: ( SPLIT-TEMP )

):

(----------)

begin ( GET-TEMP ) outer-count : = 0: ( LOOP counter ) dt : = start-dt: ( starting estimate ) r.ew-pararn: ( Compute energy balance ) last-rn-error : = rn-error: fail-converge : = false: repeat old-dt : = dt: dt : = comp-dt: i Get new estimate ) new-param: if (rn-error < last-rn-error) then ( better est ) begin outer-count : = outer-count t 1: fail-converge : = (outer-count>max-outer]: end else split-temp: ( try a series of splits ) last-rn-error : = rn-error: until ((rn-error < 0.05) or fail-converge): if fail-converge then writeln(chr(7),'Failed to converge'): END: ( GET-TEMP ) .....................................................................

PROCEDURE PRINT-RESULTS: begin ( PRINT-RESULTS ) write1n:writeln: Writeln('Resu1ts ' ) : writeln('===:==='); writeln: writeln('(Ts-Ta) ',dt:8:2,' C'): mmol/mZ/s'); writeln('Et (mass flux)',transp-moles:8:2,' writeln('Et (heat flux)',transp_heat:8:2,' W/m2'); ',convect:8:2,' W/mZ'): writeln( ' C writeln: ( PRINT-RESULTS end:

1

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Fibre Optic Microprobes and Measurement of the Light Microenvironment Within Plant Tissues

THOMAS C. VOGELMANN, GREG MARTIN, GUOYING CHEN' AND DANIEL BUTTRY'

Botany Department, P. 0. Box 3165, Chemistry Department', University of Wyoming, Laramie, W Y 82071, USA

I. 11.

111.

IV. V. VI.

. .

. . . .

.

Optical Fibre , . . . . . , . . . . . . . . . A. General Characteristics of Optical Fibre . . B. Types of Optical Fibre , , . . . . , . . . C. Transmission Characteristics . . . . . . .

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

. . . .

Introduction

. . . . . . .

,

,

.

,

. . .

256

. . . .

Microprobe Fabrication . . . , , . . . . . . . . . . . A. Heating and Stretching Versus Chemical Etching. . . . . B. Sputter Coating followed by Truncation of the Probe Tip . . C. Grinding and Polishing the Probe Tip followed by Coating with Evaporated Metal . , . . . . . , . . . . . . . D. Measurement of Probe Sensitivity and Acceptance Angle . E. Factors that Affect the Optical Properties of Probes. . . .

. .

Experimental Apparatus Terminology

,

.

.

,

.

,

.

.

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. .

,

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. .

. . .

.

.

.

,

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,

,

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260 260 262

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263 266 268 270

,

.

.

Experimental Measurements . . . . . . . . . . . . . . . A. Effect of Probe Orientation on Light Measurements within Thick Samples , , . . . . . . . . . . . . . . . . B. Effect of Probe Acceptance Width on Light Measurements . C. Strongly versus Weakly Absorbed Wavelengths of Light . D. Isotropy of Scattered Light . . , . . . . . . . . . . . E. Tissue Effects . . . . . . . . . . . . . . . . . . . F. Signal Interpretation: Reality or Artifact? . . . . . . . .

Advances in Botanical Research Vol. I8 ISBN &12-005918-5

257 257 258 259

.

272 273 273 277 277 278 283 283

Copyright 01991Academic Press Limited All rights of reproduction in any form rescrved

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THOMAS C. VOGELMANN el a / .

VII.

Prognosis and Future Applications . . . . . . . . . . . . . .

289

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

292

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

293

Acknowledgements

I. INTRODUCTION Knowledge of the light regime that exists within plant tissues is necessary for an understanding of how photosynthesis occurs within intact leaves. It is also necessary for understanding how plant growth and development is controlled by a variety of light-mediated environmental cues. The optical properties of plants have been described in several reviews (Fukshansky , 1981; Osborne and Raven, 1986; Vogelmann, 1986,1989). From a physical standpoint, plants are extremely complicated optical systems and the light regime within their tissues is determined by a number of processes such as absorption, light scattering, and the focusing of light by epidermal cells. Although various mathematical approaches have been applied to describe the light environment within plant tissues, it is difficult to correct for all the optical phenomena and only simplified models have been developed (see e.g. Seyfried, 1989). Given this and the fact that optical properties vary widely among plants, it is desirable to be able to measure experimentally the light that exists within cells and tissues and to be able to quantify the amount of light, its spectral quality and direction of travel. Fibre optic microprobes have been developed with this goal in mind and numerous improvements have occurred since the technique was originally described (Vogelmann and Bjorn, 1984; Vogelmann e l al., 1988). The idea behind this technique is relatively simple: optical fibre can be heated and stretched to a fine tip which can be sealed optically to allow light entry only into the extreme tip. The resulting fibre optic microprobe is relatively small (ca. 2 pm diameter), durable and has high spatial resolution. The probe can be inserted into plant tissues and has been used to estimate the total amount of light, and to measure its spectral quality and the direction of travel within different tissues and organs (Vogelmann and Haupt, 1985; Bornman and Vogelmann, 1988; Martin et al., 1989; Vogelmann et al., 1989; Donahue et al., 1990; Cui etal., 1991). In addition, the probes have recently been used to measure the amount of chlorophyll fluorescence within leaves (Bornman et al., 1991). Whereas a few years ago resolution restricted use to relatively thick organs (1-4 mm), recent refinements have made it possible to measure the light environment within leaves only 100 pm thick (Vogelmann et al., 1989; Cui et al., 1991). Development of the improved spatial resolution of the probes has been accompanied by the task of interpreting signals and distinguishing between real phenomena and artifacts. Although used prima-

FIBRE OPTIC MICROPROBES AND MEASUREMENT

257

rily in plant tissues thus far, these probes could also be used in many other experimental systems where it is of interest to measure light at the microscopic level. In this review we summarize the current status of the fibre optic microprobe technique. We describe: (a) how the probes are fabricated, (b) associated instrumentation, (c) measurements obtained with the probe, and (d) special problems in signal interpretation.

11.

OPTICAL FIBRE

A . GENERAL CHARACTERISTICS OF OPTICAL FIBRE

A number of varieties of optical fibre are available (Elion and Elion, 1978; Lacy, 1982) and many have optical properties suitable for making fibre optic microprobes. Generally, optical fibre consists of a solid core that is surrounded by an outer cladding (Fig. 1). The fibre is usually protected by an outer buffer layer made of acrylate, silicone or some other polymer. The ability to guide light internally through an optical fibre depends upon a difference in refractive index ( n ) between the core and cladding. This difference is rather subtle: typical values for refractive index of fused silica fibre are 1.48 for the core and 1.46 for the cladding. Rays that enter a fibre at an angle below a certain angle (0) will be reflected between the core and cladding (Figs 1 and 2) and can be transmitted with relatively little loss in energy over long distances. In contrast, light that enters the fibre at angles greater than 0 will pass from the core to the cladding where it will be dissipated by absorption and scattering by the buffer interface (Fig. 1). The range of angles over which waveguiding occurs within an optical fibre determines the numerical aperture (NA) of the fibre.

Fig. 1 . Guiding of light through an optical fibre. Step index fibre is usually composed of a core and cladding which is enclosed in a protective buffer layer. Light is reflected between the core and cladding which have different refractive indices (n,and nz). As long as the rays of light are within a critical angle the light will be guided internally by total internal reflection. Rays that fall outside a critical angle will pass from the core into the cladding where they are dissipated by light scattering at the cladding-buffer interface.

25 8

THOMAS C. VOGELMANN

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Fig. 2. Acceptance angle of an optical fibre. The acceptance angle (0) is a measure of the total angle over which internal reflection will occur within the core.

This is related to the difference between the refractive index ( n ) of the core ( n l )and cladding (n2). NA=

G;

The relationship between numerical aperture and the acceptance angle (8; Fig. 2) of the fibre is given by the equation: 8 = sin-' NA

(2)

The total included acceptance angle (TIA) of the fibre is: TIA=2x8

(3)

B. TYPES OF OPTICAL FIBRE

Three different classes of optical fibre are commonly available: multimode step index, single mode and graded index fibre. Step index fibre consists of a core that has a uniform refractive index throughout (Fig. 3). Core diameters vary but, in general, the larger the core the more light the fibre can carry and the greater the sensitivity the fibre optic microprobe will have. In addition, a

Fig. 3. Types of optical fibre based upon different core-cladding configurations. (a) Multimode step index fibre. Step index fibre has a core of uniform refractive index. The large core allows internal reflection at many angles, thus allowing many modes of propagation through the fibre. (b) Single mode step index fibre. The small core restricts the number of modes of propagation. (c) Graded index fibre. The core consists of many layers in which there is a gradual change in refractive index so that rays of light tend to be confined within the inner region of the core.

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relatively large core (Fig. 3a) will be able to guide light that is reflected internally both at shallow and steep angles (multimode propagation) so that this fibre will have a larger acceptance angle than fibres with cores of smaller diameter. Although available in different sizes, single stranded optical fibre with an outer diameter of 125 prn is the most common and has sufficient rigidity for most experimental applications. The acceptance angle of probes made from multimode step index fibre can be controlled so that it is narrow (15") or broad (120", Section 1II.E). Multimode step index fibre is best for most experimental applications. In contrast to multimode fibre, single mode fibre (Fig. 3b) has a relatively narrow core (5-8p.m) in comparison to the diameter of the cladding (110 p.m). The narrow core diameter restricts the number of angles through which light can be reflected internally. Thus, these fibres have relatively narrow acceptance angles (ca. 5") and lower sensitivity to light. Probes made from single mode fibre are highly directional sensors. A third type of optical fibre is graded index in which there is a gradual decrease in refractive index as one progresses from the central to the outer regions of the core (Fig. 3c). These fibres are useful for telecommunication purposes but do not appear to have any special advantage when used to make fibre optic microprobes. C. TRANSMISSION CHARACTERISTICS

Most optical fibre consists of silica that is doped with various oxides such as G e 0 2 , B203or P 2 0 5which impart a specific refractive index to the core or cladding. These compounds also determine the melting point and in part the spectral transmission characteristics of the fibre (Wolf, 1979). Optical fibre that is made of borosilicate glass has a low melting temperature so that it is easy to heat and stretch the fibre using electrically heated wire coils. But the relatively lower transmission in comparison to fused silica tends to lessen its usefulness when used in microprobe applications. Fused silica fibre is more difficult to work with because it has a high melting point. However, the greater sensitivity of the resulting microprobe more than offsets the difficulties encountered during the heating and stretching process. Although most of the visible spectrum is transmitted well through fibre made of borosilicate glass, light below 450 nm is rapidly attenuated by the oxide dopants and by Rayleigh scattering (Fig. 4). Thus, the blue represents the lower boundary for measurements with a fibre optic probe and measurements within the ultraviolet are not possible. In contrast, fused silica fibre transmits well into the ultraviolet and throughout the biologically active UV-B region of the spectrum (Fig. 4). Above the visible range, there are strong absorption bands at 910, 1250 and 2100 nm due to absorption by hydroxyl groups within the glass so that fibres based on silicon dioxide lose much of their usefulness for applications

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in the infra-red (Izawa and Sudo, 1987). However, for this spectral region it is possible to use fibre made of more exotic materials such as fluoride or chalcogenide glass which have high transmission from 1000 to 5000 nm and 1000 to 11000nm, respectively.

111. MICROPROBE FABRICATION A . HEATING AND STRETCHING VERSUS CHEMICAL ETCHING

The major steps in probe fabrication are summarized in Fig. 5. These include creating a taper on one end of an optical fibre, truncating the extreme tip, and coating the tapered region with metal to seal it optically. The order of these steps can vary. In some cases it is preferable to create a taper, coat with metal and then truncate the probe tip, whereas in others the order is: stretch, truncate and then coat with metal. The merits and disadvantages of these sequences are described below. A taper can be created by heating and stretching, or by etching one end of the optical fibre in concentrated hydrofluoric acid. For multimode step index fibre (Fig. 6a), heating and stretching is the best method for creating a taper (Fig. 6b). Etching is unsuitable because it removes the cladding (Fig. 6c) which degrades the waveguiding properties of the fibre and the sensitivity of the fibre optic microprobe. First the protective buffer coat is removed with solvent (Table I). The fibre is then mounted vertically under tension by

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Fig. 5 . Major steps in the fabrication of a fibre optic microprobe. (a) Step index fibre is heated and stretched (b) to a fine point. The extreme tip is ground and polished (c). then the tapered region is coated with chromium (d). Coating with metal is necessary to prevent light entry into the fibre over the tapered region, thus degrading the spatial resolution of the probe.

I

f

Fig. 6 . Probes made by etching in comparison to heating and stretching. Heating and stretching a multimode step index fibre (a) preserves a favourable coreicladding ratio (b). Etching a multimode fibre in hydrofluoric acid can give a desirable taper but results in loss of cladding (c) which degrades the light guiding properties of the fibre. In contrast, heating and stretching a single mode fibre (d) is unsatisfactory because the reduced core diameter accepts very little light (e). Etching is a preferred route for fabrication of probes from single mode fibre because core diameter is maximized for light interception while maintaining enough of the cladding for adequate light guiding (f).

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TABLE I Composition of common buffer coatings on opticalfibre and ways to remove them ~

Buffer Acrylic Silicon Polyimide

Removal agent Methylene chloride" Sulphuric acid Nitric acid, heat

T h e buffer coat must be nicked with a razor blade prior to immersion in solvent.

attaching a mass of 100 g to one end. A small portion of the fibre is exposed to the intense heat from a hydrogen-oxygen jewellers torch. The torch is mounted on a motorized shaft that moves the flame horizontally across the fibre at an adjustable rate. The torch has interchangeable nozzles of different apertures for varying the flame size and the temperature can be adjusted by controlling the rate of gas flow to the torch. By adjusting flame size, torch travel rate, torch temperature, and the tension of the fibre, it is possible to stretch fibres to the desired tapers. This involves some trial and error but once the appropriate settings are achieved, many probes with nearly identical properties can be made. For single mode fibre, etching is preferable to heating and stretching. The latter is unsuitable because it shrinks the diameter of the core (Fig. 6 d,e) to dimensions similar to that of visible light itself. This decreases the sensitivity of the probe to vanishingly small levels. Chemical etching is done by touching one end of an optical fibre to the surface of a solution of hydrofluoric acid (48% vh). The acid creeps up the initial 1cm and dissolves the cladding but leaves the core intact (Fig. 6f) leaving a maximum core surface area for capture of light. The process creates a uniform taper and is usually complete within 30 min. The success of etching is often dependent upon the chemical composition of the optical fibre and some fibres etch better than others. Better probes may be obtained from some single mode fibres by heating and stretching the fibre and cutting the tip (see Section 1II.B) so that it is 20-50 p,m in diameter prior to etching. It is important to preserve a layer of cladding near the probe tip, otherwise the light guiding properties of the fibre will be degraded. B. SPUTTER COATING FOLLOWED BY TRUNCATION OF THE PROBE TIP

After creating a taper at one end of a fibre, the probes are coated with metal. This can be done via one of two different routes: sputter coating or vacuum evaporation. For sputter coating, it is possible to build a chamber so that 10 or more probes can be mounted at a time under a target of metal foil. It is not usually necessary to clean the fibres because the oxygen-hydrogen torch

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burns away surface contaminants. But it is necessary to coat the probes immediately after they are stretched; otherwise, contaminants will collect on the tapered region. The probes are placed under a platinum metal target and sputter coated so that a layer 10nm thick is deposited on the tapered region. Since a sputter coater produces an omnidirectional stream of metal particles. it is not necessary to rotate the probes during the coating process. One problem with sputter coating is that the metal targets are limited to those metals that have relatively low bonding energy such as gold or platinum. Gold is unsuitable because it is mildly hydrophobic and adheres poorly to glass. Platinum adheres more strongly to the probes but it is relatively soft (4.3 on the Moh hardness scale, where talc is 0 and diamond is 10) and it is eventually abraded away when the probe is used repeatedly. Harder metals such as chromium (9.0) can be deposited using an evaporation technique but this requires more specialized equipment (see Section 111.C . 3 ) . To allow light entry into the probe tip, the probes are mounted individually on a micromanipulator and the extreme tip truncated on the edge of a diamond knife. Although this procedure is simple, it takes practice to make probes with 3-5 pm tip diameters, a range necessary for measurements of light within leaves. Probe sensitivity to light and a uniform acceptance angle depend upon clean cleavage of the probe tip. A balance must be struck between creating a probe with the tip size and taper geometry required for penetration of plant tissue, and maintaining the sensitivity and acceptance characteristics needed for reliable light measurements. Unfortunately, the trial and error nature of this technique and the limited number of times a poor probe can be retruncated results in only one usable probe out of three. C. GRINDING AND POLISHING THE PROBE TIP FOLLOWED B Y COATING WITH EVAPORATED METAL

A second route of probe fabrication gives probes of higher quality but the procedure takes longer and requires more specialized equipment. After heating and stretching, the probe tips are ground and polished to a flat surface. Then the tapered region is coated with a unidirectional stream of evaporated chromium. The advantages are that grinding and polishing gives probes of exceptional optical quality and their acceptance functions and sensitivity to light are uniform and reproducible. In addition, metals of exceptional hardness such as chromium can be evaporated and deposited on the probes to create a durable coating.

I. Grinding und polishing of jibre optic microprobes After the fibres are pulled down to the desired taper, they are loaded vertically into hollow screws in a fibre holder (Fig. 7) so that the tapered

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Fig. 7. Holder for grinding and polishing fibre optic probes. Thirty or more probes are loaded into a hollow screw so that their tips protrude through an attached microgrid. The fibres are fixed in place with melted paraffin wax and the height of the screw adjusted so that the probe tips lie just beyond the final plane of polishing established by the square supports. The extreme end of the probes are ground by coarse and then successively finer grits until the probe diameters reach 5 pm.

ends protrude through the holes of a 1000 mesh electron microscopy grid (15 pm openings). The tails of the fibres are confined within a glass tube that is mounted vertically just above the holder. A stereo dissecting microscope aids the loading process. When all fibres are loaded, the heights of the screws are adjusted to the desired level. The finished probe diameter will correspond to the taper position intercepted by the plane defined by the supporting squares on the holder surface (Fig. 7). Paraffin wax is then used to immobilize the fibres and to fix the heights of the screws. A heat gun with a fine nozzle is useful for this procedure. After the wax hardens, the tails of the fibres are bundled, fastened to the tail guide and the glass tube removed. The fibre holder is then inverted and the pointed tips of the probes are fully embedded in paraffin wax.

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Initial grinding is done with No. 600-grit silicon carbide abrasive (Buhler Inc., Lake Bluff, IL. USA). About 0.5 g of the abrasive is mixed with water to make a slurry that is spread on a circular glass plate (diameter 8cm, thickness 0.64 cm) that is mounted horizontally on a grindedpolisher which rotates at 33 rpm. The fibre holder sits on the glass plate with its own weight and its horizontal position is loosely confined. Grinding is continued until the thickness of the paraffin layer that surrounds the probe tips is about 0.5mm. The glass plate and holder are then thoroughly flushed with tap water and a slurry of No. 1200 silicon carbide abrasive is added. This time, grinding is continued until the remaining thickness of the paraffin wax is about 0.2mm, and the scratches from the previous grit are removed. The next step is polishing and this is done in a similar manner to grinding, except that alumina micropolish is used as the abrasive; and a sheet of polishing cloth (Buhler, Lake Bluff, IL, USA) is attached to the glass plate to hold the abrasive slurry. In the first polishing step 1.O pm abrasive is used, followed by 0.3 pm grade. If exceptional surface quality is necessary, 0.05 pn abrasive must be used. During the polishing process, water must be added frequently to maintain adequate slurry viscosity. If the slurry turns dark, fresh abrasive is added. It is also critical to rotate the holder occasionally during the polishing, or parallel grooves will develop on the probe tips. Microscopic examination of the tip surface must be done periodically to decide when to switch to the next polishing step. At these times, the water flush must be done thoroughly, and the polishing plate must be replaced. Finally, it is important to avoid over polishing, since this creates a convex instead of flat probe surface. 2. Cleaning of fibre optic microprobes When examination shows that polishing is done, the fibre holder is flushed with tap water. Fibres are then removed from the screw wells by melting the wax with a heat gun. The paraffin is removed from the probes by immersing them in hot xylene and then in pentane. Sonication facilitates cleaning at both steps. This procedure is repeated until microscropic examination shows the probes to be free of dust and debris. The glass surfaces must be exceptionally clean; otherwise the metal will not adhere tightly and will flake off during use. A monomolecular layer of contaminant is sufficient to prevent adhesion of the chrome to the glass.

