Advances in Botanical Research, Volume 5

Advances in

BOTANICAL RESEARCH VOLUME 5

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Advances in

BOTANICAL RESEARCH Edited by

M. W WOOLHOUSE Department of Plant Sciences, The University, Leeds, England

VOLUME 5

1977

ACADEMIC PRESS London NewYork San Francisco A Subsidiary of Harcourt Brace Jovanovich,Publishers

ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road, London NW1 7DX

US.Edition published by ACADEMIC PRESS INC. 1 1 1 Fifth Avenue New York, New York 10003

Copyright 0 1977 by Academic Press Inc. (London) Ltd.

All Rights Reserved No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers

of Congress Catalog Card Number': 62-21144 ISBN: 0-12405905-3

Printed in Great Britain by Whitstable Litho Ltd., Whitstable, Kent

CONTRIBUTORS TO VOLUME 5 GOTZ HARNISCHFEGER, Lehrstuhl fur Biochemie der Pfinze der Universitat Gottingen, 34 Gottingen, Germany (p. 1). J. A. RAVEN, Department of Biological Sciences, University of Dundee, Dundee DDl4HN, Scotland (p. 153). DAVID G. ROBINSON, Pfanzenphysiologisches Institut der Universitat Untere Karspale 2,D-34 Gottingen, Federal Republic of Germany (p. 89). MICHAEL A. VENIS, Shell Research Ltd., Woodstock Laboratory, Sittingbourne Research Centre, Sittingboume, Kent, ME9 8AG, England (p. 53).

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PREFACE There has been a welcome extension of the use of physical techniques in the investigation of biological problems in recent years. This development does however have its dangers, on the one hand the biologist with a limited physical background is inclined to take the findings on trust whilst many physicists who turn to biology take insufficient account of the state of the material which they are studying. In the first article in this volume G. Harnischfeger provides a valuable article which should help to meet this sort of difficulty in respect of low-temperature fluorescence studies in photosynthesis. The physical background is clearly presented and the problems of low-temperature artefacts are explored. Progress with respect to the mechanisms of action of plant hormones has lagged far behind that of comparable studies with animals. The work of Hertel and colleagues in Germany and Venis in England has been prominent in rectifying this situation during the past five years. In this volume Venis surveys recent progress in this field and considers some of the requirements for rigorous work on this subject. Most studies in the evolution of land floras are inevitably concerned with detailed anatomical descriptions and comparative studies from the fragmentary fossil record. It is comparatively rare however for a plant physiologist to address himself to this subject; J. Raven attempts this task in the present volume, by considering the constraints imposed on the long distance transport systems of plants in the course of adaptation to the terrestial environment. By bringing together information from physics, plant anatomy and the earth sciences Raven challenges palaeobotanists to take a wider view in the interpretation of the material which they describe. A consideration of recent progress in the study of tile biosynthesis of plant cell walls is given by D. G. Robinson; this subject assumes increasing importance for plant pathologists and botanists concerned with problems of growth at the cellular level. I am indebted to the authors of the chapters in this volume for the care which they have taken in their work which has lightened the editorial task. I am greatly indebted to Mrs. J. Long for preparation of the Subject Index and Miss Jean Denison for Secretarial Assistance. H. W. Woolhouse Leeds 1977

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CONTENTS CONTRIBUTORSTOVOLUME5 PREFACE . . . . . .

. . .

. . . . .

.

.

.

.

.

.

v vii

The Use of Fluorescence Emission at 7TK in the Analysis of the Photosynthetic Apparatus of Higher Plants and Algae GOT2 HARNISCHFEGER I. 11.

111.

IV. V. VI. VII.

Introduction . . . . . . . . . . . . . . . . . . Theoretical Considerations of Pigment Photochemistry . . . . A. General Concepts of the Interaction between Light and Matter . . . . . . . . . . . . . . . . . . B. General Influence of Low Temperature on Fluorescence Properties . . . . . . . . . . . . . . . . . C. Special Features of the Chloroplast System . . . . . . Spectral Distortions, Artefacts and their Prevention . . . . . A. Some Notes on Instrumentation . . . . . . . . . . B. Spectral Distortions Unconnected to Organelle Integrity . . C. Spectral Distortions Due to Organelle Destruction . . . . D. Some Points Regarding the Evaluation of Published Spectra in Liq. N, Fluorescence Spectroscopy . . . . . . . . Structure and Composition of th,” Photosynthetic Apparatus as Determined by Fluorescence at 77 K . . . . . . . . . . Orientation of Pigments within the Photosynthetic Apparatus . . Interactions between Photosystems: Qualitative Aspects and Kinetic Analysis . . . . . . . . . . . . . . . . . Synopsis and Outlook . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

2 2 3 5 5 1 7 10

12 21 22 31 31 48 49 49

Receptors for Plant Hormones MICHAEL A. VENIS I. 11.

Introduction . . . . . . . . . . . Sites of Hormone Action . . . . . . . A. Effects on Macromolecular Synthesis . B. Rapid Effects . . . . . . . . . C. Evidence for Two Sites of Auxin Action

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

53 54 54

56 58

CONTENTS

X

111.

IV .

The Search for Hormone Receptors . . . . . A. Model Systems . . . . . . . . . B. Direct Interaction with Enzymes . . . C. “Soluble” (Nuclear/Cytoplasmic) Receptors D . Membrane-bound Receptors . . . . . Concluding Remarks . . . . . . . . . References . . . . . . . . . . . . .

. . . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . . .

59 59 60 61 71 84 85

Plant Cell Wall Synthesis DAVID G. ROBINSON I.

Introduction . . . . . . . . . . . . . . . . . . 89 Structural Considerations . . . . . . . . . . . . . . 91 A. Nan-Cellulosic Components . . . . . . . . . . . 91 B . Cellulose. . . . . . . . . . . . . . . . . . 96 I11 . The Electron Microscopy of Cell Wall Formation . . . . . . 99 A. Sites of Synthesis . . . . . . . . . . . . . . . 99 B . The Orientation of Cellulose . . . . . . . . . . . 105 IV . Cell Fractionation Studies . . . . . . . . . . . . . . 111 A . Preparation of Cell Fractions and Their Identification . . . 11 1 B. Analysis of Fractions from Pulse-Chase Experiments . . . 1 18 C. Transport of Synthesized Materials . . . . . . . . . 125 V. In Vitro Synthesis . . . . . . . . . . . . . . . . 136 A. Higher Plant Cellulose . . . . . . . . . . . . . 136 B. Bacterial Cellulose . . . . . . . . . . . . . . 138 Chitin . . . . . . . . . . . . . . . . . . 138 C. D. Non-Cellulose Materials . . . . . . . . . . . . 139 Lipid Intermediates . . . . . . . . . . . . . . 140 E. VI . Conclusion . . . . . . . . . . . . . . . . . . . 142 Acknowledgements . . . . . . . . . . . . . . . . . . . 143 References . . . . . . . . . . . . . . . . . . . . . . 143

I1.

The Evolution of Vascular Land Plants in Relation to Supracellular Transport Processes J . A . RAVEN I. I1. I11. IV .

Introduction . . . . . . . . . . . . . . . . . . The Progenitors of Vascular Land Plants . . . . . . . . . The Structure of Early Vascular Plants . . . . . . . . . . The Xylemand Liquid-phase Water Transport . . . . . . . A. The Transpirational Flux of Water . . . . . . . . . B. The Xylem as a Low-resistance Pathway for Mass Flow of Water . . . . . . . . . . . . . . . . . . C. The Significance of Lignification . . . . . . . . .

154 155 162 170 170 172 177

CONTENTS

Transport in the Gas Phase . . . . . . . . . . . . . The Problem of H 2 0 Loss as a Concommitant of PhotoA. synthetic C 0 2 Fixation: Poikilohydry . . . . . . . . Homoiohydry in Vascular Land Plants and the Intercellular B. Space-cuticle-stomata Complex . . . . . . . . . VI. Transport of Dissolved Solutes . . . . . . . . . . . . A. X y l e m . , . . . . . . . . . . . . . . . . B. Phloem . . . . . . . . . . . . . . . . . . C. Symplast and Apoplast . . . . . . . . . . . . . D . Excretion . . . . . . . . . . . . . . . . . VII . The Evolution of Vascular Land Plants; an Hypothesis . . . VIII . Appendix A: Secondary Plant Products in Relation to Vascular Plant Evolution. with Particular Reference to Lignification . . . IX . Appendix B: Role of Intercellular Gas Spaces in Respiratory Gas Exchange of Vascular Land Plants . . . . . . . . . . . X. Summary and Conclusions . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . Author Index . . . . . . . . . . . . . . . . . . . . Subject Index . . . . . . . . . . . . . . . . . . . . V.

xi 181 181 182 192 192 193 197 198 199 206 207 210 211 211 221 233

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The Use of Fluorescence Emission at 7 T K in the Analysis of the Photosynthetic Apparatus of Higher Plants and Algae

GOTZ HARNISCHFEGER Lehrstuhl fur Biochemie der Pflanze der Universitat Gottingen, 34 Gottingen, Germany

I. 11.

111.

IV. V. VI.

VII.

Introduction . . . . . . . . . . . . . . . . . Theoretical Considerations of Pigment Photochemistry . . . . A. General Concepts of the Interaction between Light and Matter . . . . . . . . . . . . . . . . . . B. General Influence of Low Temperature on Fluorescence Properties . . . . . . . . . . . . . . . . . C. Special Featuresof the Chloroplast System . . . . . . Spectral Distortions, Artefacts and their Prevention . . . . . A. Some Notes on Instrumentation . . . . . . . . . . B. Spectral Distortions Unconnected t o Organelle Integrity . . C. Spectral Distortions Due to Organelle Destruction . . . . D. Some Points Regarding the Evaluation of Published Spectra In Liq. N, Fluorescence Spectroscopy . . . . . . . . Structure and Composition of th,” Photosynthetic Apparatus as Determined by Fluorescence at 77 K . . . . . . . . . . Orientation of Pigments within the Photosynthetic Apparatus . . Interactions between Photosystems: Qualitative Aspects and Kinetic Analysis . . . . . . . . . . . . . , . . . Synopsis and Outlook . . . . . . . . . . . . . . . Acknowledgements References . . . . . . . . . . . . . . . . . . . ~

1

2 2 3 5 5 7 7 10 12

21 22 31 37 48 49 49

2

GOTZ HARNISCHFEGER

I. INTRODUCTION

Since Dewar (1894a, b) first reported, that organic dye compounds emit light more strongly at 77°K than at room temperature, fluorescence measurements at low temperature have become commonplace. Dewar's experiments were quickly followed up and enough evidence had accumulated around the turn of the century, that Nichols and Merrit (1904) could publish a first systematic compilation of molecular emission properties obtained with this method. In the following decades, the work of Borisow, Kautsky and Pringsheim, to name just a few, highlighted a widely expanding field. Today low temperature fluorescence spectroscopy is a well established analytical tool in organic and physical chemistry. This particular aspect of the topic has been reviewed in depth by Meyer (1971). Investigations of the spectral properties of biological mattrial at low temperatures started with the work of Hartridge (1921) on haemoglobin. The emphasis was mostly on absorption rather than fluorescence, but many basic samplehandling techniques were developed in the course of these studies. Worth noting is the work of Lavin and Northrop (1935) who developed the glycerol-glass method, and Keilin and Hartree (1949), who described many of the spectral phenomena due to cooling of biological material. First reports using fluorescence techniques at low temperature in photosynthesis appeared in the late fifties (Tollin and Calvin, 1957). The discovery of the intense emission band around 720 nm in Chlorella at 77'K by Brody (1958) established the technique firmly as a valuable tool for obtaining information about the nature and structural arrangement of photosynthetic pigments. The subsequent research efforts of, among others, Butler, Goedheer, Krasnovsky, Litvin and Govindjee are considerable contributions to our present understanding of the light-harvesting apparatus of algae and higher plants. This article will describe the present state of the art and focus as well on shortcomings and artefacts inherent in the method. Due to the complexity of the material only the results applying to higher plants and green algae will be treated in detail. A review of the use of fluorescence techniques in the elucidation of the light-harvesting apparatus o f bacteria and blue-green algae would exceed the intended scope. Although the pigment composition and the physical arrangement in this case is quite different from that observed in higher plants, the basic techniques discussed apply just as well and the same criteria should be used in evaluating the data. 11. THEORETICAL CONSIDERATIONS OF PIGMENT PHOTOCHEMISTRY

A detailed physicochemical analysis of fluorescence phenomena in general is not intended, but, nevertheless, some basic concepts and interpretations pertinent to the problem need to be introduced. A thorough treatment of the underlying principles can be found in the monographs of Seliger and McElroy (1965) and Clayton (1965,1970).

FLUORESCENCE OF PS-SYSTEMS AT 77°K

3

A. GENERAL CONCEPTS OF THE INTERACTION BETWEEN LIGHT AND MATTER

The electron in the outer shell of an atom or the outer orbital of a molecule can occupy various energetically defined levels. Absorption or loss of energy leads to transitions from one state to another which are governed by fairly rigid rules derived from quantum mechanics. Each electron can be visualized as a spinning charge. It possesses an angular momentum associated with this rotation, the spin, whose individual value is either t 1 / 2 or -1/2. In the ground state of a molecule, the spins of all electrons are paired and thus do not contribute to its overall magnetic moment. Addition of the spin vectors results in a total spin of zero, yielding a spin multiplicity 2 s t 1 = 1 . This molecular state is termed a “singlet”. If the total spin of the molecule is greater than zero, it adds vectorially to the magnetic moment of the particular molecule. The result is a “doublet” in the case of radicals (total spin 1/2) and a “triplet” for molecules with a total spin of 1. The photochemical properties of light-absorbing molecules are determined by the energies and chemical reactivities of these quantum states together with the relative transition probabilities between them. Figure 1 shows typical states and types of such transitions. Absorption of a photon leads to the transfer of an electron from the lowest filled n-orbital (the singlet ground state) to an unfilled n*-orbital of higher energy (transition S,, + S,,* in Fig. 1). The “spin-conservation rule” forbids any transition accompanied by a change in multiplicity, i.e. from a total spin of zero to a total spin of 1, the singlet -+ triplet transition. Thus, such an event is very unlikely with a probability of about 1 0-6 of that for a singlet +triplet transfer. The corresponding absorption band is very weak, The usual strong absorptions are always due to singlet -+ singlet transitions. Quantum mechanical considerations show, that a transition is vectorial with respect to the molecular co-ordinates. Prerequisite for absorption is the coupling of the dipole moment of the molecule to the magnetic field of the incident light wave. Thus, an ordered arrangement of pigments, e.g., chlorophylls, will absorb and emit radiation preferentially in the direction of the transition moments, it will be polarized. As a consequence, the degree of polarization is closely connected t o molecular organization. The de-excitation, i.e. transition of an electron into the ground state from a singlet state with concomitant emission of light is defined as fluorescence. Fluorescence is thus the emission of light which has been absorbed and should not be confused with scattering, i.e. emission of light which has not been absorbed (Raleigh scattering without, Raman scattering with change of wavelength). The basic features of fluorescence are:

1. It occurs only from the lowest excited state obtainable by absorption (Vavilow’s law). 2. The emission maximum is shifted relative to the absorption maximum to

4

GoTZ HARNISCHFEGER

Fig. 1. Jablonski diagram showing molecular energy levels and the transitions between them. The vertical axis marks the various energy levels (Eo, . . . , Ei).,The notation represents the singlet ground state, S1 .;’, the excited singlet, and TAG!, the triplet states. The direction of the spin of the cor%ponding electrons is indicated in the brackets. The electronic states are shown by thick horizontal lines, the thin lines represent vibrational sublevels. The vertical thick lines indicate electronic transitions (a: S-S absorption; c: fluorescence; e: S-T absorption; f phosphorescence, h: T-T absorption), while wavy lines represent radiationless transitions (d: intersystem crossing; b, g: decay with loss of heat).

SR

longer wavelength due t o loss of energy by thermal relaxation in the excited state (Stokes shift). 3. The measured lifetime of the excited state is short ( 5 x lo-’ s for chla in solution, Brody and Rabinowitch, 1957). De-excitation may also occur by radiationless transition expressed through the wavy line in Fig. 1. Such transitions account for the lack of observable fluorescence from higher excited states; between these higher excited states with much shorter intrinsic lifetimes and the lowest excited singlet they are far more probable. The downward transition TRn*+ S,,, a highly unlikely event, is defined as phosphorescence. The term “delayed fluorescence” denotes a de-excitation of

FLUORESCENCE OF PS-SYSTEMS AT 77°K

5

T,,* by energy uptake to yield S,,* and a consecutive transition to S,, with concomitant emission of light. Every one of the indicated energy levels contains a set of sublevels due to non-chemical interaction and energy exchange of the atoms within the molecule. The largest splitting of the main level is caused by vibrations of the individual atoms within the molecule with respect to each other. The vibrational substates are divided up further into rotational states deriving from the motion of the nuclei. Interactions with the surroundings, i.e. the solvent or neighbouring molecules, influence the distribution of electrons in a particular molecule as well and cause a shift in the positions of the various energy levels. A consequence of these properties is the observation, that in a molecule like chlorophyll both absorption and fluorescence do not occur at one specific wavelength only but rather in a broad band whose intensity reflects the probability of the transitions from the respective sublevels. B. GENERAL INFLUENCE OF LOW TEMPERATURE ON FLUORESCENCE PROPERTIES

The characteristic influence of low temperature on fluorescence emission derives mainly from two causes. First, the thermal energy of the molecule is almost entirely lost and, thus, thermal motion is severely restricted. This is equivalent to a reduction of the number of rotational states and results in a subsequent appearance of fine structure due to the better resolution of vibrational levels. Consequently the latter are more densely populated with electrons, a property which enhances the probability of emission and, thus, the intensity of the bands. A close connection exists also between the number of rotational states and the loss of energy by radiationless transitions. Lowering the temperature diminishes both, thereby giving an additional increase in the intensity of the competing fluorescence. The second factor influencing the fluorescence characteristics derives from the solid environment induced by low temperature. The “glass” due to the frozen solvent, or the solid non-fluorescing cell organelle membrane in the case of biological specimens, forms a cage around the fluorescent species. Interactions with the solvent in the excited state, leading to the loss of some energy before emission, becomes less likely. Thus, the most probable excited vibrational level, the Frank-Condon state, is effectively preserved at low temperature. This can amount to an apparent blue shift relative to the emission at room temperature (Wehry, 1967). It also results in a sharpening of the emission band. C. SPECIAL FEATURES OF THE CHLOROPLAST SYSTEM

The photosynthetic pigments of higher algae and plants are arranged as units (Emerson and Arnold, 1932; Gaffron and Wohl, 1936a, b; Schmid and Gaffron, 1968), giant conglomerates of various chlorophylls, carotenoids and xanthophylls. The majority of the pigments (collector or antennae pigments) funnel

6

GOT2 HARNISCHFEGER

absorbed light to very few molecules (traps) which convert the radiation into chemical energy. Excitation transfer between these pigments is generally assumed to be by resonance (see review by Knox, 1975). The excitation of the singlet state is passed on t o a second molecule, according to the equation Slnn* + S2nn

+

Slnn + sznn*

The term resonance is derived from the interpretation of the excited state as an oscillating electric dipole (Forster, 1951). The oscillation migrates over an entire range of closely spaced molecules. Resonance transfer is most likely when both a proper orientation between the electric dipole of the excited molecule and the potential dipole of the second emitting molecule as well as an appropriate energy is present. In terms of spectra this requires a close overlap between emission of the first and absorption of the second molecular species. Another way of viewing energy transfer derives from the consideration that the arrangement of photosynthetic pigments in at least part of the thylakoid system constitutes a semicrystalline, packed solid. This approach, the so-called semiconductor model, has been introduced by Arnold (Arnold and Azzi, 1968) to explain delayed fluorescence and charge separation in chloroplasts. An extended model was used by Tributsch (Tributsch, 1971, Tributsch and Calvin, 1971) to cover chlorophyll excitation and energy-coupling processes. The semiconductor model focusses on the crystal character of the photosynthetic unit. In a crystal the electronic states of single molecules lose their individual character and merge into the various conductance and valence bands, which for the photosynthetic pigment array equates with a delocalization of molecular energy levels within the chlorophyll lattice. Energy transfer under these circumstances should preferentially proceed by excitons. In the semiconductor theory it is the semicrystalline arrangement of the pigments which defines the physical limit of the photosynthetic units. This notion predicts that no bandshift of emission should occur upon lowering the sample temperature, provided physical damage to the crystal array during cooling is prevented. The final conversion of radiant energy into a form which can be used chemically involves a charge separation and formation of a reducing and an oxidizing species followed by the regeneration of the pigment through an electron donor. The special form of c h l s involved (trap), supposed to possess slightly lower energy levels than the surrounding antennae pigments, has so far not been isolated in pure form in spite of elaborate attempts. One may reasonably suppose that these traps are just an energy sink determined by the environment. The semiconductor model views them as impurities in the otherwise flawless crystal, trapping and converting the exciton energy. The redox reactions of photosynthesis are in turn controlled by the rate of subsequent enzymatic processes. The latter do not proceed at liquid nitrogen

FLUORESCENCE OF PS-SYSTEMS AT 77°K

7

temperature. At 77°K the kinetics and midpoint potentials of oxidoreduction can, therefore, be separated from those of electron transport (Dutton and Wilson, 1974). Since the redox pools fill up through prolonged light exposure and since other ways of energy dissipation are severely restricted at this temperature, the intensity of fluorescence is magnified and only dependent on energy transfer characteristics. 111. SPECTRAL DISTORTIONS, ARTEFACTS AND THEIR PREVENTION

Many of the undesirable side effects encountered in liquid nitrogen fluorescence work are closely related or even a consequence of the optical arrangement and type of sample mounting. A second form of distortion arises from the “inner filter effect”, a concentration-dependent, selective reabsorption of the emitted light. The third source of artefacts is the unwanted destruction of membrane integrity, intimately connected to the method of freezing with its concomitant ice crystal formation. Unless proper precautions are taken, the various types of distortion may act synergistically, obfuscating the results to a large extent. After a discussion of the relative merits of the various instrumental devices currently in use, the other sources of error will be treated in two sections. The artefacts not connected to thylakoid or algal integrity will be presented next, those originating from alteration of membrane properties will be treated subsequently. It has to be mentioned, however, that the above distinction is quite arbitrary. The errors due to the various sources overlap and cannot be separated in most cases. A. SOME NOTES ON INSTRUMENTATION

The instrumentation used for the measurements of fluorescence emission at 77°K varies from laboratory t o laboratory. Since little commercial apparatus is available, it is generally of the Heath-Robinson type i.e. non-commercial assemblies to save costs and to fit the purpose. The exciting light is narrowed to the intended excitation band or wavelength by a monochromator or an assembly of interference filters, the ensuing fluorescence signal is processed, after removal of stray light by appropriate blocking filters, using the now common wizardry of electronic devices. Their necessary details can be looked up in the appropriate handbooks and are not of interest here. The critical point in evaluating published spectra regarding artefacts involves the design of the cuvette, including its mounting and cooling arrangement. This assembly largely determines the influences due to light leakage as well as the amount of necessary corrections and gives a fair estimate of the cooling process with its resulting imponderabilities. Thus, some frequently used devices will be briefly introduced. The most common and widely used form of sample mount is the type

8

COT2 HARNISCHFEGER

familiar from absorption measurements, originally developed by Bonner (1 961). Figure 2 depicts this device. The chloroplast or algal suspension is placed in a trough formed by two Plexiglas windows interspersed by a brass, Y-shaped fork, whose tongue is in contact with liquid Nz in a Dewar flask during measurements. The filled sample-holder is normally frozen by immersing it in liquid Nz. A disadvantage of this type of assembly is that freezing is relatively slow and that cracks form within the frozen suspension which act as mirrors to distort spectral features and which cannot be controlled easily. French (French and Koerper, 1967) has developed another type of device. In

sample holder

f dewar VeSSQl

light--path

spacer and cooling fork (sc) Fig. 2. Standard sample-mount and cuvette compartment for low temperature spectroscopy. The various parts of the mount for specimen in solution, originally introduced by Bonner (1961), are given on the right. The arrangement for measuring the spectra, used commercially in a number of spectrophotometers, is shown in cross-section at the left.

this case the specimen suspension is frozen in a groove of a metal block cooled by liquid N2. This method although much simpler than the previous one possesses most of the same disadvantages. A refinement of the device described by Cho et al. (1966) was used in my own investigations. As shown in Fig. 3 the chloroplasts or algae are adsorbed on cheese-cloth which is subsequently mounted between two polypropylene rings and quickly frozen by immersion in liquid N2. This simple device has the

FLUORESCENCE OF PS-SYSTEMS AT 77°K

upper ring

9

cheesecloth with adsorbed sample

k4 t - - - - - -- - -

lower ring

c

/

brass + spacer

acrylic w i ndow plates

I

fastening screws

light path

.L protective dewar sample

I

translucent Fig. 3. Sample-holder and measuring arrangement for cheese-cloth method. The simple clamping device is shown at the top, while the measuring arrangement is given schematically at the bottom. An adaptation of the standard method, used in this specific setup for comparative purposes, is also represented (the so-called “lollipop”).

advantage that scattering is nearly constant and reproducible and that the organelles are in direct contact with the cooling agent, resulting in higher freezing rates. Goedheer (1964) used for his spectra chloroplasts adsorbed on filter paper for similar reasons. For comparative purposes with the conventional method mentioned before the “lollipop” of Fig. 3 was devised. The spacing between the windows could be vaned through use of brass rings of appropriate thickness.