3. Coating with evaporated chromium Coating is done by mounting the probes so that they are tilted at 30" within the chamber of a metal evaporator (Model E306A, Edwards Coating System, Manor Royal, West Sussex, UK) which is evacuated to between 8 X lo-' and 8 x lO-'mtorr. The probes are located about 15 cm above a tungsten metal coil that contains a piece of chromium (ca. 70 mg) which is evaporated when the coil is electrically heated. The metal travels upwards

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Fig. 8. Coating a probe tip withchromium. The probes are positioned at approximately 30" within a metal evaporator and chromium cvaporated from a source below them. The chrome travels unidirectionally and is deposited upon the tapered region of the probe but not the polished tip which lies in a plane outside the direction of travel of the chromium.

unidirectionally and is deposited upon the tapered region of the probe but not the polished tip which is inclined away from the direction of travel of the chromium (Fig. 8). The probes must be rotated periodically and the coating procedure repeated so that all of the tapered region becomes covered with a chromium layer about 30 nm thick. Approximately 30 probes are coated at a time and the procedure requires an entire day. Probes which have been truncated on a diamond knife (see Section III.B), rather than polished may also be coated in this manner.

D. MEASUREMENT OF P R O B E SENSITIVITY A N D ACCEPTANCE A N G L E

Acceptance angles are measured for each probe while they are in air or water. This is necessary to understand the transitions in the light readings that occur as the probe passes from air into the plant tissue (see Section VI.F.1) and to estimate the total amount of light (see Section V) within the tissues. Acceptance angles are measured by attaching the probe to the end of a rod that is positioned within a beam of light that passes through the transparent wall of a tank that can be filled with water (Fig. 9). The rod is rotated through 360" and the amount of light that enters the probe is measured as it points at angles towards or away from the directional light source. Typically, probes have a slightly narrower acceptance angle in water than in air (Fig. 10) and they accept more light when they are in water. This

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FIBRE OPTIC MICROPROBES AND MEASUREMENT

optical fibre

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Fig. 9. Tank for measurement of acceptance angles of fibre optic probes. The tapered end of a fibre optic probe is attached to the end of a shaft of a variable speed stepping motor which extends into the interior of a tank which is filled with water. The walls of the tank are transparent so that directional light can be transmitted upwards through the bottom. The acceptance angle is measured by rotating the probe through 360" and measuring the amount of light that enters the fibre with a computerized detector. By raising and lowering the water level it is possible to measure the acceptance angle of the probe when it is in air or water.

1 .o

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60

100

(O)

Fig. 10. Acceptance angles of a fibre optic microprobe. (- - - -) Acceptance angle in air; (-)water.

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arises from the closer match in refractive index between the probe ( n = 1.45) and water ( n = 1.33) as opposed to air ( n = 1.00) so that there is less reflection of light from the surface of the probe tip. In addition, water reduces the amount of light scattering from surface inhomogeneities at the probe tip. The acceptance angle is expressed as the 50% acceptance half width which is the degree interval over which the probe accepts 50% or more of the light. For the probe in Fig. 10 the 50% acceptance half widths were 41" for air and 28" water, respectively. The acceptance width and the shape of the acceptance functions depend upon the type of optical fibre (Fig. 11). Single mode fibre gives probes with relatively narrow acceptance angles ( S O ) whereas multimode step index fibre gives a wide range (1CL120"). Each type of probe has sufficient sensitivity for most experimental applications. Probes made of step index fibre have acceptance functions that approximate a Gaussian distribution. This is used subsequently to estimate the total amount of light (internal fluence rate) within samples that scatter light intensely (see Section V).

E . FACTORS THAT AFFECT THE OPTICAL PROPERTIES OF PROBES

A number of factors affect probe sensitivity and acceptance angle. Sensitivity is determined largely by: (1) the optical quality of the probe tip

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Light direction (") Fig. 11. Range of acceptance widths of different microprobes. The probe with the narrow acceptance angle (-)was made of single mode optical fibre. The other probes were made from multimode step index fibre. The 50% acceptance widths were 6". 32" and 65".

FIBRE OPTIC MICROPROBES AND MEASUREMENT 0.201

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Acceptance width (50%) Fig. 12. The relationship between acceptance angle and probe sensitivity. (m) Measured probe sensitivity: ( 0 ) calculated sensitivity. Probe sensitivity was measured for probes of different acceptance widths by measuring the ratio of the amount of light that entered the probe from a diffuse light source to that from a collimated source. The calculated probe sensitivity data was derived by approximating the probe acceptance angle with a Gaussian distribution, and calculating the area under Gauss curves of different 50% widths. (-) Linear regression between the diffuse to collimated ratios and probe 50% acceptance widths.

surface, (2) the diameter of the probe tip, (3) the diameter of the core, and the (4) acceptance angle. Given two probes with different acceptance angles and equal sensitivity to collimated light, they will have different sensitivity to diffuse light. Even though peak sensitivity to collimated light is similar the probe with the narrower acceptance angle will capture less diffuse light because more light will fall outside the acceptance angle of the fibre. In fact, experimental measurements of probe sensitivity under diffuse light show a linear relationship between sensitivity and acceptance width (Fig. 12). All else being equal, doubling the acceptance width doubles the sensitivity. Probe acceptance width can be controlled to some extent and broad acceptance functions are potentiated by: (1)using fibre with a large diameter core. This favours many modes of propagation of light through the fibre;.(2) using fibre that has a relatively large difference in refractive index between the core and cladding. This allows internal reflection of more oblique rays of light between the core and cladding (see Section 1I.B); (3) creating a steep taper when heating and stretching the fibre. A steep taper favours the capture of oblique rays and translates their direction of travel within the optical fibre from angles that would normally allow the light to escape into the cladding to directions that are fully contained within the core (Fig. 13). By careful selection of the type of fibre and by controlling the taper at the probe tip it is possible to make probes that have total included acceptance angles that range from 5" to 120".

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Fig. 13. Increasing the acceptance angle by reflectioii of light within the probe tip. Heating and stretching optical fibre increases its acceptance angle. The greater acceptance is caused by the tapered tip which translates the direction of a ray of light from one that would normally enter the cladding and be lost (e.g. Fig. 1) to a direction that is confined to the core.

IV. EXPERIMENTAL APPARATUS The probe is threaded through the eye of a needle and glued in place with silver conducting paint (Ladd Research Industries, Burlington, VT, USA). The needle is clamped to the shaft of a horizontal stepping motor (Steppermike Model 18515, Oriel, Stratford, CT, USA) that has variable advance rates from 1.5to 1920 km SKI. The probe-motor assembly is mounted on the stage of an x , y , z translator so that the probe can be easily positioned at any point in space (Fig. 14). The opposite end of the optical fibre is mounted into the entrance port of a monochromator which contains a photomultiplier tube (Vogelmann and Bjorn, 1984).

M-PMT

-

SM

XYZ

-

SMC

-

Fig. 14. Diagram of the fibre optic microprobe system. Light issupplied by a 150or 1000 W xenon lamp (XL) and is directed towards a sample stage (SS) where a leaf can be clamped in place between two plastic coverslips through which there is a small hole (0.45 mm). The fibre optic probe (OF) is attached to the shaft of a high resolution stepping motor (SM) which is bolted to an x , y , z translator. Both the stepping motor and translator stage have 2 k m positioning resolution. Travel rate of the stepping motor is determined by a controller (SMC) which is activated by a computer (COM). Light that enters the fibre optic probe is measured by a photomultiplier tube (M-PMT) and the readings logged by a computer.

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The sample, such as a leaf, is clamped in place between two plastic coverslips, through which a small hole has been drilled, and irradiated with light from a xenon arc lamp (Hanovia 901C-1, l50W).The light is usually filtered through 5cm of water to remove excess heat. The light is easily controlled so that the sample can be irradiated with collimated, diffuse, white, or monochromatic light. The probe can be positioned at any orientation and advanced through the sample from the shaded towards the irradiated surface, or vice versa. As the probe travels through the sample, typically at 6 pm s-', light readings are taken at convenient intervals, such as at every 2 pm, by a computer which stores the values for later analysis. The probe can be positioned at the desired location near the sample surface by viewing with a microscope equipped with a long distance working lens (Model 101A, Gaertner Scientific Corp., Chicago, IL, USA). After activation of the stepper motor, the travel of the probe can be visually monitored as it approaches and enters the tissue. Although microscopic observation is adequate for some purposes, the small size of the probe tip makes it difficult to determine exactly when the probe enters the tissue. For exact determination of the entrance point, two leads from an electrical entrance indicator (Fig. 15) are connected to the apparatus; one lead is attached to the sample and the other to the needle which supports the probe. The circuit is closed as soon as the probe tip touches the sample which activates a light emitting diode. This system has an additional advantage in

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Fig. 15. Electronic entrance indicator. The circuit was based on an OPA128 Difet electrometer-grade operational amplifier. When used as a current-to-voltage converter, this operational amplifier provides excellent low level signal handling capabilities. As soon as the probe touches the leaf surface (L), a 3 V A.C. source creates an A.C. current through the contact point. This current flows through a feedback resistor (RZ).producing an A.C. voltage which, in turn, activates a light-emitting diode (LED). The A.C. source was used to keep the effects due to electrical polarization within the cells of the experimental sample t o a minimum.

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that the entrance indicator fails when the probe loses its chrome coating near the tip. Loss of this coating causes serious loss in spatial resolution of the probe so that the entrance indicator serves as a warning system indicating when it is necessary to change probes.

V. TERMINOLOGY Terminology and units for measurement of light have been described in detail elsewhere (Holmes, 1984; Vogelmann, 1986) and are summarized only briefly here. Whereas light is usually quantified as irradiance, which is measured with a flat cosine corrected sensor, this is inadequate for quantification of diffuse light that exists within biological samples where there is a high amount of light scattering. Diffuse light is more appropriately quantified by measuring the space irradiance (Grum and Becherer, 1979) or fluence rate (Rupert, 1974) which can be expressed as micromoles per square metre per second (pmol m-* s-l). This is the amount of light that strikes the surface of an imaginary sphere that has a unit cross-section. It is possible to measure the fluence rate using a spherical sensor, but this

0"

Fig. 16. Estimation of internal fluence rate within a light scattering sample from relative steric energy flux measurements. Pointing the probe at O", 30" and 150" measures the amount of light shown in the shaded circles on the surface of an imaginary sphere. The circles correspond to the 50% acceptance angle of the fibre optic probe. Internal fluence rate is defined as the amount of light striking the surface of a sphere with a unit cross-section. Internal fluence rate can be estimated by calculating the surface area based upon the individual relative steric energy flux measurements. For a sample irradiated with collimated light, most of the light that travels in the forward direction through the sample falls within the 0" measurement. The 30" measurement captures the most of the remainder of forward travelling light, whereas the 150" measurement is used to calculate the backscattered component. Integration is used to calculate swaths of 30" and 150" light as well as the remaining surface area.

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is not the case with a fibre optic probe which measures light only within a limited angle. However, such a probe can be used to estimate fluence rate by making measurements in representative directions (Fig. 16). What is measured can be represented by a small circle, one for each measurement, on the surface of an imaginary sphere. Values for the rest of the surface area can be estimated from these representative measurements by integration so that an approximation for the total surface area (fluence rate) is made (Vogelmann and Bjorn, 1984). In practice, each measurement with the fibre optic microprobe is corrected for the difference in probe sensitivity that exists between air and water and is standardized against the measurement of light incident upon the sample. This measurement is called the relative steric energy flux and is a dimensionless unit because it is a ratio of what is measured by the probe divided by the amount of incident light. Values for relative steric energy flux usually range between 1.0 and 0, but can be higher than 1.0 when there is significant trapping of light within the sample by light scattering or focusing of light by epidermal cells.

VI.

EXPERIMENTAL MEASUREMENTS

A. EFFECT OF PROBE ORIENTATION ON LIGHT MEASUREMENTS WITHIN THICK SAMPLES

Fibre optic microprobes are directional sensors and the light distribution curves within plant tissues will depend upon (1)orientation of the probe with respect to the light source, (2) the acceptance angle of the probe, and (3) optical properties of the tissues. A detailed understanding of these points allows reconstruction of the light gradient across plant tissues. Within etiolated tissues, the light distribution curves are determined largely by scattering and are not complicated by absorption of light by pigments. Using 7-day-old etiolated Cucurbitupepo cotyledons, the internal light fluxes at 0",30", 70", 110" and 150" (figure inserts in Figs 17-19) were measured with a fibre optic probe at 750 nm when the cotyledons were irradiated with collimated light. This wavelength was chosen because there is little, if any, absorption. The shapes of the curves were related to probe sampling orientation. There were also features that they had in common. First of all, entry into and exit from the cotyledon was usually marked by a transition in the light readings (arrows in Figs 17 and 18). These transitions arise in part from the fact that probes are more sensitive when they are in an aqueous medium (i.e. a plant cell) then when they are in air. In addition, plant tissues trap light so that internal fluence rates within the tissues may be substantially higher than outside. Light trapping will occur in any medium that has a higher refractive index than air and in which there is a

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Depth (pm) Fig. 17. Measurement of infra-red light travelling through an etiolated cotyledon of Cucurbita p e p . A cotyledon (7 days old) was irradiated with collimated white light with the adaxial surface facing the light. A fibre optic probe was inserted from the shaded surface and advanced directly through the cotyledon at 0" while measuring the amount of 750nm light. A peak in the light readings (arrow) marks the point at which the probe exited the irradiated surface.

large amount of light scattering. Under these conditions, internal fluence rates may approach four times the irradiance of incident light (Seyfried and Fukshansky, 1983). Contrary to common opinion, this is not: a violation of the law of conservation of energy. The phenomenon of light trapping within plants has been discussed previously in a number of articles (Seyfried and Fukshansky, 1983; Seyfried and Schafer, 1983; Vogelmann and Bjorn, 1984, 1986; Kaufmann and Hartmann, 1988) and is not described further here. The shape of the curve measured at 0" is a function of probe acceptance width and the absorption and scattering properties of the tissue (Section V1.B). Outside the cotyledon, most of the light was travelling initially at 0" because the light source was collimated. As this light entered the cotyledon, it was scattered and the direction of travel changed from 0" to other directions. Inserting the probe from the shaded surface of the cotyledon and advancing it towards the irradiated surface gives a measure of the light scattering properties of the tissues (Fig. 17). When the probe was near the irradiated surface of a cotyledon (150 pm, Fig. 17), much of the 750 nrn light was travelling undeflected at 0". Most of this light fell within the acceptance angle of the probe so that the light readings were relatively high in comparison to other locations within the cotyledon.

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Fig. 18. Measurement of the distribution of scattered infra-red light within Cucurbitupepo cotyledons. The probe was advanced through a cotyledon in orientations shown by the figure inserts. Conditions are similar to those described in Fig. 17. Arrows mark the point at which the probe entered or exited the irradiated surface of the cotyledon.

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Fig. 19. Measurement of infra-red light in Cucurbitu pep0 cotyledons with probes of different acceptance widths. (a) Light at 750 nm that travelled directly through the cotyledon (0") was measured by advancing the probe from the shaded toward the irradiated surface. Data were collected with probes whose acceptance widths are shown in Fig. 11: (- - - -) 6" (probe 50% acceptance width); (- - - - - -) 32"; (-) 65". (b) Measurement of scattered 750nm light with a probe with a 50% acceptance width of 32" (- - - - - -) and 65" (-).

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As the light moved further into the cotyledon it became more diffuse and was distributed more evenly over all possible directions. Thus, when the probe was near the shaded surface of the cotyledon (1500pm, Fig. 17) proportionally less light fell within the probe's acceptance angle so that the readings were much lower. Theoretically, there could be similar amounts of light at 150 pm and 1500 pm and the difference in the light readings could arise solely from the change in direction of the light within the tissues from 0" to other directions. Consequently, internal light fluxes must be measured at other orientations within the tissue to obtain a more complete picture of internal light distribution. By changing the orientation of the probe to 30", it is possible to measure the amount of light that travelled through the cotyledon in the forward direction at angles separate from 0" (Fig. 18a). This measurement is important because light may be scattered preferentially in forward directions by large particles (air spaces and organelles) within biological tissues (Latimer, 1982;Latimer and Noh, 1987). It is important to exclude light travelling at 0" and a probe within a sufficiently narrow 50% acceptance width must be used (i.e. 25" or less). At this sampling orientation, there was a near-linear increase in the amount of 750 nm light as the probe travelled from the shaded towards the irradiated surface of the cotyledon. Often, the light readings declined slightly as the probe approached the irradiated surface because a certain amount of tissue was required to scatter the 0" light to 30" where it could be measured by the probe. Often, there were anomalous peaks in the light readings near the irradiated surface that appear to result from the abrupt change in the direction of light from 0" to 30" by intercellular air spaces or other scattering inhomogeneities. Advancing the probe through the cotyledon at other orientations (70", 110" and 150"; Fig. 18b,c,d) gave similar results. In general, there was a linear relationship between relative steric energy flux and probe distance within the tissue. One consistent feature is that relative steric energy flux increased 10-20% immediately beneath the irradiated surface reaching a maximum near 170pm (see e.g. Fig. 18c). The reason for this peak well beneath the irradiated surface is that it takes up to 170 pm of tissue to scatter 0" light maximally to other orientations within the sample. This distance is comparable to the randomization of light measured at 0" (Fig. 17). Similar trends have been obtained using other thick plant organs such as the modified stems of Crassula (Vogelmann and Bjorn, 1984), Helianthus hypocotyls, and Phaseolus hypocotyls and epicotyls (Vogelmann, unpublished). It is interesting to note that this distance corresponds with the thickness of most leaves so that it is reasonable to expect that most light migration within leaves will have a predominant directional component in the forward direction. Light at 0" represents only a very small portion of the total solid angle and the linear decrease in light fluxes measured at 30", 70", 110" and 150"

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indicates that the light gradient at 750nm across an etiolated Cucurbitu cotyledon is linear or very close to it. The slope measured at 750 nm is within 10% of the calculated light gradient at 730 nm (Seyfried and Fukshansky, 1983). These calculations are based upon the Kubelka-Munk equations for propagation of light within intensely scattering media in which isotropic scattering occurs. Given the similarity between theoretical and experimental results for Cucurbitu cotyledons, it may be possible to determine the major features of the light gradients within thick samples by calculation and to corroborate theoretical and experimental results. Seyfried (1989) has discussed the applicability and limitations of calculating light gradients from optical parameters and has made a convincing case that more research is needed in this area. B. EFFECT OF PROBE ACCEPTANCE WIDTH ON LIGHT MEASUREMENTS

Since light gathering is proportional to the width of the acceptance angle of the probe, it is reasonable to expect that the shapes of the light distribution curves may show some dependence upon acceptance angle width. Comparing measurements obtained with probes that have 50% acceptance half widths of 6", 30" and 120" show that the shape of the light distribution curve at 0" is strongly affected (Fig. 19a) and the steepness of decline is proportional to acceptance width. For a probe with a 50% acceptance width of 6", the depth within the tissue at which relative steric energy flux decreased to 50% of its initial value was 30 pm. Corresponding decreases for probes with 50% acceptance widths of 30" and 120" were 45 and 200 pm, respectively. Thus, by using probes of different acceptance widths it is possible to measure the thickness of tissue required to scatter light out of a specific solid angle and obtain a measure of the scattering efficacy of the tissues. Measurement of scattered light at other sampling orientations (e.g. 150", Fig. 19b) showed that there was little effect of acceptance width upon the shape of the curves. Generally, probes with wider acceptance angles gave less noisy data because they collect more light and are thus less sensitive to local inhomogeneities of the radiation field within tissues. C. STRONGLY VERSUS WEAKLY ABSORBED WAVELENGTHS OF LIGHT

Using greened cotyledons and measuring the distribution of the more strongly absorbed 680 nm wavelength at similar orientations gave different curves. The light distribution curves for 0" showed that light decreased more rapidly in green than in etiolated tissues (Fig. 20a). This measurement is less useful than in etiolated tissue because it is dependent upon two processes, absorption and scattering instead of scattering alone. Thus, the rate of decline is more difficult to interpret. Measurement of scattered light at 150"

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2

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Fig. 20. Comparison between light distribution curves in etiolated versus green cotyledons of Cucurbitupepo. Cotyledons were 7-10 days old, and internal light was measured at 680 nm.

(Fig. 20b) showed a change from a linear distribution of light within etiolated tissues to an exponential decline within green tissues. Figure 20 shows normalized data to emphasize the differences in the shape of the light distribution curves but the relative amount of 680nm light measured in green tissues actually declined to a few percent of that found in etiolated tissue. This curve is similar to those obtained using other probe orientations that measure the distribution of scattered light (Knapp et al., 1988). D. ISOTROPY OF SCATTERED LIGHT

1. Thick samples Comparing relative steric energy flux measurements made at 0" and 150" within a Cucurbita cotyledon (Fig. 22) showed that approximately two times more light migrated through the cotyledon in the forward than backward direction. This is consistent with the interpretation that the fluxes of scattered light within the cotyledon may be relatively uniform (isotropic) but that there is a net migration of light toward the shaded surface. From other measurements (Fig. 18) it appears to take 150km or more of tissue to translate 0" light maximally to 150". These and other data suggest that the light is rather directional within the initial 150 krn of the cotyledon, but is relatively diffuse throughout the remainder of the cotyledon. It should be noted that these cotyledons have numerous storage bodies which appear to scatter light intensely (Fig. 21a). Measurements that confirm the relatively uniform distribution of scattered light are important because isotropic scattering is requisite for the calculation of light gradients using Kubelka-Munk theory (Seyfried, 1989). The application of this theory to Cucurbita cotyledons appears to be valid

Fig. 21. Leaf cross-sections. (a) Etiolated Cucurbitapepo cotyledon, 7 days old. (b) Sun leaf of Spinacia oleracea L. Samples were prepared by fixation, dehydration to 100% ethanol, freezing in liquid nitrogen and fracturing the sample. Upon thawing they were critical point dried. Scale bar = 100 pm.