10

GOTZ HARNISCHFEGER

B. SPECTRAL DISTORTIONS UNCONNECTED TO ORGANELLE INTEGRITY

These are mainly connected with the frozen medium surrounding the suspended specimen. During freezing a multitude of cracks is formed in the sample which act like mirrors and can, together with any ice crystals present, increase the scatter considerably. Butler (1964) has treated the scattering problem and ensuing artefacts for absorption at liquid N2 temperature, but some effects manifest themselves also in fluorescence emission work. The problem is similar to that posed by scatter in reflectance spectroscopy (Kortum, 1969). Scatter leads to an increased path length of the light within the sample and, therefore, increases the probability of a differential absorption of the emitted light by the various chlorophyll species. An appropriate correction is difficult since the formation and the amount of cracks cannot be controlled. It differs from sample to sample. Usually the error is reduced through the use of a suspension medium which forms few or n o cracks on freezing. The introduction of a strong, optically inert scatterer like CaC03 (Butler and Norris, 1960) which leads to an improvement of absorption spectra is not recommended in fluorescence work since it increases also the probability of differential reabsorption of emission. An example is shown in Fig. 4. The effect of adding CaC03 as scatterer upon the spectrum is clearly visible. The data are normalized to the long wavelength emission maximum which was set arbitrarily as 100. In doing so it is assumed that no pigments are present which absorb above 715 nm and that the data are corrected against an identical control not containing the algae. Closely related to the effect of scatter, and superimposed on it, is the distortion due to pigment concentration (inner filter effect). Since higher chlorophyll content equals higher particle or organelle concentration, the spectral influence is a combination of scattering errors with mutual shielding. It leads to increased reabsorption and an apparent decrease of the signal in the short wavelength region of emission. Since the photosynthetic apparatus consists of several pigments whose absorption and emission spectra overlap considerably between 670 and 7 15 nm, the fluorescence emitted at shorter wa\~elengthwill be largely reabsorbed by the other pigments in concentrated solutions of algae or chloroplasts. One expects, therefore, an apparent shift of these emission maxima towards longer wavelength. The distortions due to chlorophyll concentration have been described first by Govindjee (Govindjee and Yang, 1966; Cho and Govindjee, 1970a; Govindjee, 1972). The rule of thumb, that “if it looks green it’s too concentrated”, received its justification in these studies. Figure 5 shows the effect of increasing amounts of plastids on the spectra. The cheese-cloth technique was used in this experiment to minimize damage by freezing (see Section 1II.C). The abovementioned distortions are clearly observed.

FLUORESCENCE OF PS-SYSTEMS AT 77'K

11

My own experiments indicate that a chlorophyll amount below 5 pg/cm2 sample area largely avoids concentration artefacts. One has to remember, however, that an elimination of concentration-dependent distortion by extrapolating to zero pigment concentration is meaningless, since the pigment concentration within a single thylakoid presents the lower limit of correction.

6

650

700

750

nm emission Fig. 4. Influence of added scatterer (CaC03) on the fluorescence emission at 77°K of the ChZoreZZu mutant 520 (Claes). The algae were suspended in a sorbitol-borate-glycerol medium which forms a clear glass upon freezing. Excitation occured at 435 nm (recalculated from French and Koerper, 1967).

According to Woken and Schwerz (1954) this is considerable-around 2.5 x lo-* M. An adequate correction for that portion of the inner filter effect originating in the chloroplast structure is, therefore, impossible. The reabsorbed energy might even be emitted as secondary fluorescence. However, Szalay et aZ. (1967) showed that the absolute spectral contribution of this effect is negligible in CbZoreZZa and maximal around 5-6%in chloroplasts.

12

GOTZ HARNISCHFEGER

/8“9 a

\8 \B

\

4.\

8

I &

660

1

I

1

I

1

700 nm emission

I

I

I

I

I

750

Fig. 5. Influence of chlorophyll concentration on the emission properties at 77°K of spinach chloroplasts: (0-)4 pg chl/cm2 area; (0-)6.4 pg chl/cm2 area; (A-) 7.6 pg chl/cm2 area. Cheese-cloth mounting; excitation with blue light (Bahlzers K-2). The chloroplasts were prepared and suspended in 0.4 M sucrose-0.1 M phosphate buffer pH 6.8, 1 mg/ml BSA. The spectra are normalized, setting the emission intensity at 740 nm arbitrarily as 100 (normalization point indicated as 0). C. SPECTRAL DISTORTIONS DUE TO ORGANELLE DESTRUCTION

The concept of the photosynthetic unit illustrates the need for an intact membrane matrix system in the spectroscopic measurements. It emphasizes a close physical proximity of the pigments to facilitate energy transfer in the direction of the trapping and conversion centres. Any procedure which disturbs or degrades the intricate structure of the thylakoid membrane has, consequently, an effect on the spectral characteristics of both the fluorescence excitation and emission spectrum.

FLUORESCENCE OF PS-SYSTEMS AT 77'K

13

Figure 6 is a good example for the close connection between thylakoid integrity and spectral properties. The gradual dissolution of the membranes by galactolipase action results in a loss of the long wavelength emission band. Judicious degradation of chloroplast structure has, therefore, been frequently used to study pigment interaction within the photosynthetic apparatus (e.g. Harnischfeger and Gaffron, 1970). A considerable and unwanted source of damage t o the integrity of the pigment array can originate in the freezing process itself. In fact, the method of freezing

nm emission Fig. 6: Fluorescence emission spectra at 77°K from Riceus chloroplasts incubated with a galactolipase containing extract of Ricinus leaves. a, b, c, d, e, indicate the spectra taken after 0, 5, 10, 20 and 60 min of incubation at 330°K. Excitation was at 435 nm (redrawn from Brody et ul., 1969).

introduces most of the artefacts encountered in such studies. Since algae and chloroplasts contain more than 90%water, freezing can damage the membranes and their arrangement of pigments through both ice crystal formation and partial dehydration. Consequently all preparative methods have to minimize or preferably exclude ice crystal formation completely. A thorough understanding of the mechanics involved in the freezing process, its effect on the photosynthetic membranes and the possible prevention of ensuing artefacts is, therefore, prerequisite for a valid analysis. These aspects will be treated in detail in the following sections.

14

GOT2 HARNISCHFEGER

1. Genera2 Description o f the Freezing Process, Its Accompanying Parameters

and Their Influence on Membrane Integrity The time-course of freezing for pure water is shown in Fig. 7. Upon lowering the temperature below the melting point, one encounters first a period of supercooling. Crystal formation starts by introducing suitable crystallization nuclei into the labile system. Below a temperature of 233°K the water molecules themselves act as nucleii for crystallization. Since during crystal formation energy is released (heat of crystallization) the system warms up to the melting

Fig. 7. Schematic representation of the temperature decrease encountered during the cooling of water to very low temperatures. See text for further explanation (redrawn from Moor, 1973).

point. The mixture of ice and water will remain around this temperature until all the water is transformed to ice. The system cools down to lower temperatures only after complete solidification. If crystallization is avoided, e.g. by cooling water vapour on surfaces at liquid N2 temperature, one obtains “amorphous ice”. Upon gradual heating this amorphous ice crystallizes above 143’K. Below 263°K the increasing viscosity of liquid water impedes the rearrangement of the molecules to large crystals (Fig. 8). The formation of ice crystals within cell membranes with their high intrinsic water content leads to degradation of their structure, if the crystal size exceeds

FLUORESCENCE OF PS-SYSTEMS AT 77°K

15

8'0 200

250

150

OK

Fig. 8. Temperature dependence of the rate of crystallization of w'ater: (--) calculated; measured by Lindenmeyer; ( 0 ) measured by Mazur, both cited by Riehle (1968) (redrawn from Riehle, 1968).

(0)

a

100-200 (Moor, 1964). This is the overriding sourc:e of damage to biological specimen. Thus, it becomes clear that the main objective in preventing or minimizing crystallization damage is a high freezing rate. The quicker the crystallization range between 273°K and 143°K is passed the less damage results. Although in biological specimens the freezing point is normally lowered by several degrees and the recrystallization temperature increased to 193"K, this much smaller range cannot be bridged by supercooling alone in order to avoid crystallization. The relationship between ice crystal size and freezing rate is depicted in Fig. 9.

2

3

5 6 log cooling rate (OKls)

4

Fig. 9. Influence of the cooling rate ("K/s) on the size of the ice crystals in various aqueous solutions. The solutions contained: ( 0 - ) glycerol 10%; ( 0 - ) glycerol 5%; (a-) sucrose 5%; (0-)NaCl 5%. The influence of the added components on the critical freezing rate VK is clearly distinguishable (redrawn from Riehle, 1968).

16

G o T Z HARNISCHFEGER

The aim should, thus, clearly be for a rate around or considerably higher than the “critical freezing rate”, V k t in Fig. 9. Moor (1964) has estimated that this rate should be in the order of 10 000°K s-l for physiologically active cells and organelles. The frozen state obtained under such conditions at 77°K is called the vitreous state. Electron micrographs show no ice crystals in membranes frozen in this way. The ice crystals are either absent or, more likely, their size is less than 100 A due to impeded growth. It is important to realize that the freezing rates actually obtained are limited by the fact that only the surface of the cuvette is in contact with the liquid N2. The aqueous medium surrounding the specimen in the cuvette is a poor conductor of heat and decreases, therefore, the freezing rate considerably. If the algae or chloroplasts are in direct contact with the liquid N2, their actual shape and size is of importance. Riehle (1968) estimated that the maximum freezing rate is only preserved within 2-3 p from the contact surface. The shape of the organelle becomes thus important. The geometry of cylindrical objects and spherical objects allows vitrification up to several times this limit. The emphasis, so far, has been on membrane degradation, and its prevention, due to the size of the ice crystals formed. Although this accounts for most of the observed disruptions, the processes accompanying solidification can constitute a source of considerable damage as well, even in the absence of crystallization. According t o Litvan (1972) intracellular water, mostly adsorbed t o and located within membranes, remains liquid-like well below 175°K. Consequently its vapour pressure is greater than that of the ice in the surrounding medium. Thus, besides an electrolyte gradient (see next paragraph), a difference in vapour pressure builds up during cooling w h c h increases with decreasing temperature. This leads to spontaneous desorption and redistribution of water with a concomitant dehydration and denaturation of the membrane. During rapid freezing (at rates not leading immediately to vitreous ice) the quick redistribution of water is impeded because of the limited permeability of the membranes and rupture due to ice crystals within the membrane occurs. It follows that an increase of membrane permeability should, therefore, increase the cold tolerance of organisms. This was indeed shown by Williams and Merryman (1970) for isolated grana whose membranes had been rendered permeable t o electrolyte fluxes. Incidentally, winter hardy and frost-resistant plants also show an increased cell permeability. An additional aspect is worth noting. When an aqueous solution of a salt is cooled down, the majority of the water will crystallize first, leaving the salt and other solutes present to concentrate. If cell organelles are suspended in such a medium, the above mentioned redistribution of water will occur. Further f At and above the critical freezing rate, crystallization leads to a minimum of the size of ice crystals (amorphous or vitreous ice).

FLUORESCENCE OF PS-SYSTEMSAT 77°K

17

cooling results in a final solidification accompanied by the formation of a salt matrix of eutectic composition (Van den Berg and Rose, 1959). In fact, when the cooling rate is appropriately controlled, the “freezing-out’’ technique can yield super-pure water (Shapiro, 1961). Since the entire process is equivalent to a gradual removal of both water and ions from the liquid, large alterations of pH can be expected before solidification is accomplished. The extent to which these pH-shifts denature membrane components and influence membrane structure is unknown at present.

2. Artefacts Due to Freezing Damage The consequence of destruction or disarray of pigment complexes within the membrane matrix are distortions of the fluorescence emission spectrum. In this case the damage is caused by the combination of ice crystal formation within the thylakoid and dehydration due to an increase of the electrolyte concentration of the medium (water is removed from the system by ice formation). This so-called two factor hypothesis was first elaborated by Mazur et al. (1972). The critical importance of the freezing rate is thus understandable. Taking the value of 1 0 000°K s- as the necessary rate for preserving biological specimens (Riehle, 1968), the photosynthetic organelles should be brought from room temperature t o liquid N2 temperature in less than 21 ms. The cheese-cloth mounting is the only method presently in use w h c h approaches this value, since here the specimen is in direct contact with the liquid N2. Such high rates of freezing are unattainable when suspended organelles are used. The aqueous surrounding medium possesses a low thermal conductivity and acts as insulator, thus considerably lowering the freezing rate. The Plexiglas cover on the trough in the conventional sample-holder constitutes an additional thermal barrier. Figure 1 0 is an illustration of these effects. The freezing rate was lowered by increasing the distance between the “lollipop” covers through appropriate spacers. The amount of chl/cm2 area was kept constant using appropriate dilution. Algae adsorbed on cheese-cloth served as control. The results clearly indicate a differential effect of the freezing rate on the spectrum and the numerical relation of the intensity of the bands to each other, as recorded previously by Cho and Govindjee (1970a). Th~sis depicted in the insert of Fig. 10. It must be mentioned, however, that a certain amount of scattering error is superimposed on the results. Internal ice formation and cracks cannot be completely avoided during the freezing process. Addition of glycerol prevents among other things the formation of cracks and results in a clear “glass” (see next section). Nevertheless, the increase in the ratio of F732 : F685 is still noticeable. A further increase of the freezing rate can be achieved by immersing the cheesecloth-mounted sample in melting nitrogen. The Leidenfrost phenomenon

18

GOTZ HARNISCHFEGER

is abolished in this case. However, no further improvement of spectral quality as judged by the ratio of long t o short wavelength emission could be obtained. The close relation between emission signal and damage due to ice formation and recrystallization within the thylakoid membrane has been previously

0

1 mm

2

i

\

0

b\ 8 \

8

b

nm emission Fig. 10. Influence of the distance between cuvette covers (a freezing rate) on the low temperature fluorescence emission spectrum of Chlorella: (0-) cheese-cloth method (control); (*-I 1-mm spacing in the "lollipop". The algae were fully synchronized and in the autospore phase. In all instances the chlorophyll concentration was 2.1 pg/cm2 area. Normalization of the spectra at F,,,, which was set as 100. Excitation occurred at 595 nm.

elaborated by Cho and Govindjee (1970a). Using the quick-freeze cheese-cloth method they examined the emission spectra of Chlorella at various end temperatures. After cooling down the sample to 77°K and subsequent warming at a rate of 5-10°Kmin-' they observed spectral changes which were most pronounced at the temperature of phase transitions of ice (e.g. at 150-155"K, Fig. 11).