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x

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Depth (pm) Fig. 22. Relative amount of light travelling through a cotyledon of Cucurbiru pep0 in the forward versus backward direction. The cotyledon was irradiated with collimated light incident upon the adaxial surface. Light at 750 nrn was measured when the probe was advanced through the cotyledon at 0" (forward direction (-)) versus 150" (backward direction (- - - -)).

but needs to be examined in more detail for other thick samples. Bjorn (personal communication) has recently examined photon migration in leaves using picosecond laser spectroscopy and has shown that light migrates preferentially in the forward direction even in leaves 4 mm thick. Scattering is not isotropic within thin leaves ( C 300 p,m thick) and the Kubelka-Munk theory does not appear to be valid for this case (see Section VI.D.2). 2. Thin samples Measurements of light scattering in thin samples such as leaves are influenced by several optical effects that arise at the cellular level. In this case, the internal light measurements are influenced by epidermal focusing, nonuniform distribution of chloroplasts, specular reflection within the intercellular air spaces and differences in the optical properties of adjacent tissues such as the mesophyll versus the veins. The contribution of these effects is more important to the overall light microenvironment in leaves than in thicker samples because of the finer scale. Scaling down from samples that are several millimetres in thickness to leaves (100400 p,m) demands improved spatial resolution and the ability to recognize these special optical effects as distinct signatures in the light measurements (see Sections V1.F. 1 and VI.F.2). Measurements in leaves of Medicugo sativa (Vogelmann et al., 1989) and Spinacia olearacea (Fig.2lb) (Cui e f ul., 1991) and Brassica (Bornman and Vogelmann, 1990) indicates that scattered light is forward oriented

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Depth (Pm) Fig. 23. Relative amount of light travelling through a sun leaf of Spinacia oleracea in different directions. The leaf was irradiated with collimated light incident upon the adaxial surface. A fibre optic microprobe with a 50% acceptance width of 26" was advanced through the leaf and 550nm light measured at O", 30" and 150". Adapted from Cui et al. (1991).

(Fig. 23). In spinach leaves the probe was oriented at 0",30" and 150" to compare the amount of light that was travelling through the leaf in the forward (0" and 30") and backward (150") directions. Approximately 20 times more light at 550 nm was measured at 0" than 30", and 50 times more than at 150" (Fig. 23). The probe used for these measurements had a 50% acceptance width of 26". Applying a weighting factor to the 30" and 150" relative steric energy flux measurements to estimate all the light that travelled in these directions (30" and 150" swaths in Fig. 16) indicates that all the light travelling at 17-43" (30" probe orientation) amounted to 45% of the light measured at 0". All the light within the total solid angle from 137" to 163" (150" probe orientation) was 35% of the amount of light at 0". Two conclusions are apparent. Although a spinach leaf is only 15-20 cells thick, it appears to scatter light intensely; and light migration through the leaf appears to occur primarily in the forward direction. This is even more apparent in leaves of Medicago sativa which are typically 150pm thick (Vogelmann et al., 1989). Integrating over the remainder of the solid angle to calculate the internal fluence rate indicates that near the irradiated surface there is up to 2.5 times more light within the leaf than outside of it (Fig. 24). This falls within the range of values estimated for leaves using light scattering equations derived from Kubelka and Munk for Cucurbita pep0 (Seyfried and Fukshansky, 1983; Seyfried and Schafer, 1983) and several other leaves using megsurements made with a spherical fibre optic sensor (Kaufmann and Hartmann, 1988). The distribution of specific wavelengths within the PAR have been measured in several leaves with a fibre optic microprobe and

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Depth (Pm) Fig. 24. Light gradients across a sun leaf of Spinacia oleracea. The leaves were irradiated with collimated light which was incident upon the adaxial surface. Most of the light was attenuated by the palisade layer which comprised the initial 150 pm of the leaf. Adapted from Cui et al. (1991).

there appear to be significant differences in light penetration based upon leaf anatomy and pigmentation (Bornman and Vogelmann, 1988, 1990; Cui et al., 1991; Vogelmann et al., 1989). Curiously, wavelengths that are most strongly absorbed by chlorophyll, e.g. 450 and 680 nm, are absorbed by 90% or more within the first few cell layers of a leaf and the ambient spectral environment within a leaf is dominated by green and long wave red light (see Vogelmann et al., 1989).

Fig. 25. Cross-section of an etiolated 5-day-old Helianthus annuus hypocotyl. Scale bar = 100pm.

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Plant tissues have different optical properties and, frequently, transitions in the light readings occur as the probe passes from one tissue type into another. These specialized signatures depend upon the anatomy of the particular plant organ. For example, in etiolated sunflower hypocotyls, the cells and intercellular air spaces are much smaller in the vascular cylinder than in the cortex (Fig. 25) and there were small but identifiable discontinuities in the light signal as the probe passed through the boundary between these tissues (Fig. 26a). These largest discontinuities occurred when scattered light was measured (Fig. 26b), probably because of more intense scattering within the vascular cylinder. Other signatures were observed in maize coleoptiles where there were a sharp transitions between tissue and air layers within the coleoptile (Vogelmann and Haupt, 1985). On a finer scale, discontinuities have also been observed when the probe passes between the epidermis and palisade of a leaf or when the leaf is infiltrated with oil and the probe passes between the palisade and spongy mesophyll (Donahue, unpublished results).

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Fig. 26. Distribution of scattered 550 nm light across a 5-day-old Helianthus annuus hypocotyl. (a) Measurement of scattered light that passed through the hypocotyl in a forward direction (SO') as opposed to the backward direction ((b) 130"). Arrows indicate the approximate point at which the probe passed between the cortex and vascular cylinder. In (a) fluctuations in the light readings near the irradiated surface were due to speckles of light caused by reflection between cells and the intercellular air spaces.

F.

SIGNAL INTERPRETATION: REALITY OR ARTIFACT

1 . Local changes in refractive index As with any other experimental technique, it is necessary to distinguish between reality and artifact. For example, as a probe travels through tissues, fluctuations in the light readings commonly occur. These changes could be

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caused by local inhomogeneities in the amount of light at the cellular level or, alternatively, they could be caused by local changes in refractive index which affects probe sensitivity. It appears more likely that these fluctuations are caused by the presence of light flecks in and between cells. In the case where fluctuations in light readings could occur as a result of change in refractive index, the maximum change would occur when the probe passes between an intercellular air space ( n = 1.00) and the cell cytoplasm (n = 1.33). Probes are typically 10% more sensitive when they are in water as opposed to air and their 50% acceptance width decreases by about 18% (e.g. water 26" and air 32"). In this case, calculations indicate that there would be an 11% difference in the light readings within the tissue each time the probe passed from cell to air space, or vice versa. In fact, fluctuations on this order of magnitude are more typically obtained when the probe enters or exits tissues. The relative absence of these changes within tissues suggests that probes remain wetted as they pass through the intercellular air spaces. Smaller fluctuations could reflect minor changes in local refractive index but it is difficult to separate these effects from others such as shading of the probe by chloroplasts or other organelles. The most extreme example of light reading fluctuations observed within a tissue to date involve measurements of forward transmitted light. For example, in etiolated sunflower hypocotyls large spikes in the light readings occurred when the probe approached the irradiated surface (Fig. 26a). These spikes appeared to be caused by specular reflection within the intercellular air spaces. These bright spots were readily apparent when the tissues were examined microscopically. The absence of these spikes near the shaded surface of the hypocotyl suggests that they were not caused by alternate wetting and drying of the probe as it travelled through the tissues. Additional evidence for the relative unimportance of local refractive index changes comes from measurements of scattered light within sunflower hypocotyls (Fig. 26b). The increased cellular density within the vascular cylinder and relative absence of intracellular air spaces should increase the average tissue refractive index. In turn this should cause narrowing of the probe acceptance angle and a decrease in the light readings as the probe passed from the cortex into the vascular cylinder. Instead, an increase in the light readings occurred (Fig. 26b) suggesting that light scattering within the vascular cylinder predominated over effects caused by refractive index.

2. Lens signatures Plant epidermal cells usually have a convex shape (Fig. 27) and they will act as lenses, focusing light within the underlying tissue. The focal plane of these cells varies but it is usually located 2-3 epidermal cell diameters below the leaf surface (Martin et al., 1991). Maximum focal intensifications range from 1-6 times incident irradiance (Martin etal., 1991; Poulson and Vogelmann, 1990). The epidermis appears to be important for distributing light to the

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Fig. 27. Epidermal cells on the adaxial leaf surface of Oxalis sp. Convexly shaped epidermal cells are typical of many plants and can focus light into the underlying tissue layers. This causes characteristic rises in the fibre optic probe measurements similar to that shown in Fig. 28. Scale bar = 100 pm.

Fig. 28. Measurement of epidermal focusing with a leaf of Medicago sativa. The leaf was positioned so that the adaxial surface faced a collimated light source and the probe advanced directly through the leaf from the shaded toward the irradiated surface. Light at 550nm was measured resulting in maximum values 60 prn beneath the irradiated surface. Irradition of the leaf with diffuse light or removal of the epidermal focal properties by coating the cells with mineral oil prevented occurrence light peaks within the palisade. Adapted from Martin et al. (1989).

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underlying chloroplasts (Martin et a l . , 1989; Poulson and Vogelmann, 1990). Although noted early in this century by Haberlandt (1914), the possible physiological consequences of epidermal focusing have only recently attracted attention (Bone ef al., 1985; Lee, 1986; Martin etal., 1989, 1991; Poulson and Vogelmann, 1990). Epidermal focusing causes characteristic patterns in the light readings where the amount of light rises as the probe approaches a focal point, usually located within the palisade, followed by a decline as the probe gets closer to the epidermis (Fig. 28). The shapes of these curves will vary depending upon the location of the probe with respect to the central axis of the focal point. Such patterns can be reproduced by advancing a probe towards a planoconvex glass lens (Fig. 29). The shapes of these curves (Fig. 30) vary as the probe is moved laterally away from the central axis of the lens because there is a complex interaction between the converging rays from the lens and the acceptance angle of the probe. The exact features of the light curves for plant epidermal lenses will also vary, depending upon the focal properties of the cell and the position of the probe with respect to the cell. These cellular lens signatures do not occur when the sample is irradiated with diffuse light, or the surface coated with a layer of water or immersion oil (Martin et a l . , 1989). A fibre optic microprobe has also been used to measure the focal intensification and azimuthal distribution of light around a cylindrical cell of a Phycomyces sporangiophore where the focusing of light by the cell plays an important role in phototropism (Dennison and Vogelmann, 1989).

3. False lens effects Measurements that are caused by epidermal focusing should not be confused with the artifacts that arise when the probe distorts the outermost cell wall when it exits into the air. Formation of a convexly shaped film over the tip of the probe will cause a spike in the light readings because this deformed cell wall focuses light into the probe. False lens artifacts have been observed only when the probes were excessively large (ca. 40 pm) or in samples that had a heavily cutinized epidermis.

Fig. 29. Approximate position of the probe with respect to the focal spot created by a plano-convex lens. Advancing the probe toward the lens resulted in the data shown in Fig. 30.

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00

Distance (kim)

Fig. 30. Amount of light measured by a fibre optic probe positioned at different locations within a focal spot created by a plano convex lens. (a-d) Progressive positioning of the probe closer to the centre of the focal spot where (a) was lateral to and (d) the centre of the focal spot. The probe was positioned at each location shown in Fig. 29 and advanced towards the lens so that the probe approached and passed through the region where light was concentrated. Maximum relative steric energy flux and the shape of the curve varied with probe position. Note the resemblance between b-d and the curve measured beneath an epidermal cell lens (Fig. 28).

These effects can be distinguished from legitimate epidermal lens effects by the position of the maximum light reading. Real epidermal lens effects exhibit maximum readings at locations corresponding to locations about a cell diameter or so beneath the epidermis, whereas false lens artifacts occur at the surface itself. In either case it is important to compare the light distribution measurements with cross-sections of the sample observed with a microscope. It is possible to obtain an estimate of tissue thickness from the light readings and this should correlate with actual measurements made ander a microscope. By comparing actual leaf thickness measurements with observed or recorded probe entrance points (see Section IV) it is possible to establish a correlation between light readings and probe position within the leaf. In the case where the probe encounters surface resistance when exiting the sample and creates a false lens artifact, there will be poor correlation between the true thickness of the sample and the exit point of the probe as indicated by the light distribution plot. 4. Surging Non-uniform progression of the probe through the sample, or surging artifacts, can also occur, especially when the sample is thin or the cells loose some of their turgor. The thinner the sample the greater the likelihood of encountering this artifact and extra care must be taken to securely mount the sample, especially when making measurements within leaves (ca. 125 km thick). Surging can occur during probe entry (Fig. 31), exit, o r when it is within the sample. Surging during entry is easily observed through a microscope but at other times is not so easily discerned. All forms of surging result in foreshortening of the light distribution measurements and cause abrupt changes in the light readings.

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"

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Depth (PW Fig. 31. Artifact caused by surging of the probe into a leaf of Medicago sativa. Light at 680 nm was measured in a control leaf (- - - -) in which the adaxial surface was irradiated with collimated light. The point of probe entry into the shaded surface (arrow) was identified directly by microscopy and indirectly by the leaf entrance indicator. In a second trial (-) the probe was observed to touch the epidermis, compress the tissue by about 35 pm, and then surge into the leaf at 100 pm depth. Two consequences were foreshortening of the light distribution profile and an abrupt transition in the light readings at 100 pm. The alternate rise and fall of both lines are caused by internal light flecks within the leaf. This fine structure results from epidermal focusing, an unequal distribution of chloroplasts, or reflection of light from an intercellular air space.

This artifact can also be recognized by comparing the distance travelled by the probe through the tissue with thickness measurements of cross-sections of the sample made with a microscope. The comparison is relatively easy toC make because the distance that the probe travels through the tissue is marked by transitions when the probe enters and exits the sample. Examination of the anatomy of the sample also shows an evaluation of whether abrupt transitions in the light readings are reasonably based upon cell size, content and organization. In some organs reproducible transitions in the light readings mark when the probe enters specific tissues (see e.g. Figs. 25 and 26) or air spaces (Vogelmann and Haupt, 1985). 5. Loss of coating from the probe tip

Any loss of metal coating at the tip seriously degrades the performance of the probe and results in loss of the high spatial resolution needed to measure light distribution within thin leaves. Under ideal conditions an individual probe can be used to collect 100 scans or more within a leaf, but the metal coating can flake away during prolonged use. Loss of coating can be recognized by several features in the light distribution measurements. When measuring 0" light (Fig. 32a), the probes appear to become more sensitive as coating is lost and light is able to enter the probe along the tapered flanks as well as the terminus. This also causes a loss of fine structure in the scans

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Fig. 32. Degradation of spatial resolution by loss of metal coating from the probe tip within a leaf of Medicagosariva. (a) Comparison of 450nm measurements taken with a probe with the metal coating intact (-) or removed (- - - -). (b) Comparison of similar measurements with the probe oriented at 150". The adaxial leaf surface was irradiated with collimated light. Loss of coating in the 0" sampling orientation had three effects upon the data. First, there was an apparent increase in probe sensitivity by about 10-fold, resulting from entry of light into all regions of the probe tip instead of the ground and polished terminus. The relative steric energy flux values were normalized to 1.0 for comparison of light distribution so that this difference is not apparent from the plot. Second, loss of coating resulted in the disappearance of fine structure caused by internal light flecks ((a) (-)) between 30 and 70 pm within the leaf. Third, measurements with a probe without adequate metal coating gave an erroneous overestimate of the distance light penetrated into a leaf. Loss of spatial resolution is especially apparent in measurements of scattered light (b).

which is detected only when the spatial resolution of the probe is high. Einally, such probes give an over-estimate of the distance that light penetrates into a leaf. When measuring the distribution of back-scattered light within samples (Fig. 32b), loss of coating flattens the scans. especially at a wavelength where there is strong absorption (e.g. 450 nm). Because the distribution of back-scattered light within the sample is weighted proportionally more when estimating internal fluence rates (see Section V1.D . 2 ) , degraded spatial resolution is especially serious. Although experience can aid in recognition of aberrant light profiles, loss of metal from the probe tip causes failure of the entrance indicator which can serve as an additional warning system. The indicator relies upon completion of an electric circuit when the probe touches the sample. This circuit is not completed when electrical contact is prevented by loss of metal coating, which can be verified by examining the probe under a microscope.

VII. PROGNOSIS AND FUTURE APPLICATIONS The worth of any new experimental technique is determined solely by the biological problems that it may help solve. Now that the probe fabrication

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procedures have been developed, a number of experimental problems that require light measurements with high spatial resolution at the cell and tissue level may be approached. Some possible applications and current areas of investigation are described below. In photomorphogenesis, light gradients are important for interpreting action spectra which can be distorted when light gradients vary with wavelength. Knowledge of light gradients can help reconcile the lack of correlation of an action spectrum with the absorption spectrum of a photoreceptor such as phytochrome. In many etiolated tissues, blue light is attenuated more rapidly than red or far red light. This leads to a relative loss of physiological activity in the blue. Although action spectra for phytochrome-mediated responses often show close correlation with phytochrome absorption spectra within the red and far red, they show poor correlation within the blue. This has been termed “blue blindness” and has been explained on the basis of the fact that blue light gradients are steeper than gradients in the red or far red (Kazarinova-Fukshansky et al., 1985). Light gradients may also be used to identify more precisely just which tissues participate in phytochrome-mediated responses. Germination of the small seeds of most native plants is often under some form of phytochrome control. Relatively little is known about light penetration through seed coats and into seeds or how their optical properties affect light activation of germination. Interestingly, the coats of Taraxacum and Lactuca seeds transmit more far red than red light (Widell and Vogelmann, 1985)which may predispose them to germination only in a light environment that is especially rich in red light. It remains to be seen whether this is a deliberate survival strategy or whether it is solely a consequence of th$ cellular construction of the fruitheed coat. It is possible to measure light penetration into imbibed seeds (Widell and Vogelmann, 1988) and this approach may be useful in studies that attempt to identify the photoactive site(s) that trigger germination. In fungi, lower and higher plants, phototropism is mediated by blue light. Yet little is known about blue light gradients across various organs, or which tissues respond to a unilateral blue light stimulus. The unicellular Phycomyces sporangiophore appears to perceive light direction by focusing it on the cell wall distal to the light source. Measurement of the azimuthal profile of light with a fibre optic microprobe showed that the sporangiophore can focus light two times over ambient levels (Dennison and Vogelmann, 1989). The blue light gradient has been measured across a maize coleoptile (Vogelmann and Haupt, 1985) and has been used to construct a model that attempts to explain observed phototropic behaviour (Iino, 1987). More research in this area is necessary to test the predictions of this and other models especially in view of the fact that some plant organs, such as leaves, are able to perceive directional light vectorially (Koller, 1990). Although there is much circumstantial evidence to suggest that light gradients are

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important for light perception in phototropism (Dennison, 1979; Donahue et al., 1990) it remains to be seen whether or not other plant organs are capable of perceiving vectorial light. If they are capable of doing so, then this may indicate only that the photoreceptor is membrane bound. O n the other hand, it could be a fundamental mechanism for perception of directional light, in which case the classical view of the role of light gradients in phototropism may have to be revised. Another intriguing research area concerns light propagation within leaves. These light-harvesting organs have been present in one form or another for the last 400 million years and it would be indeed surprising if there were not some optimization between leaf anatomy, light migration into the leaf, and photosynthesis. Yet, only recently have the optical properties of various leaf tissues been examined with respect to possible consequences for photosynthetic light-harvesting. With the exception of a few studies (Bone et al., 1985; Martin et al., 1989; Poulson and Vogelmann, 1990) seminal observations by Haberlandt (1914) that the epidermis of leaves can focus light within the underlying photosynthetic tissue have not been evaluated from a photosynthetic standpoint. Initial observations indicate that these cells may concentrate incident light two- to six-fold over incident levels of irradiance within the leaf (Martin et al., 1991) and that abolition of the lens properties of the epidermis decreases light-harvesting efficiency (Poulson and Vogelmann, 1980). More studies need to be done to examine how this cell layer can serve to channel light to areas of the mesophyll under different ambient light conditions. Progressing to the interior of a leaf, there has been much speculation over the functional role of the palisade layer. This layer of columnar cells tends to be more developed in sun leaves, grown under high light, than in their shade counterparts. A parallel development is that sun leaves are thicker than shade leaves which raises questions about the distribution of light energy in the two leaf types. Recent measurements with a fibre optic probe have shown that, despite similar chlorophyll content on a volume basis in the palisade of sun and shade leaves, light penetrated further into a sun leaf (Cui et al., 1991). Thus, the palisade may compensate for increased leaf thickness by propagating light deeper into the interior of the leaf. The spongy mesophyll may have a different optical role. In contrast to the palisade, this cell layer appears to scatter light more intensely (Terashima and Saeki, 1983) thus randomizing the direction of travel of light within the leaf. This not only increases the pathlength, maximizing the probability for absorption but also tends to trap light within the leaf, Light that arrives at the inner leaf surface from an oblique direction will be reflected back into the leaf interior by the difference in refractive index of the cell wall and air. Thus, whereas the palisade may control light penetration, the spongy mesophyll may serve to increase the light-trapping capacity of leaves. Understanding the details of light propagation within leaves will allow refinements

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THOMAS C. VOGELMANN ef al.

of models of reflection of radiation from canopies. Such models are necessary for intepreting remote sensing images (Myneni et al., 1989). Despite the fact that leaf photosynthetic tissues may play a critical role in the propagation and distribution of light within the leaf, very little attention has been directed towards evaluating the photosynthetic performance of leaves when light comes from different directions. Throughout the course of a normal day, when the sun is unobscured leaves are exposed to directional light, but they are exposed to diffuse light when it is cloudy or when leaves are within or under a canopy. The directionality of light will determine how it is propagated through the leaf tissues so that the light microenvironment within the leaf will be different when leaves are irradiated with collimated versus diffuse light. The relative lack of knowledge about light direction and structural control of light migration into leaves was pointed out by Osborne and Raven (1986). Although little has changed since that time, it is now possible to measure features of the light microenvironment within leaves so that research questions are approachable experimentally. Although it is clear that leaf tissues have unique optical properties that may be rather plastic according to their development under different growth conditions, chloroplast positioning and photosynthetic specialization may further optimize light absorption and utilization within a fully developed leaf. Indeed, it has been shown that chloroplasts appear to have more sun characteristics when they are located near the adaxial surface of a leaf and more shade characteristics when near the abaxial surface (Terashima and Inoue, 1984). This specialization, taken in combination with the light gradient across the leaf, may help explain the dorsiventral photosynthetic response curves of many leaves and the overall photosynthetic capacity ofLa leaf (Terashima, 1989). Finally, in addition to these and other research areas, mathematical modelling of light propagation through plant tissues is a necessary counterpart to the experimental measurements. Unfortunately, progress has been slow in the development of physical models so that there is little corroboration between scattering theory and measurement within tissues. Approaches such as the Kubelka-Munk theory, which may be valid for exceptionally thick tissues that scatter light intensely, may not be valid for thin samples. New approaches and ideas are clearly needed to integrate our understanding of how plant tissues interact with light, and to elucidate how plant optics may be merged with physiological processes.

ACKNOWLEDGEMENTS Thanks are extended to Sedley Josserand and David Myers for their helpful comments and editorial criticisms of the manuscript. This research was

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supported by grants from the National Science Foundation (R11-8610680, DCB-8908328 and DIR-9012729) and the US Department of Agriculture (86-CRCR-1-2048).

REFERENCES Bone, R. A., Lee, D. W. and Norman, J. M. (1985). Epidermal cells functioning as lenses in leaves of tropical rain-forest shade plants. Applied Optics 24, 14081412. Bornman, J. F. and Vogelmann, T. C. (1988). Penetration of blue and UV radiation measured by fiber optics in spruce and fir needles. Physiologia Plantarum 72, 699-705. Bornman, J. F. and Vogelmann, T. C. (1991). Effect of UV-B radiation on leaf optical properties measured with fiber optics. Journal of Experimental Botany 42, 547-554. Bornman, J. F., Vogelmann, T. C. and Martin, G. (1991). Measurement of chlorophyll fluorescence within leaves with a fiber optic microprobe. Plant, Cell and Environment in press. Cul, M., Smith, W. K. and Vogelmann, T. C. (1991). Chlorophyll and light gradients in sun and shade leaves of Spinacia oleracea. Plant, Cell and Environment in press. Donahue, R. A., Berg, V. S. and Vogelmann, T. C. (1990). Assessment of the blue light gradient in soybean pulvini as the leaf orientation signal. Physiologia Plantarum 79, 593-598. Dennison, D. S. and Vogelmann, T. C. (1989). Intensity profiles in Phycomyces sporiangiophores: Measurement with a fiber optic probe. Plantu 179, 1-10, Elion, G. R. and Elion, H. A. (1978). In “Fiber Optics in Communications Systems” (H. Elion, ed.), pp. 7-66. M. Dekker, New York. Fukshansky, L. (1981). Optical properties of plants. In “Plants and the Daylight Spectrum” (H. Smith, ed.), pp. 2140. Academic Press, London. Grum, F. and Becherer, R. J. (1979). In “Optical Radiation Measurements” pp. 14-15. Academic Press, New York. Haberlandt, G. (1914). In “Physiological Plant Anatomy”, 4th edn, pp. 613-630. Macmillan, London. Holmes, M. G. (1984). Radiation measurement. In “Techniques in Photomorphogenesis” (H. Smith and M. G. Holmes, eds), pp. 81-87. Academic Press, London. Iino, M. (1987). Kinetic modelling of phototropism in maize coleoptiles. Planta 171, 110-126. Izawa, T. and Sudo, S. (1987). In “Optical Fibers: Materials and Fabrication” (T. Okoshi, ed.), pp. 1-49. KTK Scientific Publishers, Tokyo. Kaufmann, W. F. and Hartmann, K. M. (1988). Internal brightness of disk-shaped samples. Journal of Photochemistry and Photobiology 1, 337-360. Kazarinova-Fukshansky, N., Seyfried, M. and Shafer, E. (1985). Distortion of action spectra in photomorphogenesis by light gradients within the plant tissue. Photochemistry and Photobiology 41, 689-702. Knapp, A. K., Vogelmann, T. C., McClean, T. M. and Smith, W. K. (1988). Light and chlorophyll gradients within Cucurbita cotyledons. Plant, Cell and Environment 11, 257-263. Koller, D. (1990). Light-driven leaf movements. Plant, Cell and Environment 13, 615-632.

294

THOMAS C. VOGELMANN ef al.

Lacy, E. A. (1982). “Fiber Optics” Prentice-Hall, New Jersey. Latimer, P. (1982). Light scattering and absorption as methods of studying cell population parameters. Annual Review of Biophysics and Bioengineering 11, 129-150. Latimer, P. and Noh, S. J. (1987). Light propagation in moderately dense particle systems: a re-examination of the Kubleka-Munk theory. Applied Optics 26, 514-523. Lee, D. W. (1986). Unusual strategiesof light absorption in rain-forest herbs. In “On the Economy of Plant Form and Function” (T. J. Givnish, ed.), pp. 105-126. Cambridge University Press, Cambridge, UK. Martin, G., Vogelmann, T. C. and Josserand, S. (1989). Epidermal focussing and the light microenvironment within leaves of Medicago sativa. Physiologia Plantarum 76, 485-492. Martin, G., Myers, D. A. and Vogelmann, T. C. (1991). Characterization of plant epidermal lens effects by a surface replica technique. Journal of Experimental Botany 42,581-587. Myneni, R. B., Ross, J. and Asrar, G . (1989). A review on the theory of photon transport in leaf canopies. Agriculture and Forest Meteorology 45, 1-153. Osborne, B. A. and Raven, J. A. (1986). Light absorption by plants and its implications for photosynthesis. Biological Reviews 61, 1-61. Poulson, M. E. and Vogelmann, T. C. (1990). Epidermal focussing and photosynthetic light-harvesting in leaves of Oxalis. Plant, Cell and Environment 13, 803-81 1. Rupert, C. S. (1974). Dosimetric concepts in photobiology. Photochemistry and Photobiology 20, 203-212. Seyfried, M. and Fukshansky, L. (1983). Light gradients in plant tissue. Applied Optics 22, 1402-1408. Seyfried, M. and Schafer, E. (1983). Changes in the optical properties of cotyledons of Cucurbitapepo during the first seven days of development. Plant, Cell and Environment 6, 633-640. Seyfried, M. (1989). Optical radiation interactions with living tissue. In “Radiatic& Measurement in Photobiology” (B. L. Diffy, ed.), pp. 191-223. Academic Press, London. Terashima, I. (1989). Productive structure of a leaf. In “Photosynthesis: Proceedings of the C. S. French Symposium” (W. Briggs, ed.), pp. 207-212. A. R. Liss, New York. Terashima, I. and Inoue, Y. (1984). Comparative photosynthetic properties of palisade tissue chloroplasts and spongy tissue chloroplasts of Camellia japonica L.: Functional adjustment of the photosynthetic apparatus to light environment within a leaf. Plant Cell Physiology. 25, 555-563. Terashima, I . and Saeki, T. (1983). Light environment within a leaf. I. Optical properties of paradermal sections of Camellia leaves with special reference to differences in the optical properties of palisade and spongy tissues. Plant Cell Physiology 24,1493-1501. Vogelmann, T. C. (1986). Light within the plant. In “Photomorphogenesis in Plants” (R. E. Kendrick and G. H. M. Kronenberg, eds), pp. 307-337. Nijhoff and Junk, Wageningen. Vogelmann, T. C. (1989). Penetration of light into plants. Photochemistry and Photobiology 50, 895-902. Vogelmann, T. C. and Bjorn, L. 0. (1984). Measurement of light gradients and spectral regime in plant tissue with a fiber optic probe. Physiologia Plantarum 60. 361-368.

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Vogelmann, T. C. and Bjorn, L. 0. (1986). Plant as light traps. Physiologia Plantarum 68, 704-708. Vogelmann, T. C., Bornman, J . F. and Josserand, S. (1989). Photosynthetic light gradients and spectral regime within leaves of Medicago sativa. Proceedings of the Philosophical Transactions of the Royal Society of London 323, 411-421. Vogelmann, T. C., Knapp, A. K . , McClean, T. M. and Smith, W. K. (1988). Measurement of light within thin plant tissues with fiber optic microprobes. Physiologia Plantarum. 72, 623-630. Vogelmann, T. C. and Haupt, W. (1985). The blue light gradient in unilaterally irradiated maize coleoptiles: measurement with a fiber optic probe. Photochemistry and Photobiology. 41, 569-576. Widell, K. 0. and Vogelmann, T. C. (1985). Optical properties of Lactuca and Taraxacum seed and fruit coats: Their role as light filters. Physiologia Plantarum 64, 34-40, Widell, K . 0. and Vogelmann, T. C. (1988). Fiber opticstudiesof light gradients and spectral regime within Lactuca sativa achenes. Physiologia Plantarum 72, 706712. Wolf, H. F. (1979). In “Handbook of Fiber Optics: Theory and Applications” (H. Wolf, ed.), pp. 43-152. Garland STPM Press, New York.

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AUTHOR INDEX

A Aarnes, H.. 111, 119 Adams, R.M., 49, 51, 68, 105 Adams, W.W.. 19. 20,105 Addison, P.A., 9, 13, 22, 117 Agrawal. M., 88. 96, 106 Agrawal, S.B., 105 Aiga, I . , 120 Al-Johore, A.. 175 Albe, K.R., I71 Alexander, K., 118 Allen, L.H., 199. 204,235,240 Allen, O.B., 120 Allen, R.D., 240 Alscher, R., 11, 18, 19. 24, 106, 108, 109 Alscher-Herman, R., 122 Ames, G.F.-L.. 13, 165 Aihiro, B.D., 59. 61, 106 Amthor, J.S., 54. 68, 106 Amundson, R.G.. 13,24. 45,47, 54, 63,73,76, 81, 90, 100, 106, 109, 111,116, 122 Anbazhagan, M., 88,93,94, I06 Anderson, L.E., 20,106. 122 Anderson, M.P.. 155. 165, 170 Anderson, W.C., 62,106 Andreeva, N., 139, 165 Anthon, G.E., 142, 165 Antoniw, L.D., 141. 165 Antoun, H., 149, 165, 174 Aoki, K., 85, 120 Appels, M.A., 144, 165, 187 Appleby, C.A., 152, 161, 165,167, I85 Archibald, R.M., 234,245 Argillier, C., 160, 165 Arias, A., 150, 166, 168 Arima, Y . , I75 Arkin, G.F., 204.240 Arndt, U., von 56, 59,63, 66,106,115, 116,124

Arwas, R., 147, 150, 166, 169. 170 Ashenden, T.W., 6, 13,30. 45, 48, 90, 101. 106 Ashmore. M.R.. 13, 74, 75, 76, 102, 106,127 Asrar, G., 294 Assmann, S.M., 26,106 Astwood, P.M.. 179 Asylyng, H.C., 218,240 Atherly, A.G., 180 Atkins, C.A., 143, 155, 156, 158, 159, 161, 166, 168, 177, 178, 181 Atkinson, C.J., 16, 20, 24. 27. 29, 33, 57,107 Ausubel, F.M., 169 Avissar, Y . , 175 Awonaike, L.O., 154,166 Ayazloo, M., 33,107 Aycock. M.K., Jr., 121

B Baer, C.H.. 125 Baeten, H., I I O Baker, C.K., 4, 118 Bal, A.K., 137, 161, 166,180, 184 Baldocchi, D.D., 198,203, 204,240 Ball, E., 117 Ball, M.C., 217. 240 Banks, J.M., 178 Barnes, D.K., 184 Barnes, J.D., I10 Barnes, R.L., 52, 55, 61,63, 64,65, 67, 68, 107 Barton, J.R., 11, 18, 23,107 Bassarab, S., 138, 166,175,185 Bauer, F., 103,107 Bauer, H., 217,243 Bauer, K., 140,166 Bazzaz, F.A., 31,109 Becherer, R.J., 272,293 Becker, R.R., 168, 175

297

298

AUTHOR INDEX

Beckerson, D.W., 5, 14, 15, 63, 72, 73, 76,107 Been, C 167 Bell, J.N.B., 15,33,49,107,126,127 Belot, Y., 4, 11, 22, 24, 109 Bender, J., 124 Bengtson, C., 112, 125 Benhamou, N . , I74 Bennett, H., 95,116,166 Bennett, J.H., 10, 17,21,35, 36, 38, 46,47, 61,77,79, 107,113, 127 Bennett, M.J., 154, 167 Benny, A.G., 155,167 Benoit, L.F., 89,99,107,126 Benz, R., 140,166 Berg, V.S., 293 Bergersen, F.J., 132, 134, 151, 152, 154, 161,166, 167, 171,177, 185 Bergmann, F., 89, 100.107,124 Berndt, W.B., 175 Bernhard, L., 108 Berry, J.A., 217,240 Besford, R.T., 40, 107 Beversdorf. W., 118 Bhagwat, K.A., 106 Bialobok, S . , 87, 91, 120 Biggs, A.R., 9, 16, 22,24, 31, 107, 108 Bingham, G.E., 51, 66, 69, 110 Birch, J.B., 127 Birkenhead, K.. 148, 167, 176 Biro, R., 241 Birot, A.M., 183 Biscoe, P.V., 9, 22, 108, 126, 197,240 Bisseling, T., 138, 139, 167 Bjorn, L.O., 256,270,273, 274, 276, 280,294,295 Black, C.R., 10,22, 108 Black, V.J., 9, 10, 17, 18, 20, 22, 23, 24,26,70, 108,114, 120 Blank, L.W., 102,108.124 Bleuler, P., 126 Blevins, D.G., 167, 175, 178, 183 Blevins, R.D., 208,241 Blumwald, E., 137, 138, 167 Bodley, F., I79 Body, D.E., 113 Boesten, B., 167 Bogner, J.C., 116 Boland, M.J., 155, 156, 158,167, 174, 175,178 Bolton, E., 147,167 Bone, R.A., 286,293 Bonte, C., I08

Bonte, J., 11, 22, 23,24, 108 Booth, J.A., 125 Bordeleau, L.M., 165 Bornman, J.F., 256, 280, 282,293,295 Bostrom, C.-A., I25 Botkin, D.B., 53, 108 Botsford, J.L., I74 Bouet, C . , 172 Bower, J.L., 106 Boyce, S . G . ,234,241 Boyer, J.N., 76,108 Boylan, K.L.M., 184 Braam, J., 236,241 Bradbury, I . , 242 Bradley, D.J., 137, 138, 167,241 Bradley, E.F., 197, 199,200,223 Braegelmann, P.K., I21 Brandle, J.R., 234,241 Brandt, C.J., 116 Brangeon, J., 155,167 Brennan, E., 51, 53,62,111,121, I25 Brenner, M.L., I15 Brenninger. C., 13, 18, 20,108 Brewin, N.J., 138, 147,167, 170 Briarty, L.G., I 7 7 Brinckmann, E . , 117 Brown, C.M., 153,167 Brown, H.M., 116 Brown, H.W., 218,241 Brown, K.A., 51,108 Bruggink, G.T., 37,40, 108 Brunold, C., 126 Bucher, J.B., 96,108,116,125 Bucher-Wallin, I.K.. von 56, 63.68, 96,108 Buck, A.L., 241 Buck, M., 102, I21 Bull, J.N., 44, 46, 109 Bullivant, S., 179 Bunce, J.A., 222, 223,241 Burris, R.H., 141, 149, 151, 182,183 Bus, V.G., M., 108 Butcher, G.W., 167 Butin, H., 113 Butler, L.K., 59,109 Butler, M.H., 171 Buttry, D . , 255-93 Bytnerowicz, A . , 14, 73, 77, 88, 93, 109,120,125,126 Bytnerowicz, M.B., 100, 101 C Caemmerer, S., von 19,109

AUTHOR INDEX

Cai. X., 155, 167 Campbell, G . S . , 217,241 Campbell, J.C.. 38, 39, 41, 122 Camut, S . , 183, 184 Cannell, M.G.R., 206,241,244 Canny, M.J.. 185 Canvin, D.T., 172. 173, 181 Cape, J.N., 6, 89, 101, 102, 103. 104, I l l , 127 Capron, S.J.M., 35. 36, 40. 109 Capron. T.M.. 36, 38,109 Caput, C., 4, 11, 22, 24, I09 Carlson. R.W., 10, 18, 29, 31, 43, 44. 46,47, 72,75. 78, 109 Carr, M.K.V., 218,241 Carrayol, E., 143, 169 Carter, K.R., 153,168 Carter, M.B., 173 Castillo. F.J., 77. 88, 94. 95, 109 Cat, W., de 110 Cecil, R., 20. 109 Cerovic, G.Z., 19, 109 Cervenansky. C., 166, I68 Cervenansky, E., 150 Chan, Y.-K.. 185 Chappelka, A.H., 80. 82, 109 Charles, T.C., 185 Chatfield, C., 202, 241 Chen, F.-L.. 155,168 CQen,G.. 255-92 Chen. Y.-M., 96, 109 Chermenskaya. I.E., 179 Cherry, J.H., 84. 116 Chevone, B.I.. 12. 70, 71,80,109,110. 126.127 Chien. W.-S., 175 Chiment, J.J., 88, 94, 108, 109 Christeller, J.T., 143, I74 Christensen, O.V.. 42, 175 Christensen, T.M.. 153 Christie, J.M., 206, 243 Chu, C., 119 Cionco. R.M.. 198,203,241 Clark, S.A.. 240 Cock, J.M., 155, 168 Coker, G.T., 143,168 Cole, M., 149, 181 Collins, M.A., 171, 242 Cookson, C.. 178 Cooley, D.R., 3. I10 Coombs. J., 178 Copeland, L.. 142, 148, 151, 168, 175. 176. 181, 184

299

Corbett, T.C., 243 Cormis. L., de 22,108 Cornelius, R., von 69, 110 Comic. G.. 15. 30. 110 Cottam, D.A., 117, 118, 120 Courtois, B.. 171 Coutts, M.P.. 206,241 Cowan, I.R., 219,240,241 Cowling, D.W., 13.27, 110. 117 Cowling, E.B.. 102, 124 Coyne, P.I., 51,66,69, 110 Craig, A.S.. 177 Craker. L.E., 110 Cregan, P . B . , 183 Cribb. D.M., 118 Crocker. T.D.. 51, 68, I05 Crowther, J.M., 199, 205,241,246 Cui, M., 256, 281, 282,293 Cullimore, J.V., 154, 155. 166. 168, 170 Cumming, J.R.. 54, 68. 106 Curtis, C.R., 121 D Dakessian, S . . 206.246 Dakora, F.D., 161, 168 Dalton, D.A.. 161,168 Darrall, N.M., 2, 3. 6. 7, 10, 17, 20, 21, 86, 92, 95, 96, 101, 110. 124 Dashek. W.V.. 93. I l l Dassen, J.H.A., 108 Davey, A . G . . 161,168 Davis, D.D., 9, 12, 16, 22, 23, 24, 31, 107.108, 122 Davis, J.M., 6, 110 Davis, L.C., 141, 168 Davis, R.W., 236,241 Davison. A.W., 20, 107, 110 Dawes, E.A., 163,172, 185 Dawson, P.J., 109, 120 Day, D.A., 135. 139, 146, 162, 168, 172.177, 183 Day, H.M., 170 Dazzo, F.B., 136, 170, 183 deBilly, F., 183, 184 deBruijn, F.J.. 153, 169 Decleire, M., 95, 110 Decoteau, D.R., 98,110 deFaria, S . M . , 131, 169 deMaagd, R.A., 140,169 den Hartog, G., 245 Denmead, O.T., 197, 199, 200,241 Dennison, D.S., 286, 290,293