19

FLUORESCENCE OF PS-SYSTEMS AT 77°K

01

I

80

I

I

120

i

I

160

I

I

200

I

1

I

240

OK

Fig. 11. Fluorescence intensity at the various emission bands of Chlorellu as a function of temperature: ( 0 - ) F680; (A-) F686; (a-) F698; (v-) F,,,. Excitation occurred at 485 nm. Cheese-cloth method; the samples were quickly frozen to low temperature and warmed up at a rate of 5-1OoK/min (redrawn from Cho and Govindjee, 1970a).

3. Cryoprotective Agents and Their Use in Prevention of Freezing Damage Numerous substances have been added to the specimen suspensions in order to prevent freezing damage. The wide range of compounds and concoctions used in low temperature photosynthesis work reflects this empirical approach to selection. Consequently, few if any conscious studies have been made on the interaction of the various cryoprotective agents with thylakoid structure. An assessment has, therefore, to draw heavily on the analogies to cryobiological work involving mitochondria, red blood cells or even tissue cultures (cf. survey by Mazur, 1970). The differences between these systems and photosynthetic organisms or organelles should always be kept in mind. Generally it can be stated that any freezing rate leading to vitreous ice ( V >)'V does not damage the specimen to any large extent. The validity of this tenet from freeze-etch microscopy for liquid N2 fluorescence work was demonstrated before. Consequently, any low-temperature-induced destruction of membranes and cells can be traced to the formation of ice crystals and its

20

GdTZ HARNISCHFEGER

accompanying side reactions at freezing rates below the critical freezing rate V,. Since the added substances interfere with the crystallization of water to ice, the common cryoprotective agents are only effective under conditions where

v < v,. The action of cryoprotective agents, classified either as membrane penetrating or non-penetrating, generally leads to a reduction of the amount of ice formed and a lowering of the electrolyte gradient at each temperature during freezing. All protective additives show a high viscosity in aqueous solution at low temperatures, thus limiting the size of the ice crystals formed. Penetrating compounds like glycerol, glycol or dimethylsulfoxide (DMSO) increase the viscosity of the intracellular and membrane-bound water as well, thereby retarding its flow and diminishing dehydration. High viscosity is a recognized criterion for the formation of a “glass”, defined as a nonequilibrium, non-crystalline state having a higher vapour pressure than the crystalline ice at the same temperature. The vapour pressure gradient between inside and outside the cellular membranes can, thus, become effectively diminished by glass formation. Most cryoprotective compounds form glasses on solidification, e.g. glycerol, DMSO, ammonium acetate, polyvinylpyrrolidone (PVP) and sucrose. Litvan (1972) showed that no crystalline ice forms in glycerol solutions above 45%, DMSO above 40% and ammonium acetate above 37% concentration. Non-penetrating agents such as bovine serum albumin (BSA), PVP, dextran and sucrose act mainly through formation of an outside glass. In cytological and clinical work, PVP protected best in concentrations around 15%, if impurities accounting to about 12% of the commercially available product had been removed, while with dextran good results were obtained at 10% concentration (Ashwood-Smith and Warby, 1971). The effect of cryoprotective agents on thylakoid structure remains uncharted territory. A 40% concentration of glycerol presents a 4.3 M solution and the osmotic pressure doubtless influences the membrane properties before freezing. Most experimentation with additives was performed with emphasis on functional survival after long time storage at low temperature. The methods used do not necessarily guarantee the preservation of the native pigment arrangement within the thylakoid membrane. Gorham and Clendenning (1950) first reported freeze protection of chloroplasts by added sugars. Heber (1970) confirmed these results and observed in addition the protective properties of certain proteins against cryoinjury in chloroplasts stored for several hours at 77°K. Chloroplasts frozen and stored at liquid N, temperature in the presence of DMSO have been used in many instances. Witt and co-workers have used them routinely for biophysical investigations. In most cases, the amount of damage t o the pigment array induced by the freezing-thawing cycle cannot be assessed from the reported data. The studies on electronic interactions between pigments performed with

FLUORESCENCE OF PS-SYSTEMS AT 77°K

21

such preparations might not mirror the in vivo situation and are, therefore, open to criticism. For spectroscopic work, French and Koerper (1967) reported the use of a 1 : 1 mixture of 80% sodium sorbitol borate pH 7.8 and glycerol. Spectra which show clearly the protective action of added DMSO on the fluorescence emission of c;cllorella have so far been published only by Cho and Govindjee (1970a). They are reproduced in Fig. 12.

.... -....--.-.. Fig. 12. The influence of added DMSO on the fluorescence emission spectrum at 77°K of Chlorella (redrawn from Cho and Govindjee, 1970a).

In order to ensure spectral qualities as close as possible to the in vivo situation the use of the cheese-cloth mounting with rapid cooling around or above V, is presently the best approach. An improvement of the data obtained with the standard trough mounting through the addition of cryoprotective compounds is possible, but first the side effects of the necessarily high concentrations of these agents have to be known in more detail. D. SOME POINTS REGARDING THE EVALUATION OF PUBLISHED SPECTRA IN LIQUID N2 FLUORESCENCE SPECTROSCOPY

The above analysis of possible artefacts leads to definite criteria in the assessment of published spectra. Thus, a critical evaluation should consider the following points: 1. Does the type of sample mounting and the method of cooling allow rapid freezing up to and above 10 000°K s-?

22

G6TZ HARMSCHFEGER

The information is normally contained in the materials and methods section. The type of suspension, cuvette material and cuvette dimensions allow an estimate of the thermal conductivity and heat exchange and, thus, the freezing rate. 2. In which way are scattering artefacts prevented? Special attention should be given to the chloroplast concentration (in 1.18 chl/cm2 light exposed area) and the effect of added scatterer material. 3. If suspensions are used, which type and what amount of cryoprotective has been added? Is “glass” formation achieved? Can additional destruction of the membrane by the agent and its concentration be excluded? Normally one should find some reference to other spectra obtained by different methods which facilitate a decision on the latter question. It should be noted, however, that these criteria are of great importance only in quantitative work. The location of emission maxima is less affected by the possible distortions described before. It is self-evident that sonie more technical details should enter the assessment of the spectral quality as well. These comprise especially the necessary corrections against light leakage and photomultiplier sensitivity. Valid comparisons become possible only if these criteria are met.

IV. STRUCTURE AND COMPOSITION OF THE PHOTOSYNTHETIC APPARATUS AS DETERMINED BY FLUORESCENCE AT 77°K The fluorescence spectra of green algae and higher plants show several distinct emission bands at liquid N, temperature. The assignment of these to the various chlorophylls and photosystems is presently still tentative, in spite of considerable progress in this area. Although only one form of chl-a is found in organic solvent extracts, several modifications exist in viva. French (French, 1971; French et aL, 1971) with theoretical computer analysis distinguished up to ten spectroscopically different chl-u forms in aggregated or monomeric states in the red absorption band of chloroplasts and algae (see also Litvin and Sineshchekov, 1975). Fluorescence emission spectra constitute the sum of various components as well. A mathematical unravelling of such spectra, leading to a tentative resolution of the underlying components, was reported by Sineshchekov et al. (1973). However, when analysing such spectra, it should be kept in mind that the membrane-bound chlorophylls do not show a blue shift of emission upon cooling, a feature unlike the behaviour of simple fluorescent dyes in aqueous solvents. Shape and intensity of the chlorophyll emission band, on the other hand, change considerably. Some bands, hardly or not at all noticeable at room temperature, appear as distinct entities. Secondly, one has to recall that it is only an assumption that the various emission peaks dways originate in the same

FLUORESCENCE OF PS-SYSTEMS AT 77°K

23

chlorophyll species whether they are measured at room or liquid N2 temperature. The highly hydrophobic environment of the pigment apparatus which allows no large water-chlorophyll interaction supports this presumption, but its validity has, nevertheless, been recently challenged (Cotton et al., 1974). The emission spectrum of Chlorella normally possesses four bands at 77"K, at 686 nm (referred to as F686), 697.5 nm ( F 6 9 8 ) , 717.5 nm (F717)and 725 nm (F7Z5). F,,, is not always found at 77"K, it shows mostly as a shoulder only. In chloroplasts these bands appear normally at 685 nm (F,,,), 696 nm (F695) and 738 nm (F732).? It is generally presumed that F 7 2 5 of Chlorella and F73.2 of chloroplasts correspond to each other. The exact location of the various fluorescence emission bands depends on the plant or dgal species investigated. Litvin and Sineshchekov (1975) give the position of F725 as follows: Chlamydomonas-722 nm; Chlorella, Anabaena-726 nm; Nostoc-732 nm and Phaseolus-73 6-739 nm. The identification of the chl-a forms responsible for each emission band involves the comparison of absorption, excitation and emission spectra taken under similar circumstances. From absorption spectra at 77°K French (197 1) distinguished four major chl-a bands, located at 662,670,677 and 683 nm, two minor chl-a peaks at 692 and 705 nm and chl-b absorption maxima at 640 and 650 nm. These represent various monomers, aggregates and adducts of chlorophyll. Cotton et al. (1974) were able to simulate the absorption bands using concentrated chl-a solutions in hexane. They concluded from their studies that the antenna chlorophyll exists in a strongly hydrophobic environment. A similar analysis was performed on excitation and emission spectra of Chlorella at 77°K (Sineshchekov et al., 1973). Through computer resolution ten Gaussian components were distinguished in the excitation and at least eight in the emission spectrum (Fig. 13). It is noteworthy that the various band positions, again, could all be mimicked in model systems of chlorophyll and organic solvents at liquid N, temperature (cf. Table I in Litvin and Sineshchekov, 1975). The problem faced in the analysis of the emission spectra is. however, not that of a deconvolution of the various bands. The difficulty is rather the assignment of the emission maxima to a specific form of chlorophyll found in absorption studies, i.e. a tracing of the sequence of energy-transfer and an identification of the final emitting pigment. Though the computer studies hold great promise, the most common approach in this regard involves comparison of absorption and fluorescence spectra at 77°K of material that contains different proportions of pigments. This is the field of particle studies successfully pursued in the last decade (review by Jacobi, in press). Either ultrasound, detergents or

t Unless explictly stated the subscripts given in the brackets are used in denoting the emission bands. Thus, actual location and wavelength designation as inferred from the subscript might differ.

w

P

A: Excitation spectrum emission 713nm

640 660 680 nm excitation

700

660

680

700 720 nm emission

740

Fig. 13. Mathematical resolution of the liquid Nz temperature excitation (A) and emission (€3) spectrum of Chlorellu into Gaussian components. Curve 1 denotes the speetruni in both cases. The Gaussian components are indicatcd by the subsequent numbers. The difference between the spectrum and the summation of the Gaussian components is given by the dotted line (redrawn froin Sineshchekov e f ul., 1973).

FLUORESCENCE OF PS-SYSTEMS AT 77°K

25

pressure changes are used to obtain algal or chloroplast fractions with separated photosystems. Figure 1 4 shows the result of such an experiment with chloroplast particles. The correlation of the fluorescence emission properties with excitation parameters and partial electron transport activities in these systems led to the conclusion that F, can be assigned to photosystem (PS) I while FGS5and F695belong to PS I1 (Govindjee, 1963; review by Boardman, 1970; Goedheer, 1972). The results obtained with particle fractions from algae allowed a similar interpretation.

650

685

735

3

1

nm emission

8 50

Fig. 14. Fluorescence emission spectra at 77°K of spinach chloroplasts and particles obtained after treating the plastids with desoxycholate: (-) chloroplasts, (---) “heavy”, (-..-) “light” particles. Samples were frozen in I-mm cuvettes in a medium containing 60% glycerol (redrawn from Bril e l al., 1969).