300

AUTHOR INDEX

Derieux, J.-C., 171, 172 deRijk, R., I69 Deroche, M.-E., 143,169 deviser, R., 133, 169 deVries, G.E., 143, 146, 163,169 DeWalle, D.R., 201,243 Dietz, B., 87, 92, 110, 127 Dietz, J . , 112 Dijak, M., 53, 61, I10 Dilworth, M.J., 140, 146, 150, 153, 166,167, 169, 170,172,175, 176, 180 Dirnitrijevic, L., 177 Dittrich, W., 185 Dixon, M., 213, 214,215, 216, 222, 234,241,242 Dochinger, L.S., 107 Dohmen, G.P., von 96,110 Domes, W., 115 Dornigan, N.M., 138,169 Donagi, A.E., 85,110 Donahue, R.A., 256,283,293,293 Dondo, N., 118 Dougherty, D.E., 177 Downie, J.A., 179 Downs, R.J., 6 , 111 Drevon, J.-J., 165 Drew, M.C., 123 Drexler, D.M., 113 Dreyfus, B.L., 183 Duke, S.H., 171 Duncan, M.J., 150,169 Dunn, S.D., 153, 154,169 Dunning, J.A., 113 Duvick, D., 118 Duys, J.G., 169 Dyer, A.J., 196,241 E Eberhardt, J.C., 111 Ebling, S., 153,169 Edward, I., 245 Edwards, N.T., 113 Egli, M.A., 155,170 Eliassen, A., 7 , 34, 51,111 Elion, G.R., 257,293 Elion, H.A., 257,293 Elkan, G.H., 153,183 Elkiey, T., 61, 71,74,75,111 Ellenson, J.L., 90, 100, I09 Elliott, C.L., 81, 111 Elstner, E.F., 103, I l l , 124 Emerich, D.W., 142,165,172,177,185

Endress, A. G., 94,95, I I I , 113 Engelke, T., 147, 148, 170 Engwall, K.S., 180 Erickson, S.S., 93, I l l Erner, Y.,235,241 Espen, G., 176 Evans, H.J., 143, 145, 163,168, 175, 177,185 Evans, L . S . , 5,112 F Fa, C.H., 126 Faarnden, K.J.F., 153 Faensen-Thiebes, A., 52, 58, 59,111 Faris, M.A., 182 Farnden, K.J.F., 156, 169,175, 178, 180 Farquhar, G.D., 19,109,221,240, 241,245 Farrar, J.F., 16,29,31,33,111,112 Fasehun, F.E., 242 Fedulova, N.G., 170,174, I79 Feiler, S . , 20, 111 Finan, T.M., 147, 170, 185 Fink, S., 109 Finnigan, J.J., 201, 203, 207,242 Flagler, R.B., 121 Flamrnersfeld, U., 110, I27 Fletcher, R.A., 117 Forberg, E., 52,61,111,119 Forchioni, A., 167 Forde, B.G., 155,170 Fordyce, A.M., 179 Fortin, M.G., 136, 138, 139,167,170, I84 Foster, D.R., 234,242 Fottrell, P.F., 179 Fowler, D., 6 , 111 Fox, C.A., 120 Foyer, C.H., 77,95,111 Fraenkel, D.G., 150,169 Francis, B.J., 127 Franklin, J.E., 102,111 Franzen, J., 112 Fraser, A.I., 206,242 Freer-Smith, P.H., 15,48,111, I17 Fuhrer, J., 89,112,117 Fujihara, S., 157, 170 Fujinuma, Y . , I19 Fujita, K., 186 Fukai, K., I74 Fukshansky, L., 256,274,277,281, 293,294

AUTHOR INDEX

Fukuzawa, T., 120 Furukawa, A., 8 , 1 1 , 13, 18, 19, 22, 25, 35,36, 38, 39,50, 51, 53, 58, 59, 83,84,112,124 G Gaastra, P., 197,242 Gadal, P., 143,170, 176, 184 Gadzhi-zade, B.R., 151, I70 Gaito, S.T., 172 Galfre, G., 167 Gallagher, M.P., 172 Garbers, C., 139, 170, 175, 185 Gardiner, J.B.H., 206,242 Gardiol, A.E., 150,166, 170 Garland, J.A., 197,242 Garraway, M.O., 95,121 Garrett, J.R., 196,242 Garsed, S.G., 16, 17,33, 105, 107,112, 119 Gasch, G., 88,93,112 Gash, J.H.C., 246 Gates, D.M., 215, 217,242 Geburek, Th., 89, 99, 112 Gellespie, C., 127 Gerson, T., 155, 187 Gezelius, K., 11, 13, 19, 27, 28, 29, 30, 33,112,114 Gidal, P . , 182 Gillespie, T.J., 59, 61, 106 Glenn, A.R., 140, 146, 147, 150,166, 169,170, 172,175, 176,180 Gmur, N.F., 6,112 Gober, J.W., 136, 153, 171 Gonzales, H.G., 96,123 Gonzalez, R., I74 Goodchild, D.J., 132, 134, 171 Goodwin, T.W., 103,112 Gordon, A.J., 142,171 Goren, A.L., 85,110 Gorham, J., 119 Goudriaan, J., 145, 181 Gould, R.P., 49,112 Grace, J., 189-240,241,242,243,244, 245,246 Graham, P.H., 175 Granat, L., 114 Grandjean, A., 117 Grant, L., 110 Grayston, G.F., 179 Greenfelt, P., 34,35,112,125 Greenhalgh, B., 125 Gregson, K., 240

301

Greim, H., 124 Greitner, C.S., 56, 63, 64, 101, 112, 116 Greppin, H., 77, 88, 93, 95, 109 Gresshoff, P.M., 168,172,177,183 Griffith, S.M., 156,170,171, 175 Gross, G., 205,242 Gross, von K., 75, 102,112 Grum, F., 272,293 Griinhage, L., 112,114 Grunwald, C., 79, 94,113 Guerin, V., 151, 171 Guevara, J.G., 175 Guezzar, M.E., 150,171, 172 Gumpertz, M.L., 126 Gunderson, C.A., 126 Gunning, B.E.S., 177 Guoying Chen 255-72 Gupta, G., 40, 123 Guri, A., 88, 89, 94,113 H Haaker, H., 144,165,187 Haberlandt, G., 293 Hagedorn, C.H., 173 Hajy-zadeh, B.R., 179 Hall, A.E., 221,222,242,245 Hall, D.M., 237, 243 Hallgren, J.-E., 11, 13, 19,27, 28, 29, 30,33,112, I14 Halliwell, B., 77, 95,111 Halvorsen, A.M., 184 Hamilton, G.J., 206,243 Hammeed, S., 166 Han, S . , 185 Hand, D.W., 40,107 Hanks, J.F., 134, 159,167,171 Hanna, S.R., 234,243 Hansen, K.S., 116 Hanson, P.J., 80, 81, 113 Hanus, F.J., 168 Hardman, L.L., I84 Hardy, R. W.F., 130, 172, 178 Harmon, M.E., 111 Harper, J.E., 181 Harrington, A., 167 Harris, R.W., 234, 244 Hartel, O . , 90, 101, 114 Hartgerink, A.P., 116 Hartmann, G., 102,113 Hartmann, K.M., 274, 281,293 Harvey, G.W., 116 Hashimoto, Y . , I20

302

AUTHOR INDEX

Hasler, R., 16, 30, 33,57, 63, 64, 66. 115 Haupt, W., 256. 283, 288,290.295 Hay, G.T., 178 Hayakawa, N., 127 Heagle, A.S., 6,113,127 Heath, R.L., 3, 113 Heber, U., 116, 121 Heck, W.W., 6,113 Heckert, L.L., 162, 171 Hedley, C.L., 243 Heggestad, H.E., 107,121 Heichel, G.H., 165, 184,244 Heisler, G.M., 201, 243 Helms, J.A., 16, 30, 113 Hendrickson, R.C., 120 Henis, Y., 176 Hennecke, H., 169,183 Henson, C.A., 142, 145, 171 Herrada, G . , 139, 146.171 Herridge, D.F., 160, 171 Heytler, P.G., 130, 172 Higgins, C.F., 140, 172 Higgisson, B., 167 Higuchi, T., 160, 173 Hill, A.C., 10, 17,21,35. 36,38, 51, 52,107,113,127 Hine, A , , 178 Hinkelmann, K.H., 116 Hinrichsen, D., 102,113 Hinze, D.L., 241 Hirata, Y., 85, 113 Hirel, B., 155, 167, 172 Hitz, W.D., 178 Hoelzle, I . , 162, 172 Hoffmann, I., I1 7 Hofstra, G., 5 , 14, 15, 63, 72, 73. 76, 107,113 Hogsett, W.E., 61, 66, 126 Holbo, H.R., 207,243 Holland, M.R., 198,243 Holmes, M.G., 272,293 Holroyd, E.W., 234,243 Hong, Z . , I68 Hornez, J.-P., 147, 171, 172 Horsey, A.K., 184 Horton, P.J., 243 HOU,L.-Y., 44, 46. 113 Houpis, J.L., 16, 30.113 Houston, D.B., 108 Hov, O., 108 Howitt, S.M., 154, 172 Hozumi, K., 112

Huber, T.A., 156, 172 Hucl, P., 118 Hudman. J.F., 146. 170, 172 Huebert, D.B.. 117 Huel, P., 118 Hughes, P.R., 110 Humbeck, C., 141, 147,172 Hunt, G.A., 18, 20. 24, 26, 114 Hunt, S . , 143, 161, 172, 173 Hutchings, N.J., 199,241 Hutchinson, B.A., 203, 204,240 Hutchinson, T.C., 80, 81, 124 Hutton, W.J., 125 Huttunen, S . , 20,88, 93,114 Hyde, S.C., 172 I Iino, M.. 290.293 Ingle, M.. 125 Innes, J.L., 102. 114 Inoue, Y . , 294 In’t Veld, P., 169 Ishikawa. H., 123 Ishizuka, J., 174, 182, 186 Isoda. O . , 112 Israel, D.W., 143, 172, 174 Ito, O . , 84, 114 Ivanov, B.F.. 179 Iversen, T., 111 Iwaki, H., 112, 114 Izawa, T., 260,293 Izmailov, S.F., 165

J Jackson, F.A., 163,172 Jackson, W.A., 143,172 Jacobs, A.F.G., 196, 234,243 Jacobs, F., I72 Jacobs, M.R., 243 Jacobson, G.R., 148,180 Jaffe, M.F., 241 Jaffe, M.J., 233,243 Jagadism, M.N., 170 Jager, H.J., 3 , 32, 86,92,93, 95,96, 101,110,112,114, 115,124 James, G.B., 198,243 Jaques, D.R., 116 Jarvis, P.G., 197, 222, 231,243 Jayaram, S., 166 Jeffree, C.E., 223,243,244,246 Jeffries, H.E., 122 Jensen, K.F., 16, 73, 76, 78, 82,108, 114

AUTHOR INDEX

Jeon, K., 179 Jin, H.N., 154, 172 Johansson, C., 114 Johnson, L.E.B., 184 Johnston, J.W., Jr., 80, 81, 124, 125 Jokinen, J.. 111 Joliffe, P . A . , 125 Jolivet, E., 169 Jones, H.G., 222,243 Jones, L.H.P., 117 Jones, R.L., 237,246 Jones, T . , 16, 30, 114 Jones, W.T., I77 Jordan, D.C., 170 Jording, D., 170 Josserand, S.. 294,295 Joy, K.W., 175, 182 Jukola-Sulonen, E.-L.. 123 Jung, K.-D., 115,121 Juniper, B.E., 223,243 Justin, J., 125

K Kabel, R.L., 125 Kahn, M.L., 181 Kaji, M., 38. 39, 114 Kalezic, R., I09 Kambayashi, I., 186 Kaneko, Y . , 176,184 Kapp. D., 170 Kappen, L., 217,243 Karasuyama, M., 186 Karjalainen, R., 115 Karolowski, P., 88, 93, I l l , 114 Karr, D.B., 163, 172, 185 Kashket, E.R., 136, 153, 171 Katagiri. H., I74 Katainen, H.-S., 12, 13, 20, 26, 27, 32, 61,96,115 Katase, M., 112 Katinakis, P., 138, 139, 173, 174 Kats, G., 109, 120 Kaufmann, M., 56,63,66,106,293 Kaufmann, W . F . , 274,293 Kazarinova-Fukshansky, N., 290,293 Kazazian, V., 184 Keister, D.L., I76 Keitel, A . , 59, 115 Keller, T., 14, 16, 27, 30, 33, 57, 63, 64,66, 85. 90, 115 Kellomaki, S., 115 Kender, W.J., 118 Kenk, G . , 116

303

Kennedy, I.R., 181 Keyser, H.H., 183 Khmel’nitskii. M.L., 186 Kijne, J.W., 137. 169, 173 Killer, D., 290 Kimmerer, T.W., 12, 24, 96, 115 Kimura, I., 151, 173, 183 King, B.J., 143, 159, 161, 172, 173 King, K.M., 245 Kinnback, A . , 139,173 Kinze, G . , 117 Kitou, M . , 182 Klein, H., 15, 32, 93,114, 115 Klein-Lankhorst, R.M., 102, 115, 173 Klipp, W., 153. 173 Klucas, R.V., 153, 154, 169 Klugkist, J., 167 Knabe, W.. 112 Knapp, A.K., 278,293,295 Knight, T.J., 155, 173 Knoppik, D., 124 Kobriger, J.M., 74, 115 Koch, G., 119 Kohl, D.H., 152, 160,173,180, I81 Kohut, R.J., 106, 117 Koike, A . , 112 Kok, L.J., de 117 Koller, D., 290,293 Kondo, J . , 120, 125 Kort, R., 185 Kouchi, H., 134, 142, 144, 146, 159, 173,174, 182, 186 Koukkari, W.L., 171 Kouzai, K., 162, 182 Kowal, R.R., 176 Koziol, M.J., 3, 6 , 7 , 13, 15, 18, 27, 31, 32. 110, I15 Kozlova, G.I., 165 Kozlowski, T.T., 12, 15,24,31, 96, 102,115,120 Kramer, P.J., 120 Kraus, M., 127 Krause, G.H.M., 57,63, 102,115,116, 121 Kreeb, K.H., 92, 95,122 Kreith, F., 215,243 Kress, L.W., 85,116 Kretovich, W.L., 155, 163, 170, 174, 179 Krey, R., 173 Krinsky, N.I., 103, 116 Krishnamurthy, R., 106 Krizek, D.T., 31, 116

304

AUTHOR INDEX

Kropff, M.J., 12, 18, 19, 24, 25, 116 Krouse, H.R., 116 Krupa, S.V., 113, 120 Kuiper, J.C., 117 Kull, K., 243 Kumar, N., 45,48,116, I53 Kumar, P.S., 153, 174 Kumazawa, K., 155, 162,175,176 Kunert, K.J., 118 Kunishige, M., 85, 113 Kupper, R.S., 126 Kiippers, M., 119 L Lacy, E.A., 257,294 Lafontaine, P.J., 132, 147, 150, 174 LaFreniere, C., I74 Laine, K., 114 Laing, W.A., 142, 168, 174 Laisk, A., 33, 116, 221,243 Landholt, W., von 87,91,116,126 Landsberg, J.J., 198,243 Lane, M., 154, 174 Lange, 0 . - L . , 121 Langlois, J.R., 155, 184 Langston-Unkefer, P.J., 155, 173 Lankhorst. R.M.K.. 153.174 Lanzl, A. ,'I05 Lappalainen, T., 123 Lara, M., 168 Larcher, W., 217,243 Larkins, A.P., 167 Larson, P.R., 234,244 LaRue, T.A., 135, 145, 152, 62, 177, 178, 182 Lassoie, J.P., 57, 64, 67, 122 Latimer, P., 276,294 Laurence, J.A., 4 , 85,116,l 7 Law, R.M.. 35,116 Lawrie, A.C., 143, 174 Layzell, D.B., 172,173,185 Lea, P.J., 166, 168 LeBlanc, D.C., 102,116 Lee, D.W., 286,293,294 Lee, E.H., 95, 107, 116 Leffler, H.R., 84,116 Legg, B.J., 165, 200,244,245 Legge, A.H., 33,88,93,116 Lehnherr, B., 55, 63,64,65, 68,117 Lembi, C.A., 179 Lennox, R.L., 126 Lennox, R.W., 126 Lepo, J.E., 136,178

Lester, P.F., 116 Lewin, K.F., 112 Lewis, G.P., 169 Lewis, T.A., 154, I74 L'Hirondelle, S.J., 9, 13, 15, 22, 27, 117 Ligtenberg, A.J.M., 169 Linder, S., 114 Linzon, S . N . , 113 Littlefield, N., 51, 52, 113 Livanova, G.I., 165 Lockyer, D.R., 6,115, I17 Long, I.F., 244 Lorenc-Plucinska, G., 15, 31, 32, 37, 39,42, 117, 119 Losch, R., 221,244 Louguet, P., 23, 108 Louwerse, J., 173 Lovett, G.M., 234,244 Lucas, K., 159, I74 Lucas, P.W., 6,117,118 Lugtenberg, B.J.J., 140, 169 Liittge, U., 20, 117 Luxmore, R.J., 33,117 Lyttleton, P., 137, 153, 178,179 M Maas, F.M., 13, 31,117 McAdams, W.H., 215,244 McBean, G.M., 199,244 McCairns, E., 166 McClean, T.M., 293,295 McClure, P.R., 160,174 McConathy, R.K., 107,118 McCool, P.M., 119 McCormick, D.K., 154,175 McCully, M.E., 185 McCune, D.C., 43,49, 78,117 McDermott, T.R., 152, 163, I75 Macdowell, F.D.H., 182 McFarlane, J.C., 6,118 McGowan, M., 240 Machal, L., 176 Machler, F., 117 McInroy, S.G., 178 McIntyre, L., 132, 137,176 Mackay, C.E., 5,117,153 McKay, I.A., 149, 166, 169,170, 175 McKersie, B.D., 95. 117, 118 McLaughlin, S.B., 4 , 7 , 69,107,113, 118 Maclean, D.C., 110,116 McLeod, A.R., 4,118

AUTHOR INDEX

McNaughton, K.G., 201, 205, 231, 243,244 Macnicol, P.IK., 165 McParland, R.H., 154, I75 McRae, D.G., 139, 148, 175 Maier, R.J., 152, 161, 176 Majernick, O., 12, 13, 22, 23, 117 Makinen, E., 111,115 Malcolm, D.C., 242 Malik, N.S.A., 138, 174 Mandl, R.H., 6, II0,116,117 Manian, S.S., 167, 174, 176 Mann, L.K.. 118 Manning, W.J., 3, 110 Mannix, M., 145, 162,168 Mansfield. T.A., 12, 13, 16, 22, 23, 30, 35, 36, 38, 43, 44, 45, 46, 48, 49, 78,106,112.114,116,117,118, 120, 222.243 Marczewski, W.. 143, 174 Marie, B.A., 120 Marques, I., 106 Martin, B., 90, 101, 118 Martin, G . , 255-92,293,294 Martinez-drets, G . , 166 Martinoia, E . , 121 Marunov, S.K., 186 Mask, P.L., 178 Matschke, J., 96, 118 Matsumoto, T., 186 Matsumura, H., 119 Matsuoka, Y . , 11, 118 Matsushima, J., 51, 118 Matzner, E . , 102,118 Maxwell, C.A., 184 Mayer, H., 207, 208,209, 234,244 Mayhead, G.J., 205, 208,246 Mayo, J . , 116 Meade, J.. 174 Meaden, G.T., 244 Meckbach, R., 170 Meeks, J.C., 154, 175 Meguro, H., 151.183 Mehler 104 Mehlhorn, H., 87, 88, 89, 92, 93,98, 99, 102. 103,109,118 Mellor, R.B., 137, 138, 139,166, 270, 173,175,185 Mercer, E.I., 103, 112 Messmer, S., 169 Meyer, A., 96, 98,118 Michaelis, P., 224,244 Michaels, T.E., 92, 119

30.5

Miflin, B.J., 166, 168 Miller, J.E.. 119, 127 Miller, P.R., 109 Miller, R.W., 140, 152, 175 Miller, S.S., 165, 170 Milne, R., 207, 244 Mimmack, M.L., 172 Minamisawa, K., 156, 174, 175 Minchin, F.R., 133, 175, 178, 181, 185 Miszalski, Z . , 31, 119 Mitchell, D.F.. 171 Mitchell, J.W., 215, 244 Mitchell, M.K., 158, 175 Mitsumori, F., 114 Miyake, H., 64, 119 Modi, V.V., 145, 176 Molchanov, M.I., 179 Monk, B.C., 169 Monteith, J.L., 197, 199, 210, 212, 213, 214, 231,240,244 Mooney. H.A., 4. 11, 12, 16, 17, 18, 19.20, 22, 29, 34, 107,119, I 2 7 Moors, I., 110, 127 More, L.D., 107 Morell, M.. 142, 175, 176 Moreno, S., 176 Morett, E., 153, 176 Morgan, J., 206,241,244 MorrC, D.J., 139, 179, 182 Morrison, N.A., I53 Morschel, E., 132, 137, 163, 175, 185 Mortensen, L.M., 6, 119 Mould, R.M., 168 Mudd, J.B., 58,119 Mueller, P.W., 17, 119 Mulchi, C.L., 121, 123 Mulders, I.H.M.. 169 Mulhearn, P.J.. 207,242 Miiller, P . , 118 Muller, R.N., 10, 17, 21, 119 Murali, N.S., 6, 7, 8, 11, 17, 37, 38, 41, 52, 58, 87, 89, 91, 99. 104,124 Murray, A.J.S., 119 Murray, F., 15, 27, 30, 33, 39, 96, 129 Muschinek, G., 106 Musselman, R.C., 5, 6, 119 Myers, D.A., 294 Myhre, A., 51. 52. 61,62, 119 Myneni, R . B . , 294 N Nadler, K., 167 Nakaji. K., 142, 173, 174