26

GOTZ HARNISCHFEGER

The danger of artefacts distorting the fluorescence spectra and leading to erroneous conclusions is, however, considerable. Figure 15, taken from Mohanty et al. (1972), is a reminder of this pitfall. Although an assignment of the various emission bands to the two photosystems has been possible, there are stdl some unresolved problems about the nature of the emission maxima themselves. Thus, though the F732 band belongs definitely to PS I, its exact origin is not quite understood. Brody (1965)

660

700

740

660

700

740

780

nm omission Fig. 15. Emission spectrum at 77°K of a system I chlorophyll-protein complex. A: chlorophyll concentration 2,O pg/ml. B: chlorophyll concentration 40 pg/ml. Excitation of the samples, adsorbed on cheese-cloth, was at 430 nm (redrawn from Mohanty el al., 1972).

attributed it to an aggregated chl-a form (P700) with excitation bands at about 682 and 705 nm. Butler (1965) located the absorption maxima at the same wavelength. Litvin and co-workers (Litvin and Sineshchekov, 1975) reported that at an excitation wavelength exceeding 700 nm only the F7 band can be observed at 77°K. They put the corresponding absorption band at 710 nm, a wavelength exciting only PS I as judged by functional parameters. One major problem in the interpretation of F 7 3 2 is the observation that its fluorescence is considerable only at low temperatures. This might indicate a

FLUORESCENCE OF PS-SYSTEMS AT 71°K

27

transition of an aggregated, self quenching chlorophyll form to an essentially fluorescent monomeric one upon cooling. However, such an explanation undermines the tenet of the equality of the pigment arrangement at room and liquid N2 temperature as a basis for the extrapolation of low temperature data to the in vivo situation. More is known about F 6 9 5 . This band appears as a distinct entity at temperatures below 140°K (Goedheer, 1964), at room temperature only if bright light and DCMU is used (Papageorgiou and Govindjee, 1967). F695 is closely linked to the trapping pigment of PS 11. This was conluded from its being quenched by plastoquinone (Brody and Brody, 1963; Brody and Broyde, 1963; Broyde and Brody, 1965), its sensitivity to redox agents (Goedheer, 1966; Ke and Vernon, 1967; Boardman and Thorne, 1969), its exponential signal increase at 77°K upon PS I1 illumination (Kok, 1963), the preferential sensitization of F695 by pigments of PS I1 (Govindjee, 1963) and its sensitivity to hydroxylamine, a potent inhibitor of PS I1 (Mohanty et al., 1971). F6g5 is generally regarded as fluorescence originating in a special monomeric form of chl-cl which possesses a strong excitation band at 674 nm in vitro and in vivo (Brody and Broyde, 1963; Broyde and Brody, 1966). The third distinct band in the 77°K emission spectrum of chloroplasts, F685, has been interpreted as originating from the bulk chla. This acts as antenna chlorophyll transferring harvested light energy into photosystem 11. Main evidence for this notion comes from the above mentioned particle studies where considerable PS I1 activity was always found to be associated with the dominant presence of F685 and F695 in the emission spectra at 77°K. The origin of F7 in Chlorella is still unresolved. In contrast to the other emission bands it is not strongly influenced by phase transitions of ice during cooling (Cho and Govindjee, 1970a). Some interconversion of the chla species emitting at 695 to a form fluorescing around 720 nm has been proposed (Nathanson and Brody, 1970). There is ample evidence for the suggestion that the long wavelength emission band, normally identified with F72 5 , might actually be a composite of sub-bands, some of which become apparent only at certain temperature intervals (Litvin and Sineshchekov, 1975). Excitation spectra at 77°K provide some insight into the sequence of energy transfer between the various pigment species. Excitation of cN-b in Chlorella leads to a lower F725 and a higher F 6 8 6 , while the opposite is true when excited at 430 nm (chl-a absorption only). The excitation spectrum for these bands, shown in Fig. 16, illustrates the accessory role of chl-b in PS I1 (Govindjee and Yang, 1966). The action spectra for the various emission peaks, published by Murdta et al. (1966), concur with this notion. The energy transfer from carotenoids was assessed by Goedheer (1969). His experiments show that a high efficiency of energy transfer exists between carotenes and chla, while xanthophyll proved less effective. This observation

normalization point

150

400

450

500

550 nm excitation

600

6 50

7 00

Fig. 16. Excitation spectra for F685 ( 0 ) and F732 ( 0 ) . A thin suspension of chloroplast fragments was used in this experiment of Govindjee and Yang (1966). The data were normalized at the points indicated.

FLUORESCENCE OF PS-SYSTEMS AT 77’K

29

was interpreted as indication for a much closer connection between carotenes and chla than between xanthophylls and chla in the pigment apparatus. Both F695 and F,,, show excitation peaks around 670 and 680 nm which correspond with the absorption peaks found at the same positions (Cho and Govindjee, 1970b). This suggests that the underlying chla species should be present in both photosystems. Cho er al. (1966) investigated the effect of cooLing to very low temperature (4°K) upon the relative size of the various emission peaks. They observed that the lower the temperature the higher the increase of F6,, relative to the other bands. From these results they concluded that under these circumstances energy transfer between pigments and pigment systems becomes more and more impeded. No physical reason, however, is given for this interpretation. One aspect of these observations is again the question whether information from spectra taken at liquid N, temperature is in any way representative of the in vivo process. The results might merely indicate the changing environment produced by freezing the thylakoid membrane systems which contain a considerable amount of water. If quick cooling produces only a rigid pigment arrangement compared to the dynamic equilibrium encountered at room temperature, i.e. if it serves only as tool to preserve the most probable physical arrangement, freezing below a certain temperature should not produce any further quantitative change in emission properties. Presuming a correspondence between the pigment interactions established at liquid N, temperature with those found in vivo, the following model for the photosynthetic pigment complex emerges (Fig. 17). The various pigment species are arranged in photosystems in accordance with the results discussed above. The identification of P700 as the trapping centre of PS I was not accomplished by liquid N, spectroscopy but resulted from observations of electron transport and room temperature absorption difference spectra (Kok, 1963). Although the arrangement shown suggests a separate package for both photosystems, the similarity of the outer pigments in the two complexes should be noted. They might constitute “common ground” for both, distributing their harvested energy statistically. Butler and Kitajima (1974, 1975b) emphasized this aspect and arrived at a somewhat different model of the photochemical apparatus (Fig. 18). They distinguish three pigment complexes instead of the usual two, corresponding to the two photosystems. A third, light-harvesting (LH) complex is postulated whose radiation energy is transferred to the photosystems in varying proportions. Support for such a notion derives from the studies of Thornber and co-workers (review by Thornber, 1975) who were able to separate the photochemical apparatus into various chlorophyll-protein complexes. One of them, containing equal amounts of chlu and chl-b but no proteins associated with the trapping centres, seems to constitute the light-harvesting complex LH (Thornber and Highkin, 1974).

presence o f D C M U )

,F6871 'F 695,

(weak band a t 29OoK,77"K) (weak,290°K,77"

f o r m s o f C h i ? are destroyed)

F687=

( + v i b band at 740nm,major band at 2 9 0 ° K , 7 7 " K )

F 710-715 ( w e a k , 2 9 O 0 K ) , F 720 - 7 3 5 ( s t r o n g , 7 7 ° K )

-+F 710 - F 7 3 0

(?)

F 693 -696, tP680-690

( a t 7 7 " K , a t 2 9 0 " K in b r i g h t l i g h t or i n the p r e s e n c e o f DCMU)

Fig. 17. Hypothetical model of the distribution of chlorophyll species within the photosynthetic pigment systems. The possible source of the various emission peaks as well as methods of their detection are indicated. Note that F,,, is given as F687 (from Govindjee and Govindjee, 1975).

FLUORESCENCE OF PS-SYSTEMS AT 77°K

*

31

PSI

0 PI *I Fig. 18. Arrangement of the various pigment complexes according to Butler and Kitajima (1974). See text for further details of this tripartition model.

Butler and Kitajima interpret the emission at 695 nm as originating in PS 11, F685as coming from LH and F,32 as due to P s I. Their model allows a relatively simple theoretical assessment of photosystem interaction and the control thereof. This aspect will be treated in detail in Section VI. V. ORIENTATION OF PIGMENTS WITHIN THE PHOTOSYNTHETIC AE'PARATUS The models of the photosynthetic pigment complex discussed in the previous section account for its spectroscopic behaviour and the energetic aspects of energy transfer. They are, nevertheless, quite unspecific about the actual architecture encountered in vivo, restricting only the choice of possible spatial arrangements. Antibody studies have given some indication about the localization of photosystems and pigment blocks within the membrane (review by Trebst, 1974). The necessary information about the relative orientation of single chlorophyll molecules and aggregates within the light-harvesting unit, on the other hand, has been mainly derived from studies using polarized light. Determination of dichroism, the difference in chlorophyll absorption of plane polarized light with the analyser in parallel and perpendicular position to the electric vector (E-vector) of the incoming light, is one way to investigate the amount of orientation of the pigments present. Such studies provided the following information: 1. The porphyrin rings of the major fraction of chl-a are likely t o be arranged in parallel to the lamellar membrane (review by Kreutz, 1970). 2. Chlorophyll absorbing at longer wavelength appears more highly oriented. Thomas et al. (1967) calculated that one-third of the oriented chlorophyll belongs to ~ h l - a ~two-thirds ,~, to C h l d 6 8 0 .

32

GOTZ HARNISCHFEGER

3. No orientation occurs with chl-b. 4. A specific fraction of chla, which absorbs around 681-682 nm, is oriented at right angles to the plane of the chloroplast membrane (Gregory, 1975). A relatively low intrinsic dichroism has usually been found in the experiments, which has been interpreted as lack of overall orientation of the pigment molecules. However, Cherry et al. (1972) pointed out that this could also result from a partly oriented system where the chlorophyll transition moment makes an angle of 35" with the plane of the membrane. Miiller and Wartenberg (1971, 1972) estimated in Mesataenium an average of 16% oriented chl-a. If only photosynthetically active pigment is counted, the percentage is around 34%. This high degree of orientation has also been reported for spinach chloroplasts by Breton et al. (197 1, 1973). Investigation of the polarization of chlorophyll fluorescence in viva verified and extended the information obtained in the dichroism studies. These experiments are based on the following consideration: in systems where the individual fluorescing pigment is prevented from rotating, the degree of polarization reflects the distribution of excitation energy over the molecular array before fluorescence takes place (Knox, 1975). The incoming linearly polarized light is absorbed preferentially by those molecules whose transition dipoles are aligned parallel to its polarization vector. If no excitation transfer occurs, only those molecules that absorb a photon will emit light. Thus, linearly polarized light creates an anisotropy with the result that the fluorescence is also partly polarized. If excitation energy transfer does occur, the initial anisotropy created by the incoming radiation will decrease and, consequently, the degree of fluorescence polarization will diminish as well. No fluorescence polarization will be observed in a sample of completely random oriented molecules between which excitation transfer takes place, A non-zero polarization has to indicate, therefore, some molecular order in the pigments involved. The degree of polarization, p , is given by the equation

where the symbols Ill, I, indicate the emission intensity when the analyser is parallel and perpendicular respectively to the linearly polarized excitation. It has to be emphasized again that the polarization of a group of molecules which share excitation energy depends not only upon the extent of energy migration but also considerably upon the orientation of the pigments. The data obtained with polarized fluorescence measurements led to similar conclusions as the dichroism studies. Polarized emission of plastids oriented in a magnetic field (Becker ef aL, 1973) confirmed the high degree of orientation

FLUORESCENCE OF PS-SYSTEMS AT 77°K

33

postulated by the Mesotaenium studies. Whtmarsh and Levine (1974) concluded from their observations on Chlamydomonas that the photosynthetically active pigments are arranged in physically discreet groups and that chlorophyll molecules absorbing at longer wavelengths exhibit more relative order than those absorbing preferentially at shorter wavelengths. In order to obtain these results in chloroplast suspensions at room temperature, it is essential to correct for the depolarization due to the rotation of the plastids in solution. Experiments with modulated light (Mar and Govindjee, 1971; Whitmarsh and Ixvine, 1974) or short flashes (Junge and Eckhoff, 1973) circumvented this source of artefacts and gave basically the same results. The advantage of working at liquid N, temperature lies in the abolition of Brownian movement with its influence on p . In addition, on a molecular basis, the orientation of the individual pigment at the time of freezing is effectively preserved, eliminating errors due to its rotation during the observation period. Prerequisite for any interpretation is, however, a reasonable assurance that no alteration of pigment orientation occurs in the freezing process (see Section 111). A third advantage is the reasonable assumption that the extent of energy migration before emission remains constant. Polarization measurements at 77°K have been undertaken to investigate light-mediated alterations of chlorophyll orientation in algae and chloroplasts (Harnischfeger, 1974). They showed that the orientation of pigment molecules within the light-harvesting complex is not a static parameter. On the contrary, its degree is influenced and altered by light and photosynthetic electron transport. The following observations led to this conclusion. A brief illumination (seconds to minutes) of algae and chloroplasts before rapid cooling to 77°K results in an increase of fluorescence emission (Fig. 19). T h ~ sphenomenon was first noticed by Donze and Duysens (1969) who thought it to be associated with the nature of the primary acceptor of PS I1 in the electron transport chain. However, there is no alteration of the spectral quality associated with this emission increase, as can be seen from the same ratio between the individual emission peaks. The rise in fluGrescence depends on both light energy and exposure time of the pre-illumination and shows transient behaviour (Fig. 20). Further investigation led to the observation that the degree of polarization, p , decreased at the principal fluorescence peaks upon light exposure (Fig. 21). This gives an indication that the increase of fluorescence intensity is coupled to some reorientation of pigment. Prolonged illumination diminishes the stimulation of fluorescence and increases p (Fig. 22). Although this light-induced pigment orientation is not directly dependent on the state of the trap of PS 11, an alteration of the F,,,-band (Goedheer, 1966) should be observed if there is a definite connection to electron transport. This was concluded from the observation that the fluorescence stimulation in chloroplast suspensions is influenced by the presence of electron acceptors and uncouplers (Harnischfeger, 1974).

34

GOTZ HARNISCHFEGER

660 680 700 720 740 760 nm emission Fig. 19. Fluorescence emission spectrum of ChZoreIZa at 77°K before ( 0 ) and after ( 0 ) light exposure. Synchronized algae in the autospore phase, adsorbed on cheese-cloth, were used. Excitation at 642 nm; normalization of the data t o the filter transmission at 660 nm. Every point represents the average of five different determinations. The bars indicate the standard deviation (from Harnischfeger, 1974).

The effect of the added inhibitor to photosystem 11, DCMU, is of special interest. When incubating algae with this chemical in the dark and cooling the sample directly to 77”K, an increase of p in the short wavelength region was noticed while no effect or only a small decline was observed in the 720-nm region. This points to some direct interaction between DCMU and the pigment complex randomizing the orientation of PS I1 chlorophyll. The stimulation of emission by pre-illumination can still be seen in DCMU treated Chlorella but at

U

0-

FLUORESCENCE OF PS-SYSTEMS AT 77°K

C

.-0

E

m .-m

a,

E

t

Fig. 21. Change in degree of polarization p upon illumination of Chlorella: ( 0 - ) dark kept control; ( 0 - ) 1 min light exposure. Each point represents the average of five determinations. See Fig. 19 for further details (from Harnischfeger, 1974).

35

Fig. 20. Dependence of 1'686 on light intensity and exposure time during illumination prior to freezing: (0-1 l o 5 ergs/cm2 s; (e-) 1.6 x lo4 ergs/ cm2 s. For other details see Fig. 19 (from Harnischfeger, 1974).

36

GOT2 HARNISCHFEGER

much shorter exposure times than in the control. If exposure times and light intensities were used which lead to maximum stimulation of emission and minimum p at 77°K in the untreated controls, DCMU affected these parameters in much the same way as it diminished electron transport (Harnischfeger, 1974).

24 0

,-LE I

-. \

\

m

I

\

I

\

I

200

160 a,

In C

2 V

120 100

80

40<

0 0

I

I

I

20

40

60

I

I

I

80 100 120 sec. iight exposure

Fig. 22. Inverse relation between fluorescence intensity and degree of polarization p in Chlorellu: ( 0 - ) F684; ( 0 - ) P686; (m-) F725; ( 0 - ) P725. Each point represents the average of five spnaratp determinntinnc T P P Fio

19 fnr fnrther details (from Harnifchfeeer. 1974).

It is noteworthy, that the increase of p upon addition of DCMU has been noticed also by Whitmarsh and Levine (1974) in excitation spectra of emission >680 nm, provided the excitation wavelength was above 650 nm. They interpreted these results, obtained in experiments performed at room temperature, as a closing of PS I1 traps and, consequently, an increased number of energy transfer steps between individual pigment molecules before emission takes place. A similar notion had been forwarded previously by Mar and Govindjee (1971).

FLUORESCENCE OF PS-SYSTEMS AT 77°K

37

If one assumes that the data taken at room and liquid N, temperature reflect the same effect of DCMU on the photochemical complex-which is by no means certain-then the values for p measured at 77°K do not support the above interpretation. Under these circumstances the converting centre or trap is always in a closed position since the electron transport chain is inoperative and all available quencher completely reduced by the exciting light before measurements are started. An interference with, and an alteration of, the pigment orientation seems at present the only plausible interpretation for the action of DCMU under these conditions. In consequence, the polarization experiments at 77°K suggest that some transition of pigments from one semi-organized arrangement to another seems to take place upon illumination. Its transient nature, sensitivity to DCMU and electron I-Iill-acceptors indicates that this rearrangement is, although indirectly, connected to electron transport. The notion is supported by the sluggishness of the response, which is in the order of seconds to minutes as compared to ps for electron transport. The pigment orientation measured seems, therefore, to be a consequence of swelling/shrinking phenomena occuring within a similar time range, and/or the general redox state of the thylakoid membrane. Some further experimentation on this subject is clearly needed.

VI. INTERACTIONS BETWEEN YHOTOSYSTEMS: QUALITATIVE ASPECTS AND KINETIC ANALYSIS The experiments of Emerson (1957) provided the first evidence that the photosynthetic pigment complex might consist of different though interacting photosystems. Since then, a functional characterization of the two photosystems directly connected to electron transport (PS I, PS 11) has been accomplished largely through the use of selective donor and acceptor systems (review by Myers, 1971). The assignment of the various pigments and chlorophyll species to one of these photosystems has been discussed in a previous section. In the following, therefore, the emphasis is placed on the mutual interaction between the different pigment systems, since the distribution of energy between them constitutes an important regulatory feature of photosynthetic electron transport. (F732 : F685 in chloroplasts) taken from the emission The ratio F,,, : spectra at 77°K is generally used as a measure for the energy distribution between the pigment complexes of the two photosystems. This value, however, can serve only as an estimate. It is self-evident from the previous foregoing treatment, that cooling artefacts influence greatly the obtained ratios. Figure 23 gives a particularly good example. The distribution of harvested energy between PS I and PS 11, which changes over the synchronous cycle of Chlorella, differs according to sample thickness. A shift of the curve by several hours is seen, although exactly the same algae have been used to prepare the sample.

38

GOTZ HARNISCHFEGER

The main criticism, however, arises from the fact that the areas under the peaks and not their maximum intensity should be the parameters evaluated. Since the emission bands overlap considerably, the areas are difficult if not impossible to determine. Thus, the above ratio is usually regarded as an approximate estimate of energy distribution. The following considerations are based on this notion. A qualitative redistribution of energy can be observed when the fluorescence emission spectra at 77'K in the presence and absence of divalent cations are

3

c

0

-

0

0

L 0

4

8

12

16

20

U

hr in the synchronous C Y C ~ Q Fig. 23. Ratio F725 : F686 of a synchronous Chlorella culture measured using the cheese-cloth ( 0 ) and a suspenslon (0) method (lollipop with 1-mm spacing).

compared. As seen in the example of Fig. 24, addition of Mg2+ enhances the emission at 685 and 695 nm (PS 11) and decreases that at 732 nm (PS I). This effect, first reported by Murata (1969), is not connected with any major change in absorption properties of the chloroplast preparation. Concentrations of 3 mM sufficed for maximum efficiency, regardless whether Mg2+,Ca2+ or Mn2+ were used. Murata interpreted his results as an ion-induced change of excitation transfer between pigment systems I and 11. His data on partial reactions of electron transport supported this notion. He envisioned the control by ions at the place of energy transfer from the bulk chlorophyll of PS I1 to that of PS I.

FLUORESCENCE OF PS-SYSTEMS AT 77°K

39

The conclusion concurs with earlier findings on fluorescence induction in spinach chloroplasts at 77'K which showed that excess excitation energy in PS I1 is shuttled into PS I (Murata, 1968). According to Joliot et al. (1968) this "spillover", if present, does not exceed 30% of the total absorbed light energy. The above interpretation, that the effect of added Mg2+ is solely on the physical interaction between active pigment systems, has been disputed by a

nm emission Fig. 24. Influence of added MgClz on the fluorescence emission spectrum of spinach chloroplasts at 77°K (Murata-effect). Broken chloroplasts in 20 mM NaC1-15 mM TRIS pH 7.8. Chlorophyll concentration of the suspension, which contained 60% glycerol, was 15 ccg/ml. Lollipop sample mount with 1-mm spacing, excitation by blue light (Bahlzers K-2). Qualitative similar data can be obtained also with the cheese-cloth method and when aqueous suspension was used in the lollipop.

number of researchers. Comparing the number of light quanta harvested and the ensuing rate of electron transport they emphasize either an activation of photosynthetically inactive pigments (Rurainski and Hoch, 1971 ; Malkin and Siderer, 1974) or argue that part or all of the control might be exerted through the enzymatic properties of the electron transport chain proper (Harnischfeger and Shavit, 1974).

40

GOT2 KARNISCHFEGER

The effect of monovalent ions, both anions and cations, is difficult to characterize in this respect. While it is well known that such cations mimic the effect of divalent ions in electron transport reactions, although about ten times their concentration is required, these influences are less pronounced in the emission spectra at 77°K. Li (1974) observed an increase of the ratio F 7 3 2 : F695 upon addition of 10 mM NaCl (2.4 without, 3.8 in the presence of this salt). It is not clear whether this effect is due to the anion or the cation and whether F695 in the ratio can be taken as measure of PS 11. Chloride ion is known to enhance F 6 9 5 supposedly due to an altered redox state of the PS I1 reaction center (Heath and Hind, 1969). In addition, freezing artefacts cannot be excluded in that particular investigation. A control of energy distribution through cations is difficult to demonstrate in algae. An attempt was made by Mohanty et al. (1974) using the kinetics of

670

700

750

nm emission Fig. 25. Influence of added acetate on the emission spectrum of Chlorellu at 77°K. Excitation at 436 nm. The samples contained 15 pM DCMU and, if indicated, 0.1 M K-acetate. The algae were frozen adsorbed o n cheese-cloth (redrawn from Mohanty et ul., 1974).

FLUORESCENCE OF PS-SYSTEMS AT 77°K

41

fluorescence induction at room temperature as the indicator. The results did show an influence of added ions on fluorescence which they interpreted as an altered interaction between photosystems. In addition, they observed that acetate ions exerted the opposite effect to divalent cations, namely, a decrease of PS I1 and an increase in PS I emission (Fig. 25). A role for iron in determining the proper balance of energy distribution between the photosystems has been pointed out by Oquist (1974). He observed that iron deficiency in Anacystis nidulans leads to the collection of excitation energy predominantly in PS I1 (Fig. 26). A shift in the absorption spectrum from 679 nm to 673 nm and the appearance of a new emission band at 755 nm was ascribed to a simultaneously occurring rearrangement of pigments. The site of action of the added ions seems to be the thylakoid membrane. As pointed out by Franck (1958), chlorophyll fluorescence and the quantum yield of photosynthesis are influenced by external agents which change the distribution of water in the membrane. The effect of ions on the fluorescence

686

’

1

670

1

717

1

1

1

1

700

1

1

1

1

750

nm emission Fig. 26. Effect of iron deficiency on the fluorescence emission of Anacystis nidulans at 77°K. Broadband excitation between 350 and 500 nm. The algae were frozen adsorbed on filter paper. The spectra are normalized to equal area under the curves (redrawn from Oquist, 1974).

42

@TZ HARNISCHFEGER

emission has, thus, to be viewed in context with their ability to induce drastic changes in thylakoid structure, e.g. swelling and shrinking (Izawa and Good, 1966; Murakami et al., 1975). The chlorophyll molecules are localized within the membrane matrix and their mutual orientations and distances are, therefore, influenced by ion-induced membrane changes. An effect on excitation transfer between pigment complexes is a logical consequence of these processes. The ion-induced redistribution of absorbed light energy, observed as differences in the fluorescence emission spectra, provides the starting point for a more detailed investigation of the interaction between photosynthetic pigment complexes. One promising approach along these lines has been developed by Butler and Kitajima (1974, 1975a,b). It is based on the alteration of the fluorescence kinetics at 77"K, which is caused by the addition of cations. The importance of this approach lies in the novel theoretical concept for the subsequent experiments. Since t h s theoretical groundwork offers, in my opinion, a distinct advantage for further investigations into this matter, the work of Butler and Kitajima will be treated in some detail using their terminology and data to illustrate crucial features. Figure 27, taken from Butler and Kitajima (1975b), gives the kinetic traces measured, together with the nomenclature of the different parameters used in the analysis. It has to be emphasized that the general interpretation of this kinetic behaviour at 77°K is different from that observed at room temperature. W e at 295°K the emission increase of PS I1 to the final level is governed by the primary electron acceptor Q , this is not entirely the case at 77°K (Okayama and Butler, 1972; Boardman and Thome, 1969; Murata et al., 1973). The validity of interpreting F, in connection with Q under low temperature conditions was specifically questioned by experimental results of Yamashita and Butler (1969). Although they could restore the electron transport in TRISwashed chloroplasts through addition of the electron donor diphenylcarbazide (DPC), F, remained quenched. The interpretation that Fu(690)t might originate in spillover from PS I to PS 11, postulated from electron transport experiments by Sun and Sauer (1972), could not be substantiated. Butler and coworkers, on the other hand, showed, that the major part of Fo and F, measured at 690 nm both originate in PS 11. Fo(730) is the only signal entirely due to the PS I pigment complex. F v ( 7 3 0 )is energy shuttled from PS I1 to PS I only (Kitajima and Butler, 1975; Butler and Kitajima, 1975a). These findings led to their proposal of the tripartion model, presented in Fig. 18 and Section IV, with its bulk chlorophyll (light-harvesting complex, LH) and the pigment complexes encompassing PS I and PS 11 proper.

t F690 and F730 in the following refers to the fluorescence signal measured at these wavelengths by Butler and Kitajima (1975a, b). They use these parameters in developing and extending their theoretical model to experimental measurements. For comparative purposes their notion will be followed, but, as will become evident, these signals correspond to F685 and F732 as defined in the previous sections.

A. F690

IS0 100

80

60 40

20

100 80 60 40

20

F690

Fo F"

Mg

+

Mg

28

32

39

82

Fig. 27. Fluorescence induction curves and resulting intensities of chloroplasts at 77°K

in the absence and presence of 5 mM MgC12. Excitation in the blue spectral region. The table gives the appropriate line segments in relative units at the wavelength of actual measurement (from Butler and Kitajima, 1975b).