306

AUTHOR INDEX

Nakamura, H., 58, 119 Nandi, P.K., 106 Nash, T.H., 53, 54, 61, 123 Natori, T., 9,22, 24,44,47. 70, 71, 84, 112,119 Nautiyal, C.S., 145, 153, 176 Neel, P.L., 234,244 Neighbour. E.A., 45, 48, 49, 120 Nelke, M., 136, 183 Newcomb, E.H., 132, 134, 176,180, 184,185 Newcomb, W., 132, 137, 159, 176 Nguyen, J., 159,176,182,184 Nienhaus, F., 113 Nikaido, H., 140,176 Nilsen, S., 111,119 Nixon, T.B., 179 Nobel, P.S., 217, 222,244 Noble, R.D., 9, 17, 22,25, 76, 114, 125 Noel, K.D., 184 Noh, S.J., 276,294 Noonan, B., 167,176 Norby, R.J., 15, 31, 120 Nordin, P., 141,168 Norman, J.M., 293 Nosal, M., 116 Noti, J.D., 169 Nouchi, I . , 85, 120 Noyes, R.D., 3,9,120 Nur, I., 163, 176 Nystrom, S.D., 6. 120 0 O'Brian, M.R., 152, 161, I76 Odorico, R., 183 O'Gara, F., 148, 167, 174, 176 Ogier, G., 109 O'Hara, G.W., 154,176 Ohyama, T., 155, 162, 176 Oja, V., 243 Okano, K., 40,41,120 Okinen, J . , 115 Okon, Y . , 176 Oleksyn, J., 87, 91, 120 Oliver, H.R., 198, 199,244,246 Oliver, J.E., 170 Olszyk, D.M., 5, 12, 15,20, 25, 30, 58, 70,71,74,75,109,120, I25 Omasa, K., 3, 12,25,120 Omielan, J.A., 58, 61. 120 Onal, M., 13,74,75,76,106 Orme-Johnson, W.H., 153,168,177

Ormrod, D.P., 4,5,53,61,70,71,72, 74, 76,110,111, 120, 125 Osborne, B.A., 256,294 Oshima, R.J., 121 Oshima, Y . , 10, 54,66,131 Osmond, C.B., 117,245 Osswald, W., 103, 111,124

P Pande, P.C., 31,117,121 Pankhurst, C.E., 131, 132,177, I84 Papian, L.E., 215,217,242 Parker, G.G., 234,245 Parlange, J.Y., 212,244 Pascoe, G . A . , 168 Pate,J.S., 132, 133,166,175,177 Patel, J.J., 132, 134,177, 187 Paterson, L.S., 109,127 Patton, R.L., 95,121 Pearce, S.R., 172 Pearson, N.S., 58,121 Pedersen, P.B., 179 Pell, E.J., 51, 53, 58, 61, 62, 89, 98, 120,121,125 Pelletier, R.L., 111 Peoples, M.B., 131,160,171,177 Perrier, E.R., 204, 240 Perrot-Rechenmann, C., 176,184 Peterson, J.B., 143,145, 152, 162,177 Peterson, R.L., 176 Petolino, J.E., 95,121 Petty, J.A., 205,244 Pfanz, H., 19, 25, 39,116, 121 Pfeffer, H.U., 102, 121 Pfeiffer, N.E., 138,174,177 Pfleeger, T., 6,111,118 Philbeck, R.B., 113 Phillips, D.V., 141, 162, 177 Pierre, M., 30, 121 Pinckney, H.R., 108,126 Pitas, J.W., 154,180 Pitcairn, C.E.R., 223, 224, 234, 236, 244 Pladys, D., 138,177 Planque, K., 137, 173,184 Plate, E.J., 205, 245 Plesnicar, M., 109 Podleckis, E.V., 89, 95, 121 Poole, P.S., 170 Poole, R.J., 167 Poorter, H., 133,169 Posthumus, A.C., 85,121 Pottier, R.H., 173

AUTHOR INDEX

Poulson, M.E., 284. 286. 294 Powell, C.E., I71 Pratt. G.C., 120 Preston, G.G., 14Y, 177 Preston, K.P.. 16, 34, 121 Price, G.D., 146,168, 177, 183 Primrose, S.B., 150, 179 Prinz, B., 102, 104, 11.5, 121 Puckett, L.I., 99, 122 Puente, M . , 89, 98. I21 Piihler, A., 170, 173 Puppo, A., 161, 171, 177 Pursley, W.A., 127 Putz, F.E., 234, 24.5

Q

Queiroz, Q . , 30, 121 Quinnell, R.G., 168 Quiocho, F.A., 140, 177 Quispel, A , , 169, 184

R Raba, R.M.. 106,122 Rabe, R., 92,95.122 Raikhinshtein, M.V.. 179 Rainbird, R.M.. 130, 159,178 Raison, J.K., 217,240 Randall, D.D., 178. 183 Rao, D.N., 106 Rao. I.M., 12, 13,27.31, 122 Rao. N.V., 159, 178 Rao, S.L.N., 153,174 Rastogi, V.K., 155, 187 Ratet, P., 169 Ratsch, H.C., 126 Raupach, M.R., 196, 197. 200, 201, 2 45 Raven, J.A., 134, 178,294 Rawlings, J., 168 Rawsthorne, S . . 130. 135, 145, 162, 178 Raynal, D.J., 116 Rea, P.A., 167 Reagan, C.A., 126 Reddy, R.S., 178 Reding, H . K . . 136, 178 Reibach, P.H., 139, 141, 142, 146, 148, 162,178, 184 Reich, P.B., 54, 55, 56. 57, 63, 64, 65, 66,67, 68, 81, 87. 91, 106. 122 Reilander, H., 173 Reiling, K., 107 Reimers, J.M., 171 Reiners, W.A., 21,244

307

Relton, J.. 106, 111 Renner, C.J., 107, 110 Reville, W.J., 167 Revsbech, N.P., 185 Reynolds. K.L., 116 Reynolds, P.H.S., 156, 157,167,173, 17.5, 178 Rhodes, E.C., 116 Rich, S . . 61, 122 Richter, A , , 114 Rigaud, J . , 138, 152,171, 177, 183 Riley, I.T., 176 Rist, D.L., 12, 23, 122 Ritchie, A , , 166 Robe, S.V., 107 Roberts, B.R., 73,78,114 Roberts, T.M., 51,108,118 Robertson, A., 234,245 Robertson, J.G.. 137, 140, 148, 153, 154,169,178, 179,180 Robinson, D.C., 58,61,122,127 Rodber. K., 178 Rodecap, K.D., 98, I22 Rogers, H.H., 6 , 38. 39. 41, 110, 122 Rohr, K., 126 Roland, J.C., 137,179 Roloff, A , , 89, 99, 122 Romanov, V.I., 146, 149, 155, 163, 170,174, 179 Ronson, C.W., 140, 147, 148, 150, 179 Roper, T.R.. 51, 53, 123 Ropertson, J.G., 167 Rosenberg, N.J.. 218,241 Rosendahl, L., 143, 179 Ross, L.J., 53, 54, 61,123,294 Ross-Todd, B.M., 126 Rossbach, S . , 169 Roth, E.L., 137, 179 Rowe, P.B., 166 Rowland, A.J., 41,123 Rowley, B.I., 185 Rowney, F.R.P., 166 Ruck, B., 205, 206,245’ Ruhle, W., 110, 127 Runecles, V.C., 12.5 Rupert, C.S., 272,294 Russell, G., 234, 24.5 Russell, S.A., 168 Rutt, A.J., 106 Rutter, A.J., 105, 106, I l l , I12 Ryan, E., 156, 179 Ryle, G.J.A., 171

308

AUTHOR INDEX

S Sabaratnam, S . , 37,39,40,123 Sadowsky, M.J., 176 Saeki, T., 293,294 Saharan, M.R., 180 Saka, H., 119 Salema, M., 100, 107, 123 Salminen, S.O., 140, 141, 142, 148, 149, 155, 162, 180, 182 Salom, C.L., 183 Salsac, L., 165 Saltbones, J., 111 Sandmann, G . , 96,123 SanFrancisco, M.J.D., 148,180 Saroso, S . , 148, 149, 169, 180 Sasahara, H., 182, 183 Sastry, K.S., 178 Sato, S . , 15, 123 Sauer, D., 166 Sauvageau, R., 165 Sawhney, V., 143, 156,180 Saxe, H., 2-105,123, I24 Schafer, E., 274, 281,294 Schell, J., 169 Schellhase, H.U., 116 Schenk, S.U., 166 Schicker, S . , 98, 125 Schilling, N . , I75 Schliiter, A., 173 Schmidt, A., 134 Schmidt, E.L., 183 Schmidt, W., 92,118,124 Schmitt, F., 205,206,245 Schneider, M., 153 Schneider, R.E., 169 Schoettle, A.W., 106,122 Scholz, F., 89, 100,112, 124 Schonherr, J., 223,245 Schramm, M.J., 116 Schreiber, U., I24 Schubert, K.R., 130, 131, 143, 157, 160,167,168,171, 173,174, 178,180 Schucht, J.H., 234, 235,245 Schuepp, P.H., 212,216,245 Schulte-Hostede, S . , 3, 124 Schulze, E.D., 221,222,245 Schiitt, P., 102, 124 Schwarz, B., I25 Schweizer, B., 124 Scott, D.B., 180 Scott, M.G., 80, 81,124 Scott, R.K., 240

Seiler, J.R., 126 Selinger, von H., 82,124 Selker, J.M.L., 132,134, 160,180 Sembdner, G., 118 Semeniuk, P., 116 Sen, D., 131, 132, 134, 146, 155,180 Senaratna, T., 117 Senger, H., 94, I24 Sengupta-Gopalan, C., 154,180 Senser, M., 124 Seufert, G . , 102, 103, 104,124 Seyfried, M., 256,214, 277, 278, 281, 293,294 Shafer, E., 293 Shaffer, P.W., 175 Shantharam, S . , 139, 180 Shaposhnikov, G.L., I79 Shatters, R.G., 181 Shaw, R.H., 203,245 Shearer, G., 160, 173,180,181 Sheehy, J.E., 161,181,185 Shelp, B.J., 134, 155, 156, 158, 159, 166, 181 Shelvey, J.D., 115 Shen, W.-S., 127 Shertz, R.D., 63, 124 Shimazaki, K.-I., 19, 124 Shimizu, H., 15, 124 Shramko, V.I., 174,179 Shriner, D.S., 125 Shugart, H.H., 111 Sigal, L.L., 4 , 80, 81, I24 Sij, J.W., 11, 17, 125 Silvius, J.E., 19, 125 Simons, L.H., 169 Simpson, D., 111 Simpson, R.J., 161,168 Sinclair, T.R., 145, 181 Singh, R., 180 Sinn, J.P., 36, 38,125 Sippell, D., 176 Sisson, W.B., 10, 18, 21, 125 Skarby, L., 41,53,63,67, 68,112,125 Skelly, J.M., 107, 116, 127 Skot, L., 185 Sloger, C., 183 Smith, A.M., 142, 181 Smith, F.A., 136, 181 Smith, G . , 5,125 Smith, S.E., 136,181 Smith, W.H., 108 Smith, W.K., 293,295 Snapp, S . S . , 156,181

AUTHOR INDEX

Soikkeli, S . , 20, 114 Soleimani, A . , 113 Somerville, J.E., 153, 181 Spence. R.D., 221,245 Sprent, J.I., 131, 132, 136, 141, 165, 169. 177, 178, 181 Sprugel, D.G., 119 Srivastava, H.S., 35, 36, 39, 41, 61, 125 Stacey, G., 137,179 Stade, S . , 143, 184 Stam, H., 169 Stan, H.-J., 98, 125 Stax, D.. 183 Stewart, J.B., 246 Stone, S.R., 155, 181 Storer, P.J., 166. 181 Stouthamer. A . H . , 169 Stovall, I., 149, 181 Stowers, M.D., 130, 181 Strain, B.R., I20 Strasser, R.J., 87, 93, 125 Streeter, J.G., 129-64,172,178, 180, 181, 182 Stripf, R., 154, 182 Stumpf. D.K., 141, 182 Sturrock. J.W., 241 Suarez, S.J., 111 Suda, Y., 125 Sudo, S . , 260,293 Suer-Brymer, N . M . . 72, 76, 125 Sugahara, K . , 19. 125 Suganuma, N., 144,182 Summerfield, R.J., 178 Suter, G.W., H., 4 , 124 Sutherland. J.M., 169 Sutton, W.D., 168, 174 Suzuki, A., 155. 182 Suzuki, F., 172 Swanson, C.A., 1 1 , 17, 125 Szeto, W.W., 169 T Ta, T.-C., 156. 182 Tabner, P., 106 Tajima, S . , 142, 151, 152, 159. 162, 173, 174, 182, 183 Takemoto, B.K., 80,81, 87,92, 125 Takemoto, K., 9 , 17, 22, 25, I25 Tanaka, K., 95, 125 Tandon, S.H., 176 Taylor. G.E., Jr., 10, 16, 20, 25, 28, 61,126 Taylor, H.J., 18, 49, 59, 60, 98, 126

309

Taylor, M.P., 148,178 Taylor, O.C., 62, 73, 77,89,106, I 11, 126 Tazaki, T., 12,120,121,127 Tchermenskaya, I.E., 179 Tchetkova, S.A., 155 Telewski, F.W., 234,245 Temmerman, L., d e 110 Temple, P.J., 4 , 7 , 56, 65, 66, 81, 82, 126 Tenhunen, J.D., 221,244 Terashima, I., 221,245, 293,294 Teso, R.R., 119, 121 Thierfelder, H., 285 Thom, A.S., 164, 165, 196, 197, 198, 245,246 Thompson, C.R., 109,120 Thompson, J.R., 236,246 Thompson, K.H., 5 , 111 Thony-Meyer, L., 153,183 Thorne, D.W., 149, 151,183 Thorstenson, Y.R., 118 Throneberry, G.O., 125 Thummler, F., 142,183 Thurtell, G.W., 106,245 Thutt, G.L., 126 Tibbitts, T.W., 59, 70, 74, 7 5 , 115, I20 Timell, T.W., 234,246 Tingey, D.T., 25, 5 8 , 6 1 , 6 6 , 7 0 , 7 1 , 74,75,98,120,122,126 Tjepkema, J.D., 132, 161, 183 Tolbert, N.E., 167, 171 Tomiczek, C., 90, 100,126 Tomlinson, H., 122 Topunov, A.F., I70 Torres, C.M., 177 Torvele, H., 114 Totsuka, T., 9 , 2 2 , 24,40,41, 44,47, 70, 71, 84, 112, 114, 119, 120, 124,128 Tranquillini, W., 13, 18,20,108, 217, 224,246 Tremblay, P.A., 140, 175 Trinchant, J.C., 151, 152, 171,177, 183 Triplett, E.W., 159,183 Tritton, D.J., 205, 246 Troeng, E . , 114,125 Truchet, G.L., 132,170,183,184 Tschanz, A , , 32,126 Tseng, E.C., 54,63,66,126 Tsien, H.C., 132,183 Tsukamoto, M., 174 Tuomisto, H., 89, 99,126

310

AUTHOR INDEX

Turner, G.L., 151, 152, 165, 166, 167, 185 Turpin, D.H., 184 Turton, J.F., 170 Tuzimura, K., 151,183 U Udvardi, M.K., 146,168, 172, 183 Ulrich, B., 102,111,118 Umezawa, T., 123 Unsworth, M.H., 9 , 17,22,23,108, 120,126,244 Upchurch, R.G., 153,183 Urbach, W., 124 Urban, J.E., 136, 183 Ushijima, T., 12. 112, 121, 127 V Vaara, M., 140, 176 vandenBos, R.C., 153.173,174 VanGardingen, P., 18%240,246 vanBerkum, P., 159, 176, 183 vanBoxel, J.H., 196,243 vanBrussel, A.A.N., 140,169,184 Vance, C.P., 132, 134, 143, 156.165, 170,171, 175, 179, 181,184 VandenBosch, K.A., 132, 159, 176, 184 vanKammen, A., 167,173, 174 Vanlerberghe, G.C., 162, 184 Varshney, C.K., 88, 94,127 Varshney, S.R.K., 88,94, 127 Vasse, J., 136, 183,184 Vaughn, K.C., 159,184 Vella, J . , 142, 168, 184 Verma, D.P.S., 136, 138, 139, 142, 161,167, 172, 173,183,184 Verma, S.B., 240 VCzina, L.-P., 155,184 Vidal, J . , 143, 176, 182, 184 Vogelmann, T.C., 255-92,293,294, 295 Volk, R.J., 174 Vollbrecht, P., 112 Vornweg, A., 112 Vozzo, S.F., 57, 63, 66, 127 W

Waggoner, P.E., 122,244 Wagner, E., 112 Wagner, F.W., 174, 177 Wake, R.G., 20,109 Waldron, L.J., 206,246

Walker, R.B., I16 Wall, J.D., 177 Walmsley, L., 5 1 , 56, 63, 66, 69, 127 Walsh, K.B., 133,173,185 Warburton, M.P., 178, 179 Ward, A.C., 163, 185 Wardle, P., 224,246 Waters, J.K., 172, 185 Watson, R.J., 147, 148, 149, 155, 185, 187 Weagle, G.E., 173 Weaver, R.W., 146,180 Webb, M.A., 185 Weber, D.F., 183 Weger, H.G., 184 Weidner, M., 103, 127 Weinstein, L.H., 13, 24, 45, 47,106 Wellburn, A.R., 3 , 47, 58,61, 96, 98, 104,109,122,123,124,127 Wells, B., 167 Welty, J.R., 213,246 Wen-jun, S., I70 Wentzel, K.F., 17, 112, 127 Wergin, W.P., 116 Werner, D., 132, 137, 139, 141, 147, 149, 154, 163, 166, 170,172, 173, 175, 182,185 Whatley. F.R., 3 , 115 Wheatcroft, R . , 185 Wheeler, C.T., 143,174 White, E.H., 116 White, K.L., 10, 43,44, 46, 127 White, M.F., 246 White, R.G., 208,246 Whitmore, M.E.. 106, 117 Wicks, C.F., 246 Widell, K.O., 290, 295 Wiegel, H.-J., 114 Wientjes, F.B., 169 Wild, A . , 87, 92, 110, 127 Williams, D.R., 174 Williams, J.H., 90, 101, 106 Williams, L.E., 5 1 , 53, 123 Williams, P., 106 Wilson, C. E., 205,246 Wilson, D.O., 177 Wilson, J., 212, 215. 216, 234,246 Wilson, R.E., 246 Winner, W.E., 4 , 11, 12, 17, 18, 19, 20, 22, 29, 33, 56, 63, 64, 101, 107,112,119,127 Winter, K., 105 Witherspoon, A.M., 122

AUTHOR INDEX

Wittenberg, J.B., 161, 185 Witty, J.F., 131, 159, 161, 181, 185 Wolf, H.F., 259,295 Wolfenden, J . , 89, 103,109, 127 Wolff, J., 109, 120 Wolk, C.P., 175 Wolting, H.G., 108 Wong. C.H., 115 Wong, M.N., 187 Wong, P.P., 155, 163,167, 185 Wong, S.C., 245 Wood, E.A., 167 Wood, J.M., 170 Wood, S.M., 132, 134,176 Woodruff, N.P., 234,246 Worrell, R., 205.244 Wright, B.E., 171 Wright, E.A., 118 Wukasch, R.T., 113

Y Yamaguchi, M., 157, 170 Yamamoto, Y., 142, 144, 159,182 Yang, A.F., 132, 134,177,185 Yang, Y.-S., 12, 55,63, 68, 70, 71, 108,109. 127

311

Yarosh, O.K., 148, 185 Yasuda, T., 128 Yates, M.G., 153, 185 Yazaki, J., 127, 128, 186 Yocum, C.S., 133, 159, 161, 183 Yokoyama, M., 112 Yonemori, K., 51,118 Yoneyama, T., 41,127,128, 141, 146, 160,173,174,182, 186

Yoshida, T.. 186 Yushkova, L A . , 174

Z Zeiher, C., 177 Zelechowska, M., 170 Zemlyanukhin, A . A . , 179 Zemroch, P.J., 244 Zengbe, M., 165 Zhiznevskaya, G.Ya., 165 Ziegler, I., 19, 128 Ziegler-Jons, A., 124 Zipfel. W., 106 Zlotnikov, K.M., 152, 156, 186 Zogbi, V., 184