44

GOT2 HARNISCHFEGER

They state that the photosystems I and I1 themselves contain besides still considerable amounts of antenna chlorophyll their appropriate reaction centre couple PIAI or PIIAII.The absorbed light energy is divided into the a-fraction, light exciting PS I either directly or by transfer from LH, and the P-fraction, combining the energy exciting PS I1 with all the radiation being dissipated by LH through fluorescence and non-radiative processes. Quanta of 0 can also be used to excite PS I after energy transfer from PS 11. An energetic representation of the Butler and Kitajima model, which shows the various transfer and deexcitation reactions, is given in Fig. 28. The excited state is denoted by an asterisk, the subscripts p, F, T (t), D represent photochemical, fluorescent, energy transfer and radiationless decay reactions respectively. A refers to the

Pa. A,,

Chl an-

Chl LH

Chl aI

5. A,

Fig. 28. Kinetic representation of the tripartition model of Fig. 18. See text for further details (from Butler and Kitajima, 1975b).

primary acceptor of photosystem while P A depicts its combination with the trap pigment, the reaction centre complex. Its closed form is given by P'A-. The diagram contains various assumptions, some of which are supported by experimental evidence (Butler and Kitajima, 1975a). Firstly, there is a rapid exchange of harvested quanta between PS I1 and LH in both directions. This is equivalent to the statement, that k ~ ( 6 and ~ ~ ) k ~ ( are~ equivalent ~ ~ ) although they originate in different chlorophylls. Secondly, it restates the earlier finding of Kitajima and Butler (1975) that FO and F, at 695 nm result entireIy from PS 11. Thirdly, no fluorescence is emitted by a reaction centre itself. Also, excitation trapped by a closed reaction centre of PS I1 can be transferred back to the antenna chlorophyll, a feature not possible in PS I. To simplify the calculations, kp is normally set as %kt, i.e. the yield vl, = 1. Lastly, irradiation of PS I only has no effect on PS I1 fluorescence, i.e. no energy transfer PS I + LH + PS I1 occurs at 77°K. This supposes that far red light gives rise to only the F o ( 7 3 0 )signal, while Fu(730) denotes explicitly the energy transferred from PS I1 into PS I.

FLUORESCENCE OF PS-SYSTEMS AT 77'K

45

Considering the above restrictions and assumptions implicit in the diagram, Butler and Kitajima (1975b) arrived at the following four equations which provide a mathematical description for the fluorescence yield pF: (l)

okF(690)

(PF(690)

='iF(690) + k D I I + kT(II+I)

'

kDII kF(73 0)

qF(730) = @pT(II+I)

'a) kF(730)

(3)

-+

kTI

kT(II-+I) pT(II-I) kF(690)

(4)

I -I-A11 VTIIptII

kT(II-+I)

kDII

)

atP=l.

stands here for the fraction of oxidized acceptor, equivalent to open reaction centres of PS I1 while the expression

A11

I I-

-

A11

pTIIptII

denotes the contribution due to closed ones. The calculation of a, and 0 can be accomplished with the use of these equations in a relatively straightforward way. An example using the measurements of Fig. 27 taken by Butler and Kitajima, is given in the following. The data are also used to express the intensity shift of the bands, resulting from addition of Mg2+, in terms of the ratio a+ : a!-." The calculation is based on the assumption that 9(730) consists of a constant part due t o direct excitation or direct transfer from LH to PS I (= F o ( 7 3 0 ) ) and a fraction originating in PS 11. The latter transfers energy from its constant part @ ~ ( T I I - + I ) ~and ) its variable part ( P ~ T ( I I - + I )into ~ ) PS I. & T ( I I + I ) ~ can be set equal to Fu(730)* Since fiT(lI-*I)O _- F0(690) f i T (II-tI)u

Fu( 6 9 0)

we obtain using the appropriate line segments - FO( 6 9 0 ) fiT(II-tI)O

- ___

Fu ( 7 3 0)

Fu( 6 9 0)

Measured data are expressed as F (subscript) while their general expression is given asp; Superscript, + denotes the presence, - the absence of Mg2+.

46

GOTZ HARNISCHFEGER

For the data given in Fig. 27,

Subtracting & T ( I I . + I ) ~ from FO(730) gives directly a relative value of a, i.e. F0(730) -PPT(II+I)O

=a

in the example cited a- = 61 a+ = 51

and a-- 1.2

a+

To estimate p we assume that non-radiative decay ( k D )and chemical dissipation of energy (k,) are neghgibly small compared to fluorescence, i.e. total quanta emitted =total quanta absorbed or F0(690) t F u ( 6 9 0 ) tF0(730)

tFu(730)

=

thus in the example a+ = 0.27; ' 0 = 0.73; a- = 0.36; 0- = 0.64.

Butler and Kitajima developed more detailed expressions for a, and the various rate constants involved without recourse to the simplifications used above. Their detailed calculations can be looked up in the cited literature. Unfortunately, the straightforward logic of the equations derived from the theoretical model is difficult to test in an experimental way. Some basic obstacles, still defying adequate correction, prevent a meaningful interpretation of the calculated figures. This becomes evident from the results compiled in Table 1, which contains the averaged results of a series of independent determinations performed with the same chloroplast preparation. Inspecting Table I it is immediately noticed that the method of sample mount and freezing leads to differences in the final results. A good example is the ratio F732 : F685. Its increase with increasing width of the lollipop sample is due to the reason indicated previously (Fig. lo), namely a decrease in freezing rate, which is roughly inversely proportional to the thickness of the aqueous medium between chloroplasts and liquid N,. Secondly one observes, that the ratio Fo : Fu decreases with decreasing freezing rate. Since both this ratio and the fluorescence intensity at the

47

FLUORESCENCE OF PS-SYSTEMS AT 77°K

appropriate emission maximum enter into the calculation, a gross distortion of the real situation is obtained. This can be clearly seen in the value of a, which should be constant in a specified chloroplast preparation. Since, however, the only parameter varied in the experiment is the freezing rate, the different results clearly indicate artefacts introduced during the cooling process. It is noteworthy that the ratio ( Y ( - M ~ :) remains constant, i.e. the introduced errors must cancel each other. The value of around 1.4 is higher than TABLE I The Light Distribution between the Photosystems, Calculated According to Butler and Kitajima ( 1 975b), in Samples of the Same Chloroplast Preparation Using Different Types of Specimen Probes Probe Cheese-cloth mounting Lollipop 1-mm spacing Lollipop 2-mm spacing

Addition

F732/F685

(Fo/Fd685

a!

5 mM Mg2+

2.1 3 1.09 2.67 1.51 2.72 1.61

4.1 4.8 1.6 1.9 1.2 1.1

0.1 35 0.095 0.337 0.228 0.380 0.283

-

5 mM Mg2+ 5 mM Mg2+

“(-Mg)/&(+Mg) 1.42 1.47 1.36

Spinach chloroplasts, prepared in 0.4 M sucrose-2 mg/ml ascorbate-1 mg/ml BSA-0.1 M TRICIN pH 7.8, washed and stored at 0°C in the dark in 0.4 M sucrose-0.1 M TRICIN pH 7.8. For the measurements the suspension (1.2 mg chllml) was diluted, using this medium, to a concentration of 0.6 pg/cm2 illuminated area. Excitation with blue light between 450 and 500 nm. The emission peaks appeared at 687 nm (F685). 695 nm (F695) and 738 nm (F732). The signals at 687 and 738 nm were used in the calculations after appropriate corrections for photomultiplier sensitivity and instrument deviation. The values given are averages from 10 (cheese-cloth) to 4 (lollipop) independent determinations. Individual measurements differed not more than 10% from each other.

that given by Butler and Kitajima (1.2) but this difference can be traced to the small amount of NaCl present in their experiments. Under the latter circumstances the ratio obtained in my experiments was 1.1 6 . There are some other arguments against too far-reaching an interpretation of the theoretical model. Butler and Kitajima themselves (1975b) pointed out that the rate constants cannot be taken as descriptions of rigorously defined activities with precise physical meaning, since that requires an exact definition of the coupling between the light-harvesting complex and the reaction centre pigments. Statistical variables, necessary by the various possible spatial arrangements, enter into such calculations (Seely, 1971; 1973a, b). Nevertheless, although the experimental procedure devised is presently unable to produce clearcut results, the approach of Butler and Kitajima constitutes an important step towards a quantitative analysis of the interaction between pigment systems. Their simplified method allows at least an estimate of its mutual dependency from a physical point of view.

48

GOTZ HARNISCHFEGER

Since the distortions in the experimental determinations will, in time, become known in detail and will then be eliminated either by correction factors or different experimental design, such calculations might in the future yield results equally as important as the values obtained from assessing the photosystems in a functional way through the enhancement effect of Emerson (1957).

VII. SYNOPSIS AND OUTLOOK The study of the light-harvesting pigment complex has certainly gained new impetus through the application of low temperature spectroscopy to the problems associated with it. The obvious advantage of high resolution of the spectral bands paired with an arrest of the dynamic equilibrium between the various membrane components to give a momentary picture of the actual chromophore orientation, made the technique ideally suited for the investigation of the photosynthetic apparatus. The results subsequently obtained seemed to justify these expectations. With the help of liquid N, spectroscopy it was thus convincingly demonstrated that the chlorophylls are aggregated in various ways within the thylakoid membrane, either among themselves or with membrane components. Based on these results theories of pigment interaction and efficient control of energy transfer were devised and subsequently tested. A consequence of such considerations in connection with functional arguments is the speculation that the protein attached to chlorophyll acts as an organizer providing different pigment orientations while the membrane lipids determine the various favourable microenvironments (reLiew by Anderson, 1975). This concept leads to the postulation of a simple control mechanism of the energy distribution between photosystems. The control is simply exerted through alterations in pigment microenvironment evolving from changes of the lipid-protein matrix caused by ions. The actual observation of ion influences on the fluorescence properties at 77°K provided an experimental support of this concept. Fluorescence measurements at liquid N, temperature also expanded our knowledge of the energy transfer and distribution between the various pigment species of the photosynthetic apparatus. Sufficient information is now available to propose the models of actual pigment arrangement presented earlier and to take the first steps to calculate the energy distribution in a quantitative way. Some scepticism, however, seems to be well founded. The crucial question surrounding all the experiments is far from solved, namely, whether the various bands of fluorescence observed at 77°K and their underlying pigment aggregates are also an in vivo property of the system or constitute only artefacts inherent in the method. We know from measurements at room temperature, especially studies on dichroism, that various forms of chla oligomers do exist, but there is at present no convincing evidence that all of those inferred from fluorescence at 77°K are present in vivo. Since nothing definite is known about the actual

FLUORESCENCE OF PS-SYSTEMS AT 77°K

49

arrangement of pigments within the membrane matrix, the arguments are only speculative. As long as the possibility cannot be excluded, that alterations of the pigment system which can neither be adequately assessed or corrected for are introduced through the necessary sample treatment, spectroscopy at 77°K can only provide circumstantial evidence. Steps to analyse the underlying systemic errors and methods for their prevention have finally been taken and are actively pursued. From studies of freeze-etch procedures in electron microscopy it is known, that a quick-freezing method does not introduce visible damage to membrane structure down to a 20 A level of resolution. To extend our knowledge in this respect to the molecular level determining the spectroscopic properties seems, in my opinion, 10 be the most pressing problem for a further development of liquid N, spectroscopic techniques. Only with a yolid body of information about these systemic side-effects can the problems involving a quantitative assessment of the photophysical parameters of photosynthesis be successfully attacked. It seems ironic, that the theoretical basis for experiments in this respect is far ahead of the actual instrumental capability of the method. This is amply illustrated in the detailed quantitative models of Seely (1971, 1973a, b) and Butler and EGtajima (1974, 1975b). It is to be hoped that the practical side will gain ground in the near future, so that the full potential inherent in the low temperature techniques can be exploited. ACKNOWLEDGEMENTS The he1pft:l suggestions of Drs Jacobi, Robinson and Rurainski are gratefully acknowledged. My own work which is reported in this article was supported by the Deutsche Forschungsgemeinschaft. Thanks are due to Ms S. Forbach for excellent technical assistance. REFERENCES Anderson, J. M. (1975). Biochim. biophys. Acta 416, 191-235. Arnold, W. and Azzi, J. R. (1 968). Proc. natn. Acad. Sci. U.S.A. 61, 29-35. Ashwood-Smith, M. J. and Warby, C. (1971). Cryobiology 8, 453-464. Becker, J. F., Geacintov, N. E., van Nostrand, F. and von Metter, R. (1973). Biochem. biophys. Res. Commun. 57, 597-602. Boardman, N. K. (1970). Ann. Rev. Plant Phys. 21, 115-137. Boardman, N. K. and Thorne, S. W. (1969). Biochim. biophys. Acta 189, 294-297. Bonner, W. D. (1961). In “Haematin Enzymes” (J. E. Falk, R. Lemberg, and R. K. Morton, Eds), 479-500. IUB Symposium, Pergamon Press, Oxford. Breton, J. and Roux, E. (1971). Biochem. biophys. Res. Cornmun. 45, 557-563. Breton, J., Michel-Villaz, M. and Paillotin, G. (1 973). Biochim. biophys. Acta 314, 42-56. Bril, C. van der Horst, D. J., Poort, S. R. and Thomas, J. B. (1969). Biochim. biophys. Acta 172, 345-348.