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SUBJECT INDEX

A a-mannosidase, 139 a-tocopherol, 78 AA, see Ascorbic acid ABA, see Absisic acid Abies alba (fir), 13. 14 air pollutants bioindication, 89 bioindication, 97 dieback, 102 0 3 fumigations, 56, 57 0 3 / S 0 2exposures, 75 SO2 exposure, 30 Abiesfraseri (Fraser fir), 54, 66 Abrasion and facts/fallacies/mysteries, 237-38 Absisic acid (ABA), 59 “Abunda”, 37 ACC, 98 Acceptance angle. 258, 2 6 6 8 fibre optic microprobe, 267 light reflection, 270 measurement tank, 267 probe sensitivity, 269 Acceptance widths of microprobes, 268 Acerplatanoides, 57 Acer saccharinurn (silver maple), 72, 76 Acer saccharurn (sugar maple), 54, 71 03/S02 exposures, 78, 81 SO2exposure, 29 Acid fog, 81 Acid mist, 94, 98 Acid precipitation (AP), 104 0 3 , 79-82 Acid rain, 97, 99 Additive responses and 0 3 6 0 2 exposures, 78 Adenylates, 62 ADP and O3 exposure, 64, 68 Aerodynamic resistance, 197 Age, influence of and O3exposure, 65-6

Agricultural crops air pollutants bioindication, 87 bioindication, 91 0 3 exposure, 67 SOziNO2 mixtures, 49 Air flow and Populus leaf, 216 Air pollutants bioindication methods, 87-90 Air pollutants, specific, 104 Air pollution, 99 anthropogenic, 101 combinations, 82-5 exposure of plants, 4-7 chamberless exposure, 4-5 laboratory exposures, 6-7 open/closed-top field chambers, 5-6 prediction, 84-104 bioindication, 85-101 bioindication and resistant plant selection, 104 early detection bioindications for novel forest decline, 101-3 Air-exclusion system, 5 “Alcala SJ-2” cv., 54 Alfalfa, see Medicago sativa “Alibis” cv., 55 Allantoic acid, structure of, 157 Allantoin, structure of, 157 Almond orchard, wind velocity in, 203 “Alsweet” cv., 71 “Ambassador” cv., 36 Aminoethoxyvinylglycine, see AVG Ammonia, 93 AMP, 64 Annuals, 34 Anthopogenic tropospheric ozone, see 0 3

Antioxidant system, 94 AP, see Acid precipitation ATPase, 138

313

314

SUBJECT INDEX

Arachis hypogaea (peanut), 132, 137 nodules, 131, 134, 155 Arabidopsis, 235 Arid environment, 228 temperature differences, 232 transpiration rate, 232 “Arlington” cv., 54 Artemisia vulgaris, 69 Ascorbate, 78 see also Ascorbic acid Ascorbic acid, 77, 95 Asparagine, 131, 155-6 Astragalus alpinus, 132 ATP, 145 O3 exposure, 59, 62, 64, 68 Atriplex sabulosa , 11 Atriplex triangularis, 11 Avena sativa (oats), 37, 89, 97 NO, exposure, 35 0 3 exposure, 51 O3 fumigations, 52 AVG, 59,97,98 Azimuthal profile, 290

B Bacteroids functions and carbon processing, 146-52 functions and nitrogen processing, 153-4 nodule anatomy and terminology 139-40 transport to, 146 Band-pass filter, 208 Barley, see Hordeum vulgare Bean crop, 194 Bean plants, 76 Beech, see Fagus sylvatica “Beeson” cv., 13, 90 SO2/NOz exposures, 45 “Be1 W3” cv., 52 Betula lutea (yellow birch), 9 Betula papyrifera (paper birch), 15 Betula pendula (European white birch), 9, 15, 16, 90, 96 canopies, 101 S02/N02 exposures, 45 Betula populifolia (gray birch), 9 Bioindication air pollution prediction, 86101 anatomical/morphological analysis, 98-9 endogenous elements, 92-3

endogenous enzyme activity, 94-5 endogenous metabolites, 93-4 genetic analysis, 99 resistant plant selection, 104 Birch, see Betula pendula Bisulphite, 78, 97 Black oak (Quercus velutina), 71 “Black Valentine” cv., 9 “Blaze” cv., 10 Blue blindness, 290 Boreal forests, 81 Borosilicate glass, 259 Boundary layer conductance, 215,219,220,225, 228, 229 convection, 211 heat flux, 214 structure, 211-3 wind and energy transfer, 210-5 laminar, 211, 212,215,216 turbulent, 211, 212. 216 Bowen ratio method, 197 Bradyrhizobium japonicum, 148, 153, 162 Brassica nigra, 88, 94, 280 Broad bean, see Vicia faba Broadleaved trees, 33, 48 Bronzing, 92 Buffer coatings, 262 “Bush Blue Lake 274” cv., 72 C

C2H4, see Ethylene Ca2+,82 “California Wonder” cv., 80 Calvin cycle enzymes, 92 “Canadian Wonder” cv., 45 “Capri” cv., 74 Capsicum annuum (green pepper). 80 Carbon metabolism, 149-52 legume nodules, 144 mutants of rhizobia, 150 processing, 140-52 bacteroid functions, 146-52 host functions, 141-6 transport and metabolism, 129-64 uptake, mechanisms of, 1 4 6 8 uptake and mutants of rhizobia, 147 see also C 0 2 Carotenes, 78 Carotenoid, 92 Carya illinoensis (pecan), 10

315

SUBJECT INDEX

Cell typesitissues in nodule anatomy/ terminology, 131-4 Cells, infected in nodule anatomy/ terminology, 134-9 Cereal fields, 239 Chalcogenide glass, 260 Chamber input concentration, 18 Chamberless exposure, 4-5 “Champion” cv., 72 Charcoal filtering, 62 “Charger” cv., 53 Chemical etching, 260-62 Chemical protectants, 5 “Cherry Belle” cv., 16, 56, 57 Chestnut oak, 99 Chlorophyll content/fluorescence. 92 Chlorosis, 92 Chromium, 263 evaporated, 265-6 Cicer arietinirm. 89 Cladina rangiferinu, 80 Cladina stellaris, 80 Closed-top field chambers, 6 Clover (Trifolium repens), 54, 130, 132, 141 COz. 5 . 23 bioindication, 86, 91 carbon processing. 143 fluxkoncentration over forest, 200 NO, exposure, 35, 38, 39. 40 0 3 exposure, 59.62, 64, 66, 69 SO2 exposure, 18, 19, 32 S02/N02 mixtures, 46, 48 “C043” cv., 88 Coating fibre optic microprobes, 265-6 Coating loss, 278-9 “Comet” cv., 15 Computer models, 33 Concentration gradients. 196, 197 Conifers air pollutants bioindication, 87 bioindication, 91, 93 O3 exposure, 67 “Conquest” cv., 52 Continuously stirred tank reactors, (CSTR). 6 Contorta x banksiana, 88 Convection boundary layer conductance, 211 forced, 215 free, 215, 216 sensible heat transfer, 235 Convective energy flux, 215-7

Corn, see Zea mays Cotton, see Gossypium hirsutum Counter-gradient position, 200 Cowpea, 135 nitrogen processing, 156, 159 Crassirla stems, 276 CSTR, see Continuously stirred tank reactors Cucumber, see Cucumis sativus Cucumis sativus (cucumber), 15. 72 0 3 / S 0 2exposures, 76 Cucurbita pepo. 274, 275 cotyledons, 273. 277, 278, 281 etiolated, 279 light travel, 280 “CUF101“ cv., 15 Cuticle, 223 development, 2 2 3 4 wind damage, 224 Cuticular conductance. 220 wind and energy transfer, 2 2 3 4 Cyclitols, 141 D d values, typical, 195-6 Dark respiration, 42, 67 absolute, 68 S02IN02 mixtures, 47 Dehydroascorbate (DHA). 95 “Delaware” cv., 70 Delayed light emission (DLE). 100 Desert shrubs, 20 Detection bioindications, early, 101-3 Detection, early parameter, 93 Detoxifying response, 95 DHA, see Dehydroascorbate Dicarboxylic acids, 136, 146, 148, 149, 151, 163 transport, 140 Dieback zone, 237 Diffusive resistance, 197 air pollution combinations, other, 84 N02/S02 mixtures, 43-9 NO, exposure, 34-42 long-term, 40-2 short-term, 38-9 03,49-69 03/acid precipitation, 79-82 SO2 exposure, 7-34 long-term, 3&2 SO*/NOz exposure. long-term, 48-9 short-term, 47

-

316

SUBJECT INDEX

Diffusive resistance (cont.) SO2/O3mixtures, 69-78 Digital image analysis, 92 Diode, 271 Diplacus aurantiacus, 12, 20, 25 DLE, see Delayed light emission Dosage, 7 Dose-response, linear, 35 Dose-response functions, 8 Drag coefficients, 205 Drag force. 196, 198 Drought, 48 stress, 66 “Dylan” cv., 9, 10 03/S02fumigations, 71 E Eastern hemlock, 99 Eastern white pine, see Pinus strobus ECD, see Electrical conductivity Ecological effects and 0 3 , long-term, 69 Ecological effects and S 0 2 , long-term, 34

Ecological phenomena, 237-40 EFE, 97 Effective dose, 7 Electrical conductivity (ECD), 100 Electron microscopy grid, 264 Electronic entrance indicator, 271 Energy balance equation, 208-8, 224-32

English Mechanic and World of Science, 232 Entner-Doudoroff pathway, 149 Environment NO, exposure, 38 O3 exposure, 61, 66-7 03/S02 exposures, 74-5, 78 parameters, 222 S02,20-1, 234,29-30, 31 S02/N02 mixtures, 47 stresses, 87-90 Epidermal focussing, 286 Medicago sativa, 285 Oxalis , 285 “Essex” cv., 12

air pollutants bioindication, 88 03/S02 fumigations, 71 Etching, chemical, 260-62 Ethylene (C2H4), 59,98 emission, 103 formation from methionine, 97 see also Stress ethylene

Euonymous japonica Os/NOz exposures, 84 03/S02 fumigations, 71 SO:! fumigation, 9 S02/N02 exposures, 44 European white birch, see Betula pendula Evaporated chromium, 265-6 Evaporated metal coating, 263-6 Excess resistance, 197 Experimental apparatus for fibre optic microprobes, 270-72 Experimental measurements fibre optic microprobes, 273-89 signal interpretation, 283-9 tissue effects, 283 Exposure, 65-6 chamber, 18 long-term, 3 M 2 short term, 35-9 External dose, 7 F Facts/fallacies/mysteries, 231-40 abrasion, 237-38 ecological phenomena, 238-40 thigmomorphogenesis, 233-36 Fagus sylvatica (beech), 87, 91 air pollutants bioindication, 89 bioindication, 93, 103 Fallacies, see Facts/fallacies/mysteries False lens effects, 286-7 Far field, 201 Far red light, 290 Fatty acids, 94 “Feltham First” cv., 61 0 3 fumigations, 52 Festuca arundinacea (grass), 237 Fibre optic microprobes acceptance angle, 267 cleaning, 265 coating, 265-6 experimental apparatus, 270-72 experimental measurements, 273-89 fabrication, 261 grinding and polishing, 263-5 light measurement, 287 measurement, 256-71 probe sensitivity and acceptance angle, 266-8 probes and optical properties, 268-70

prognosis/future applications, 289-92

317

SUBJECT INDEX

system, 270 terminology, 272-3 Fibre optic sensor, spherical, 281 Field chambers, 27 Fir, see Abies alba Flacca mutant of tomato, 39 Flows, nature of near plants, 201-5 Fluence rate, 272, 273 internal, 268, 272,274, 289 Fluoride, 260 Flux gradient analysis, 197, 198,200, 201 failure, 199 one-dimensional, 240 Fog events, 81, 82,102 Forest, 239 see also Boreal forest; novel forest decline; replanting forest Fourier transformation, 207 Fraser fir (Abiesfraseri), 54, 66 Fraxinus americana (white ash), 71 03/AP, 81 O3/SO2 exposures, 78 SOz exposure, 29 Fraxinus excelsior, 13 Free proline, 93 Free radicals, 78, 94, 95 Frost injury, 67 “FS-51” CV., 51, 53 Fungi, 290 Fused silica fibre, 259 Fusicoccin, 74 G G-6-PDH, 100 “Gales County” Prov., 80 Gadheat exchange resistances, 220 Gaussian distribution, 268 g,, calculation, 213-5 GDH, see Glutamate dehydrogenase Gel electophoresis, 138 Geranium carolinianum, 10, 16, 21 SO2 exposure, 18, 25, 28 Ginkgo plant, 22 Glutamate, 152 dehydrogenase (GDH), 93,96,99, 155 oxaloacetate transminase (GOT), 99-100 synthase activity, 155 Glutamine, 93 Glutathione (GSH), 78, 95 levels, 95 reductase (GR), 94, 95

Glycine max (soybean), 13, 15 air pollutants bioindication, 88, 89, 90 bioindication, 94, 97, 101 carbon processing, 141, 142, 143, 152 carbodnitrogen transport, 130, 131, 134 infected cells, 136 legume nodule structure, 131 nitrogen oxides fumigations, 37 nitrogen processing, 154, 156, 158, 159 nodule, cross-section, 133 nodule, infectedhninfected cell, 135 NO, exposure, 38,42 0 3 exposure, 58,60,65, 66, 68 0 3 fumigations, 54, 56, 57 03/N02 exposures, 84 03/S02 exposures, 70, 76, 79 03/S02 fumigations, 71, 72, 73 SO2 exposure, 18, 25 SO2 fumigation, 9, 10, 12 S02/N02 exposures, 43,44,45, 47 strain, T219 9 Glycolytic enzymes, 148 Gold, 263 “Golden Cross Bantam” cv., 12 Golgi processing, 137 Cossypium hirsutum (cotton), 54 0 3 exposure, 56, 66 SOz/NOz mixtures, 47 GOT, see Glutamate oxaloacetate transminase “GR3” cv., 88 Graded index, 258,259 Gram-negative bacteria, 140 Grapevine, see Vitis vinefera Grashof number, 214 Grasses, 235 SOz/N02 mixtures, 48, 49 Gravity and wind in tree crown, 207 Gray birch (Berulapopulifolia), 9 “Great Green Longpod” cv., 9 Green pepper (Capsicum annuum), 80 Grinding and polishing of fibre optic microprobes, 263-5 Grinding and polishing holder, 264 GSH, see Glutathione GSSH, 94 H H’, 79 HzOz, 19,94

318

SUBJECT INDEX

H2O flux over forest, 200 H2S, 94 Hardwoods air pollutants bioindication, 87 bioindication, 91 O3 exposure, 67 “Hark” cv., 13 “Harosoy 63” cv., 72 Hartel-turbidity, 100 HCO;, 19 Heat flux and boundary layer conductance, 214 Heat flux over forest, 200 Heat transfer estimation, 196-7 Heat transfer, sensible, 235 Heat/gas exchange resistances, 220 Heating and probes, 261 Heating and stretching versus chemical etching, 260-62 Helianthus annuus (sunflower), 10, 11, 15, 276, 282 nitrogen oxides fumigations, 36 NO, exposure, 39, 40,41 O3 exposure, 50, 58 O3 fumigations, 53 0 3 / N 0 2 exposures. 84 scattered light, 283, 284 SO2 fumigations, 10, 11, 12 Helical roll vortices, hypothetical, 239 Heteromeles arbutijolia, 12, 20 HN03 NO, exposure, 42 03/S02exposures. 79 SO2/N02 mixtures, 48, 51 “Hodgson” cv., 54, 56 Hordeum vulgare (barley), 10, 13 NO, exposure, 41 03/S02 exposures, 75 Horizontal stepping motor, 270 Host functions and carbon processing, 141-6 HSO;, 79 Hydrofluoric acid, 260, 262 Hydrogen peroxide, 77

I “1-214” CV., 51 IMP synthesis, 159 Indicator plants, 85 Infected cells organization, 134-9 Infra-red light, far, 274, 275 Injuries, plant, 87-90

Interaction mechanisms and O3/SO2 exposures, 77 Interfacial apoplast, 136 Internal fluence rate, 268,272,274, 289 Invisible injury, see Bioindication Irradiance, 272 “Ives” cv., 70

J Jack pine, see Pinus banksiana Japonicum, 147 K K+ efflux, 31 K+ flux, 236 KCN, 145 “Kennebec” cv., 36 Kidney bean, see Phaseolus vulgaris Kirchoff‘s law, 210 Klebsiella pneumoniae, 153 Krebs cycle, 93 Kubelka-Munk equations, 277, 278, 280,281,292

L Labaria pulmonaria, 80 Laboratory exposures, 6-7 Lactate, 152 Lactuca sativa (lettuce), 36, 290 NO, exposure, 38,40 Lagrangian specification, 201 Laminar boundary layer, 211, 212, 215, 216 Larch plantation turbulence, 204 Larrea tridentata (shrub), 12, 15, 20 Laser spectroscopy, picosecond, 280 Leaf conductance, 219 water vapour, 222 cross-sections, 279 pigments, 92 reflectance, 101 temperature, 226, 227,228,229, 230 Legume nodules, 132,164 carbonhitrogen metabolism, 144 infected cells, 136 Leguminosarum, 150 carbon uptake, 147 Lens effects, false, 286-7 plano-convex, 286,287 signatures, 284-6 Lettuce, see Lactuca sativa

319

SUBJECT INDEX

Lichens, 61 03/AP, 81 Light adaptation index, 93 distribution curves, 273, 277, 278 gradients, 290 Spinacia oleracea, 282 measurement fibre optic probe, 287 probe acceptance width, 277 terminology, 272-3 thick samples, 273-7 migration, 292 reflection acceptance angle, 270 travel and Cucurbita pepo, 280 travel and Spinacia oleracea, 281 wavelengths, 277-8 Linear dose-response, 35 Lipid peroxidation, 68 Lipids, 68 Lipopolysaccharide, 137 Liquidamber plants, 234 Liriodendron tulipifera (yellow poplar), 72 03/AP, 80, 82 03/ S02 exposures, 78 “Little Marvel” cv., 12, 13 Loblolly pine, see Pinus taeda “Local” cv., 88 Lolium perenne (ryegrass), 10, 13, 15 nitrite reductase activity, 47 SO2exposure, 27, 31, 32 Lotus pedunculatus, 131, 132 Lucerne, 30 Lycopersicon esculentum (tomato), 36 Lycopersicon lycopersicum (tomato), 37 M MACC, 97 “McCall” cv., 71 Magnesium, 93 deficiency, 102 see also Mg2+ Maize, see Zea mays Malate, 143 dehydrogenase, 149 synthesis, 31 Malonate, 141, 149 Malus fruits, 236 Mandarin leaf stomata, 51 Mass transfer estimation, 196-7 Medicugo sativa (alfalfa), 10, 15, 58, 130,280, 281

air pollutants bioindication, 87 coating loss, 289 epidermal focussing, 285 legume nodule structure, 132 nitrogen oxides fumigations, 36 nodules, 136, 155 NO, exposure, 35 0 3 exposure, 66 S02/N02 mixtures, 43, 44 surging, 288 Meliloti, 147, 150 Meristem temperature, 218 Metabolism and transport of carbon, 129-64 Metabolism and transport of nitrogen, 129-64 Methionine, 96 Mg2+,82 Microaerobic conditions restrictions, 161-3 O2 and metabolism impact, 161-3 0 2 regulation system, 16&1 Micrometeorology, classical, 193-8 Micrometeorology, limitations, 199-208 Micropr obe acceptance widths, 268 fabrication, 26C70 fibre optic, 261 heating and stretching versus chemical etching, 260-62 sputter coating and probe tip truncation, 262-3 grinding and polishing, 263-6 Mimosa, 235 Mitochondria, 134-7 “Moapa” cv., 87 Moh hardness scale, 263 “Money Maker” cv., 36 Monochromator, 270 Monoclonal antibodies, 138 “Monosa” cv., 13 Montane environment, 224-5 temperature differences, 226 transpiration rates, 227 Mountainous needle yellowing, 102 Multimode step index, 258,259,260, 268 Mung-bean, see Vigna radiata Mysteries, see Facts/fallacies/mysteries

N NzOs, 51

320

SUBJECT INDEX

NA, see Numerical aperture NAD, 151 NADH/NAD ratios, 162 NADP, 68, 145, 151 NADPH, 68, 78 “National Pickling” cv., 72 Near field, 201 Near infra-red wavelength, 101 Necrosis, 92 NFD, see Novel forest decline NH3 assimilation, 145 NHd, 138 assimilation, 154-5 Nicotiana tabacum, 52 0 3 exposure, 58, 59 Nicotianurn, 59 Nitrate, 41 Nitric oxide, see NO Nitrite ions, 41 Nitrite reductase, 47, 84 Nitrogen content, 40 fixation, 148 metabolism, 144 oxide, see NO; NOz processing, 153-60 bacteroid functions, 153-4 transport and metabolism, 129-64 Nitrogenase, 151, 152 Nitrogenous compounds, 41 Nitrogenous gases, 34 NO, 34 bioindication, 91, 92, 97 fumigations, short and long-term, 367 NO, exposure, 35, 38, 40, 42 N02,34 bioindication, 91,97, 101, 104 NO, exposure, 35, 38,39,40, 41, 42 O3 exposure, 61,62,68 03/S02 exposures, 70, 79 SO2 exposure, 43-9 diffusive resistance, 47 long-term, 48-9 respiration responses, 47-8 short/long-term, 44-5 SO2 fumigation, 46 sunflower leaves, 83 see also 03/N02exposures; SO2 NO2 O3 NO3 and 03/S02 exposures, 79 Nodule anatomy and terminology, 131-40

+

+

bacteroids, 139-40 infected cells organization, 134-9 tissues and cell types, 131-4 Nodule, cross-section, 133 Nodule, infectedhninfected cell, 135 Northern red oak, 81 Norway spruce, see Picea abies Novel forest decline (NFD), 69, 86, 92, 93,97 air pollutants, specific, 104 bioindication, 99 Novel spruce decline, 91 NO,, 293 bioindication, 97, 102 detoxification, 84 exposure, long-term, 40-2 exposure, short-term, 38-9 03/S02 mixtures, 70 photosynthesis response and diffusive resistance, 34-42 S02/N02 mixtures, 43 stomata1 uptake, 38-9, 41-2 “Nugget” cv., 11, 53 Numerical aperture (NA), 257, 258 Nusselt number, 213,214 Nutrients, 92, 102 Nutritional status, 82 0 “1-214” CV.,11 02, 51 carbon processing, 145 infected cells, 135, 136 microaerobic conditions restrictions, 161-3 0 3 exposure, 64 see also Superoxide 0 3 , 2 , 3, 5 acid precipitation, 79-82 bioindication, 91, 95, 97, 98,99, 100, 101, 104 ecological effects, long-term, 69 endogenous metabolites, 94 fumigations short and long-term, 52-7 leaf pigments, 92 long-term response, 68 NO2 exposure, 84 NO, exposure, 39,42 photosynthesis response, 49-69 long-term, 62-9 short-term, 51-62 poplar, hybrid, 67

SUBJECT INDEX

respiration response, 67-8 short-term, 50, 62 SO2 fumigations, 71-3 long-term, 7 5 9 short-term, 70-5 SO2 mixtures, 69-78 soybean, 60 stomata1 uptake and plant response, 59-61 sunflower leaves, 83 tropospheric, 102 see also S 0 2 + N 0 2 + 0 3 Oats, see Avena sativa “Ogle” cv., 89 OH, 77 Ohms’ law, 210 Onobrychis viciifolia, 130 Open-top chambers, 27, 62-3 Open-top field chambers, 5-6 Optical fibre, 257-60 acceptance angle, 258 buffer coatings, 262 general characteristics, 257-8 guiding of light, 257 transmission characteristics, 259-60 types, 258-9 Optical properties and probes, 268-70 Organic acids, 140-5 Orthophosphate, 68 Oryza sativa (rice), 11, 88 OTC, see Open-top field chambers Oxalis, epidermal cells on, 285 Oxidant stress, 94 Oxides, 259 Oxygen regulation system, 160-1 Oxygen-hydrogen torch, 262 Oxyradicals, 58 03/s02 exposures, 77 Oxytropis arctobia, 32 Oxytropis maydelliana, 132 Ozone, 103 bronzing, 92 exposure, 95 soybean, 65 see also O3 P Paper birch (Betula papyrifera), 15 PAR, 281 Paraffin wax, 264, 265 “ P a r k cv., 37, 52 Particulates, 84

321

Pea, see Pisum sativum “Peace” cv., 51, 53 Peanut, see Arachis hypogaea Pecan (Carya illinoensis), 10 Pelargonium, 22, 23,24 Pelargonium x hortorum, 11 Penman-Monteith equation, 217 PEP, 145 concentration, 143 microaerobic conditions, 162 PEPC, 142, 143 PEPO, 145 Perennial shrub (Salvia rnellifera), 16, 34 Perennials, 49 Periplasmic space, 140 Peroxidase (PO), 95 Petunia, 75 cultivars, 74 Petunia hybrida, 71 Phaseoli bacteroids, 153 Phaseolus radiatus, 94 Phaseolus vulgaris (kidney bean), 11, 12, 13, 14,276 air pollutants bioindication, 88, 89 bacteroid functions, 151 bioindication, 94 carbon processing, 146 legume nodule structure, 132 nitrogen oxides fumigations, 36 nitrogen processing, 155 NO, exposure, 35,39 O3 exposure, 58, 59, 61, 68 O3 fumigations, 52, 53, 54 03/N02 exposures, 84 0 3 / S 0 2exposbres, 77 0 3 / S 0 2fumigations, 71, 73 SO2 exposure, 23 SO2 fumigation, 9 SO2 long-term effects, 28 SO$NOz exposures, 45, 48 Phenols, 95 Phenomena, ecological, 238-40 Phenomena, wind-speed dependent, 234 Phleum pratense (Timothy grass), 16, 30 SOz/NOz exposures, 45,48 Phloem import, 134, 164 Phosphoenolpyruvate carboxylase, see PEPC Phosphotungstic acid (PTA), 137 Photomorphogenesis, 290

322

SUBJECT INDEX

Photon migration, 280 Photorespiration, 42 O3 exposure, 68 SOzIN02 mixtures, 47 Photosynthesis diffusive resistance, 86, 91 response air pollution combinations, 82-4 curves, dorsiventral, 292 mechanisms, 58, 64 NO, exposure, 34-42 long-term, 39-42 short-term, 35-9 03,49-69 O3 and acid precipitation, 79-84 0 3 / S 0 2mixtures, 69-78 long-term, 76-8 short and long-term, 78 short-term, 70-5 s02,7-34 short-term, 17-26 SO2 and NO2 mixtures, 43-9 long-term, 48-9 short-term, 43-8 reversibility and visible symptoms, 21 stomata1 responses, 2-105 Phototropism, 290, 291 Phycomyces sporangiophore, 286, 290 Phytochrome, 290 Phytohormone, 96 Phytotrons, 6, 7 Picea abies (Norway spruce), 13, 15, 16 air pollutants bioindication, 87, 88, 89, 90 bioindication, 98,99,100, 103, 105 nitrogen oxides fumigations, 37 NO, exposure, 38 O3 exposure, 58,66 O3 fumigations, 52, 57 SO2 exposure, 30 SO2 fumigation, 11 S 0 2 / N 0 2 exposures, 45 Picea excelsa (spruce), 14,88,92 air pollutants bioindication, 90 bioindication, 93 needles, bioindication, 94, 97 0 3 exposure, 63 0 3 / S 0 2 exposures, 75 SO2 exposure, 27, 30 stressed, 100 tree acceleration, longitudinal, 209 wind, uIvIw components of, 209

Picea sitchensis, 236 Picea spp., 82, 104 Picosecond laser spectroscopy, 280 Pine, 19, 81 grafts, 76 O3 exposure, 66 SO2 exposure, 28, 29 species, 93 Pinto beans, 62 “Pinto” cv., 11, 12 O3 fumigations, 53, 54 Pinus banksiana (Jack pine), 13, 15 SO2 fumigation, 9 Pinus elliottii, 55, 65 Pinus nigra, 11 Pinus pinea, 11 Pinus ponderosa, 16, 69 SO2 exposure, 30 Pinus spp., 90 hybrid, 88 populations, 105 Pinus strobus (eastern white pine), 53, 54, 55, 65, 69,99 air pollutants bioindication, 89 O$SO2 exposures, 81 Pinus sylvestris (Scots pine), 13, 15, 16 air pollutants bioindication, 87, 88, 89 bioindication, 96, 99, 103 nitrogen oxides fumigations, 37 NO, exposure, 41,42 O3 exposure, 67 O3 fumigations, 53, 56 SO2 exposure, 27,29, 31, 32 SO2 fumigations, 11, 12 Pinus taeda (loblolly pine), 55, 65 NO, exposure, 38 03/AP exposures, 80 Pisum arvense, 132 Pisum sativum (pea), 13, 15, 130, 138 bioindication, 97 carbon processing, 144 frost injury, 67 legume nodule structure, 132 O3 exposure, 61 O3 fumigations, 52, 53 0 3 / S 0 2 exposures, 70, 74, 75 03ISO2 fumigations, 71 SO2 exposure, 25,31,32 SO2 fumigation, 11, 12 SO2/NO2 exposures, 44 Pitch Dine, 99 P1ano:convex lens, 286, 287

323

SUBJECT INDEX

Planta, 233 Plants, waving, 205-8 Plants and wind, 189-240 Plastids, 164 Platinum, 263 PO, see Peroxidase Poinsettia cultivars, 32 Polhausen relationship, 213 Polishing and fibre optic microprobes, 263-5 Polyamines, 93 Polysaccharide, 139 Poplar, see Populus euramericana Populus deltoides x trichocarpa, 16, 72 Populus euramericana (poplar), 11, 87 air flow, 216 air pollutants bioindication, 88 bioindication, 9, 95 hybrid, 54, 55 clone, 76 03,67 O3 fumigations, 57 03/S02exposures, 81 O3 exposure, 50, 51, 58 0 3 fumigations, 53 SO2exposure, 19 Populus maximowizii, 16 Populus tremuloides, 12 Potato, 36 Probe acceptance width, 269 light measurements, 277 etching/stretching/heating, 261 optical properties, 268-70 orientation, 273-7 sensitivity and acceptance angle, 2664,269 tip coating loss, 278-9 grinding/polishing , microprobe fabrication, 2 6 S 6 light reflection, 270 truncation and sputter coating, 262-3 “Processor” cv ., 13, 15 “Progress” cv., 11 Protease inhibitor, 139 Proteins, 20, 68, 94 Pseudoparmelia caperata, 53, 54 Pseudotsuga, 90 PTA, see Phosphotungstic acid “Pure Gold Wax” cv., 36 Purines, synthesis of, 158 I

Q

Quantum flux density, 20, 26, 29 Quercw velutina (black oak), 72

R Radish, see Raphanus sativus Ramalina menziesii, 53, 54 “Ranger” cv., 10 nitrogen oxides fumigations, 36 S02/N02 exposures, 44 Raphanus sativus (radish), 15, 16, 56 bioindication, 101 O3 fumigations, 56, 57 03/S02 exposures, 76 03/S02 fumigations, 72 Rayleigh scattering, 259 “Red Kidney” cv., 11 Red light, 290 Red spruce, 98 Refractive index, 258, 283-4 Remote sensing bioindication, 101 Replanting forest, 86 Respiration response NO, exposure, long-term, 42 NO, exposure, short-term, 39 O3 exposure, long-term, 67-8 O3 exposure, short-term, 61 SO2 exposure, 25-6 SO2 exposure, long-term, 32-3 S 0 2 / N 0 2exposure, 47-8 Respiration response, see Dark respiration; photorespiration Response, long term to SO;?and NO2 mixtures, 48-9 Reynold’s number, 212 Rhizobia, 140, 148 carbon metabolism mutants, 150 carbon uptake mutants, 147 cultured, 149 genotype, 143 infected cells, 136 microaerobic conditions, 162 nitrogen processing, 153 Rhizobium, 146 mutants, 139, 164 strains, 160 Rhizobium leguminosarum, 146, 153 Rhizobium meliloti, 153 Rice (Oryza sativa), 11, 88 Rime ice, 238 RNA transcription, 234 “Robusta” cv., 88 Roughness length, 195

324

SUBJECT INDEX

RuBPC alfalfa foliage, 58 bioindication, 103 carboxylation activity, 64 NO, exposure, 40 O3 exposure, 62 SO2 exposure, 19,20,26,28,29,32,33 “Russian Mammoth” cv., 10, 11, 12, 15 nitrogen oxides fumigations, 36 O3 exposure, 58 O3 fumigations, 53 Ryegrass, see Lolium perenne S

“S23” cv., 10, 13, 15 Sage scrub, 34 Sainfoin, 130 Salvia mellifera (shrub), 16, 34 “Sanilac” cv., 14, 71 Saturation vapour pressure deficit (SVPD), 224,225,226,227 stomatal conductance, 221,222 temperature, 218 transpiration, 219 “Saxa” cv., 52 SBM, see Symbiosome membrane SBS, see Symbiosome space Scaling up, 231-32, 240 Scattered light isotropy, 278-82, 283 thick samples, 278-80 thin samples, 280-82 Sclerenchyma cells, 131-2 Scots pine, see Pinus sylvestris Sedum album, 88,94, 95 Sensible heat transfer, 235 Sesbania rostrata, 132, 152 Shelter, 218 Sherwood number, 213 Shrub (Larrea tridentata), 12, 15, 20 Shrub (Salvia mellifera), 16, 34 Signal interpretation and experimental measurements, 283-9 Silica, 259 Silver conductance paint, 270 Silver fir, see Abies alba Silver maple (Acer saccharinum), 72, 76 Single mode fibre, 258,259, 262, 268 Snap bean, 47 SOz+NOz+03exposure, 84 SOz, 2, 3, 92 bioindication, 100, 104 chlorophyll fluorescence, 93

endogenous elements, 93,94 exposure, 8 bioindication, 96 diffusive resistance, 22-5 long-term, 26-33 diffusive resistance, 30-2 respiration response, 32-3 responses, 33 respiration response, 25-6 short term, 17-26 transpiration response, 8 fumigations, short and long-term, 9-16 NO2 exposure long-term, 48-9 respiration responses, 47-8 short-term, 47 shortAong-term, 44-5 NOz fumigation, 46 NO2 mixtures, 43-9 interaction mechanisms, 47 short term, 43-8 NO, exposure, 39,42 O3 exposure, 58,62,63,68 photosynthesis response, 69-79 03/N02 exposures, 84 Phaseolus vulgaris, 28 response of photosynthesis, 7-34 long-term SOzexposures, 26-34 stomatal uptake, 24-5, 31-2 sunflower leaves, 83 toxicity, 96 Viciafaba photosynthesis, 21 see also O3/SO2mixtures SO3, 79 soi, 79 SOD, see Superoxide dismutase Soil acidification, 102 Soil moisture stress, 75 Solidago canadensis, 69 “Sonja” cv., 10 Sorghum bicolor, see Sorghum Sorghum (Sorghum bicolor), 12 Soybean, see Glycine max Space irradiance, 272 Spatial resolution degradation, 289 Spherical sensor, 272 Spinach, 92, 281 Spinacia oleracea, 13, 279,280 light gradients, 282 light travel, 281 Spring barley, 31 see also Hordeum vulgare

SUBJECT INDEX

Spruce, see Picea excelsa Sputter coating and probe tip truncation, 262-3 Steric energy flux, relative, 273, 276, 28 1 Stomata1 conductance, 220 changing, 228-9 variation, 221 wind and energy transfer, 221-3 density, 32 pollutant uptake, 75 resistance, 70-5 response mechanisms, 59, 64-5 response and photosynthesis, 2-105 uptake, 67 Stress ethylene, 59, 78 formation, 104 production, 96-7 Stress injury, diagnostic methods for, 85-104 Stresses, environmental, 87-90 Stretching in microprobe fabrication, 26M2 Succinate dehydrogenase, 144 Sucrose, 145 gradient centrifugation, 134 Sugar maple, see Acer saccharum Sugars, 141-5 Sulphate, 97 Sulphite, 78, 94, 96 SO2 exposure, 20 Sulphur, 93 see also SO,; SO4 Sulphur dioxide, see SO2 Sunflower, see Helianthus annuus Superoxide, 78 Superoxide dismutase (SOD), 78,95, 96 Surface temperature calculation, 217-8 Surface temperature energy balance equation, 224-32 Surging, 287-8 SVPD, see Saturation vapour pressure deficit Swaying, 207-8 Sweet corn, see Zea mays Symbiosome membrane (SBM), 136, 137-8, 139, 146, 155, 164 microaerobic conditions, 161 Symbiosome space (SBS), 136, 139, 141, 146 Symbiosomes, 134-7 terminology/definitions, 136

325

T “T3” cv., 89 TAA, see Ascorbic acid Taraxacum seeds, 290 Taxonomic relationships, 136 TCA cycle, 145, 149 microaerobic conditions, 162 Temperate environment, 225-6 temperature difference, 228, 236 transpiration rate, 229, 230, 237 Temperature calculation, surface, 217-8 differences arid environment, 232 montane environment, 226 temperate environment, 228, 236 tropical environment, 230 energy balance equation, 224-32 flux over forest, 200 meristem, 218 vapour pressure, 220 wind and energy transfer, 217-220 Terminology in fibre optic microprobes, 272-3 Thick samples, light measurements in, 273-7 Thick samples and scattered light isotropy, 278-80 Thigomorphogenesis, 232-35 Thin samples and scattered light isotropy, 280-82 “Thompson Seedless” cv., 53 “Three Fold White” cv., 10 Thylakoid electron transport, 92 TIA, see Total included acceptance angle Time-response functions, 8 Timothy grass, see Phleum pratense Tissue effects, 283 Tissues and cell types, 131-4 “Titus” cv., 52 “TKM9” cv., 88 Tobacco callus, 62 Tocopherol, 94 Tomato (Lycopersicon esculentum) , 36 Tomato (Lycopersicon lycopersicum), 37 Tomato mutant, 25, 39 Total included acceptance angle (TIA), 258 Transfer coefficients, 196 Transfer functions, 208 Transmission characteristics, 259-60

326

SUBJECT INDEX

Transmission spectra of optical fibre, 260 Transpiration energy balance equation, 224-32 fluxes, 218-220 rate arid environment, 232 calculation, 219 montane environment, 227 temperate environment, 229, 230, 237 wind and energy transfer, 218-220 Transport of carbon, 129-64 Transport of nitrogen, 129-64 Transport role and wind regimes, 193208 micrometeorology limitations, 199208 “Trebi” cv., 10 Tree acceleration, longitudinal, 209 broadleaved, see Broadleaved trees crown, 207 SOz/N02 mixtures, 49 vibrations, spectral method of analysis, 208 wind tunnel, 206 Trehalose, 139, 142 Trichome density, 32 Trifolium pratense, 130 Trifolium repens (clover), 54, 130, 132, 141 Trifolium spp., 147, 150, 236 Triticum aestivum (winter wheat), 14, 54, 55, 72, 88 Tropical environment, 226-8, 230 Turbulence, 204 measured, 202-4 statistics, 202 Turbulent boundary layer, 211,212, 216 U u / d w components, 240 Ureide exporting nodules, 143 Ureide synthesis, 134, 157-9 UV-B region, 259 V Vapour pressure, 220 Vapour pressure deficit (VPD), 82, 222,229, 230

Vibrations, tree and spectral method of analysis, 208 Vicia faba (broad bean), 10, 13 air pollutants bioindication, 88 fumigations, 71 03/S02 mixtures, 70 SO2 exposure, 19,22, 23, 24, 26, 31 SO2 fumigation, 9, 12 SO2 inhibition, 21 Viciae bacteroids, 153 Vigna radiata (Mung-bean), 132 S02/N02 mixtures, 45, 48 Vigna unguiculata, 132, 146 Vines, 70 see also Vitis vinefera Violaxanthin-to-antheraxanthin ratio, 92 Visible injury, see Indicator plants Vitis labruscana, 70 Vitis vinifera (grapevine), 51, 53, 63 Von Karman’s constant, 193 “Vona” cv., 54, 72 VPD, see Vapour pressure deficit W Wakes of individual plants, 204-5 Water stress, 61 Water use efficiency (WUE), 64, 221-2 Water vapour, 222 Wave-regeneration forests, 239 Wave-regeneration pattern, 237 Wavelengths of light, strongly/weakly absorbed, 277-8 “Wayne” cv., 10 “Wells” cv., 10 Wheat, 93 see also Winter wheat White ash, see Fraxinus americana “White Cascade” cv., 71, 74 White oak, 38 White pine, see Pinus strobus “Williams” cv., 37, 56 Wind damage, 224 and energy transfer, 208-232 boundary layer conductance, 210-5 convective energy flux, 215-7 cuticular conductance. 223-4 energy balance equation, 208-10, 224-32 stornatal conductance, 221-3 temperature, 217-220

327

SUBJECT INDEX

transpiration, 218-220 and gravity, 207 patterns, 237 and plants, 189-240 plants, waving, 205-8 profiles above vegetation, 193-5 heathass transfer estimation, 196-7 within canopy, 198 regimes and transport role, 193-208 speed, 196 dependent phenomena, 234 mean, 193 stornatal conductance, 222-3 throw, 206 tunnel, 200, 205, 206 uIvIw components, 209 velocity, 203 “Winsor Harlington” cv., 12, 13 Winter wheat ( T r i t i u m aestivum), 14, 54,55,76 WUE, see Water use efficiency

X Xenon arc lamp, 271 Xylem export, 134, 164

Y “Yecora Rojo”, 88 Yellow birch (Betula h a ) , 9 Yellow poplar, see Liriodendron tulipifera “Young” cv., 57 Young’s modulus, 206

Z ZAPS, 4, 5 Zea mays (maizeicorn), 15 air pollutants bioindication, 88, 89 bioindication, 94 coleoptile, 283, 290 NO, exposure, 38 0 3 / N 0 2 exposures, 84 SO2 exposure, 9, 32 SO2 fumigation, 9, 11, 12 Zero plane displacement length, 195 Z , values, typical, 195-6

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