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Brody, M., Nathanson, B. and Cohen, W. (1969). Biochim. biophys. Acta 172, 340-342. Brody, S. S. (1958). Science, N.Y. 128, 838-839. Brody, S. S. (1965). Archs. Biochem. Biophys. 110, 583-585. Brody. S. S. and Brody, M. (1963). Natn. Acad. Sci.: Natn. Res. Council Publ. 1145, 455-478. Brody, S. S. and Broyde, S. B. (1963). Nature, Lond. 199, 1097-1098. Brody, S. S. and Rabinowitch, E. (1957). Science, N.Y. 125, 555. Broyde, S. B. and Brody, S. S. (1965). Biochem. biophys. Res. Commun. 19, 444-45 1. Broyde, S. B. and Brody, S. S. (1966). Biophys. J. 6, 353-366. Butler, W. L. (1964). Ann. Rev. Plant Phys. 15,451-470. Butler, W. L. (1965). Biochim. biophys. Acta 102, 1-8. Butler, W. L. and Kitajima, M. (1974). Proc. IIZ Int. Congr. Photosynth. 13-24. Butler, W. L. and Kitajima, M. (1975a). Blochim. biophys. Acta 376, 116-125. Butler, W. L. and Kitajima, M. (1975b). Biochlrn. biophys. Acta 396, 72-85. Butler, W. L. and Norris, K. H. (1960). Archs. Biochem. Biophys. 87, 31-40. Cherry, R. J., Hsu, K. and Chapman, D. (1972). Biochim. biophys. Acta 267, 5 12-522. Cho, F. and Govindjee (1970a). Biochim. biophys. Acra 205, 317-328. Cho, F. and Govindjee (1970b). Biochim. biophys. Acta 216, 139-150. Cho, F., Spencer, J . and Govindjee (1966). Biochim. biophys. Acta 126, 174-176. Clayton, R. K. (1965). “Molecular Physics in Photosynthesis”. Blaisdell, New York. Clayton, R. K. (1970). “Light and Living Matter”, Vol. I : The physical part. McGraw-Hill, New York. Cotton, T. M., Trifunac, A. D., Ballschmiter, K. and Katz, J. J. (1974). Biochim. biophys. Acta 368, 18 1-198. Dewar, J. (1894a). Proc. R. Soc. 55, 340. Dewar, J. (1894b). Proc. Chem. Soc. 10, 171. Donze, M. and Duysens, L. N. M. (1 969). Progr. Photosynth. Res. 11, Tiibingen, 991-995. Dutton, P. L. and Wilson, D. F. (1974). Biochim. biophys. Acta 346, 165-212. Emerson, R. (1957). Science, N. Y. 125, 746. Emerson, R. and Arnold, W. (1932).J. gen. Physiol. 15, 391-420. Forster, T. ( 1951). “Fluoreszenz organischer Verbindungen”. Vandenhoeck und Ruprecht, Gottingen. Franck, J. (1958). Proc. natn. Acad. Sci. U.S.A. 44, 941-948. French, C. S. (1971). Proc. natn. Acad. Sci. U.S.A. 68, 2893-2897. French, C. S. and Koerper, M. A. (1967). Yb. Carnegie Inst. Wash. 65, 492-498. French, C. S., Brown, J. S., Wiessner, W. and Lawrence, M. C. (1971). Yb. Carnegie Inst. Wash. 69, 662-670. Gaffron, H. and Wohl, K. (1936a). Naturwissenschaften 24, 81-90. Gaffron, H. and Wohl, K. (1 936b). Naturwissenschaften 24, 103-107. Goedheer, J. C. (1 964). Biochim. biophys. Acra 88, 304-3 17. Goedheer, J. C. (1966). In “Biochemistry of Chloroplasts” (T. W. Goodwin, Ed.), 75-82. Academic Press, London and New York. Goedheer, J. C. (1969). Biochim. biophys. Acta 172, 252-265. Goedheer, J. C. (1972). Ann. Rev. Plant Physiol. 23, 87-1 12. Gorham, P. R. and Clendenning, K. A. (1950). Can. J. Res. C28, 513-524. Govindjee (1963). Natn. Acad. Sci.: Natn. Res. Council. Publ. 1145, 318-334.

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Govindjee (1 972). In “Chloroplast Fragments” (G. Jacobi, Ed.), 17-45. Gottingen. Govindjee and Govindjee, R. (1 975). In “Bioenergetics of Photosynthesis” (Govindjee, Ed.), 1-50. Academic Press, New York. Govindjee and Yang, L. (1966). J. gen. Physzol. 49, 763-780. Gregory, R. P. F. (1975). Bzochem. J. 148,487-497. Harnischfeger, G. (1 974). Ber. dt. Bot. Ges. 87,483-49 1. Harnischfeger, G. and Gaffron, H. (1970). Planta 93, 89-105. Harnischfeger, G. and Shavit, N. (1974). FEBS Lett. 45, 286-289. Hartridge, H. (1920/21). J . Physiol. 54, 128-130. Heath, R. L. and Hind, G. (1969). Biochim. biophys. Acta 180, 414-416. Heber, U. (1970). In “The Frozen Cell” (G. E. W. Wolstenholme and M. O’Connor. Eds) 175-188. Churchill, London. Izawa, S. and Good, N. E. (1966). Plant Physiol. 41, 544-552. Jacobi, G. (In press). In “Encyclopedia of Plant Physiology” (A. Pirson, Ed.). Springer-Verlag, Berlin, Heidelberg and New York. Joliot, P., Joliot, A. and Kok, B. (1968). Biochim. biophys. Acta 153, 635-652. Junge, W. and Eckhoff, A. (1973). FEBS Lett. 36,207-212. Ke, B. and Vernon, L. (1967). Biochemistry 6,2221-2226. Keilin, D. and Hartree, E. F. (1949). Nature, Lond. 164, 254-259. Kitajima, M. and Butler, W. L. (1975). Biochim. biophys. Acta 376, 105-1 15. Knox, R. S. (1975). In “Bioenergetics of Photosynthesis” (Govindjee, Ed.), 183-224. Academic Press, New York and London. Kok, B. (1963). Natn. Acad. Sci.: Natn. Res. Council Publ. 1145, 45-55. Kortiim, G. ( 1969). “Reflectance Spectroscopy: Principles, methods, applications”. Springer-Verlag, Berlin, Heidelberg and New York. Kreutz, W. (1970). Advs. bot. Res. 3, 53-169. Lavin, G. I. and Northrop, J. N. (1 935). J. A m . Chem. Soc. 57, 874-875. Li, K. S. (1974). Bot. Bull. Acad. Sin. 15, 89-95. Litvan, G. G. (1972). Cryobiofogy 9, 182-191. Litvin, F. F. and Sineshchekov, V. A. (1975). I n “Bioenergetics of Photosynthesis’’ (Govindjee, Ed.), 6 19-66 1. Academic Press, New York and i London. Malkin, S. and Siderer, Y. (1 974). Biochzm. biophys. Acta 368,422-43 1. Mar, T. and Govindjee (1971). Proc. I I I n t . Congr. Photosynth, Stresa 271-281. Mazur, P. (1970). Science, N.Y. 168, 939-949. Mazur, P., Leibo, S. P. and Chu, E. H. Y. (1972). Expl Cell Res. 71, 345-355. Meyer, B. (1 971). ‘‘Low Temperature Spectroscopy”. Elsevier, New York. Mohanty, P., Govindjee and Wydrzynski, T. (1974). Plant Cell Physiol. 15, 213-224. Mohanty, P., Mar, T. and Govindjee (1971). Biochim. biophys. Acta 253, 213-221. Mohanty, P., Zilinskas Braun, B., Govindjee and Thornber, J. P. (1972). Plant Cell Physiol. 13, 8-9 1. Moor, H. (1964). Z. Zellforsch. 62, 546-580. Moor, H. (1973). In “Freeze Etching, Techniques and Applications” (E. L. Benedetti and P. Favard, Eds), 11-20. SOC.Fr. Microsc. Electron, Paris. Miiller, W. and Wartenberg, A. (1971). Z . Pfl.Physio2. 65, 365-377. Miiller, W. and Wartenberg, A. (1972). 2. PjZPhysiol. 67, 318-332. Murakami, S., Torres-Pereira, J. and Packer, L. (1 975). I n “Bioenergetics of Photosynthesis” (Govindjee, Ed.), 556-6 19. Academic Press, New York. Murata, N. (1968). Biochim. biophys. Acta 162, 106-121.

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Murata, N. (1969). Biochim. biophys. Acta 189, 171-181. Murata, N., Itoh, S. and Okada, 0. (1973). Biochim. biophys. Acta 335, 463-47 1 . Murata, N. Nishimura, M. and Takamiya, A. (1966). Biochim. biophys. Acta 126, 234-243. Myers, J. (197 1). Ann. Rev. Plant Phys. 22, 289-3 12. Nathanson, B. and Brody, M. (1 970). Photochern. Photobiol. 12,469-479. Nichols, E. L. and Merrit, E. (1904). Phys. Rev. 18, 355-365. Oquist, G. ( 1 974). Physiol. Plant. 31, 55-58. Okayama, S. and Butler, W. L. (1 972). Biochim. biophys. Acta 267, 523-529. Papageorgiou, G. and Govindjee (1967). Biophys. J. 7, 375-390. Riehle, U. (1 968). “Uber die Vitrifizierung verdunnerter wassriger Losungen”. Dissertation, ETH Zurich. Rurainski, H. J. and Hoch, G. E. (1 97 1). Roc. II Int. Congr. Photosynth. Stresa, 133-141. Schmid, G. H. and Gaffron, H. (1968). J. gen. Physiol. 52, 212-239. Seely, G. R. (1971). Proc. I l I n t . Congr. Photosynth. Stresa, 341-348. Seely, G. R. (1973a). J. theoret. Biol. 40, 173-187. Seely, G. R. (1973b). J. theoret. Biol. 40, 189-199. Seliger, H. H. and McElroy, W. D. (1965). “Light: Physical and Biological Action”. Academic Press, New York. Shapiro, J. (1961). Science, N. Y. 133, 2063-2064. Sineshchekov, V. A., Shybin, V. V. and Litvin, F. A. (1973). Dokl. Akad. Nauk. 21 1, 1226-1 229. Sun, A. S. K. and Sauer, K. ( 1 972). Biochim. biophys. Acta 256,409-427. Szalay, L. Torok, M. and Govindjee (1 967). Acta Biochim. Biophys. Acad. Sci. Hung. 2,425-432. Thomas, J. B., van Lierop, J. H. and Ten Ham, M. (1967). Biochim. biophys. Acta 143, 204-220. Thornber, J. P. (1975). Ann. Rev. Plant Phys. 26, 127-158. Thornber, J. P.and Highkin, H. R. (1974). Europ. J. Biochem. 41, 109-1 16. Tollin, G. and Calvin, M. (1957). Proc. natn. Acad. Sci. U.S.A. 43, 895-908. Trebst, A. ( 1 974). Ann. Rev. Plant Phys. 25,423-458. Tributsch, H. (1971). Bioenergetics 2 , 249-273. Tributsch, H. and Calvin, M. (1971). Photochem. Photobiol. 14, 95-1 12. Van den Berg, L. and Rose, D. (1959). Archs. Biochem. Biophys. 81, 319-329. Wehry, E. L. (1967). In “Fluorescence, Theory, Instrumentation and Practice” (G. Guilbauld, Ed.), 37-132. Marcel Dekker, New York. Williams, R. J. and Merryman, H. T. (1970). Plant Physiol. 45, 752-755. Whitmarsh, J . and Levine, R. P. (1974). Biochim. biophys. Acta 368, 199-213. Wolken, J. J. and Schwerz, F. A. (1954). J. gen. Physiol. 37, 11 1-120. Yamashita, T. and Butler, W. L. (1 969). Plant Physiol. 44, 1342-1345.

Receptors for Plant Hormones

MICHAEL A. VENIS Shell Research Ltd, Woodstock Laboratory, Sittingboume Research Centre, Sittingboume, Kent, ME9 8AG

I. 11.

111.

IV.

Introduction . . . . . . . . . . . . . . . . . . Sites of Hormone Action . . . . . . . . . . . . . . A. Effects on Macromolecular Synthesis . . . . . . . . B. RapidEffects . . . . . . . . . . . . . . . . C. Evidence for Two Sites of Auxin Action . . . . . . . The Search for Hormone Receptors . . . . . . . . . . A. Modelsystems . . . . . . . . . . . . . . . B. Direct Interaction with Enzymes . . . . . . . . . C. “Soluble” (Nuclear/Cytoplasmic) Receptors . . . . . . D. Membrane-bound Receptors . . . . . . . . . . . Concluding Remarks. . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION Plant hormones-auxins, gibberellins, cytokinins and abscisins-are involved in the orderly regulation of growth and development. The spectrum of responses elicited by the compounds is wide, and there is considerable interplay between the different groups in the overall regulatory process as shown, for example, by auxin-cytokinin interaction during differentiation of callus tissue. Nevertheless, each class of plant hormones is chemically distinct, and physiological and biochemical responses unique to each group are well documented. The development of synthetic analogues, particularly of the natural auxin 3-indolylacetic acid (IAA), has yielded compounds which continue t o be of major agricultural and horticultural importance and which frequently afford considerable experimental advantages in terms of chemical and metabolic stability. The availability of both active and inactive analogues has led to the formulation of defined structural requirements for activity. Plant hormones act at low concentrations, they are apparently active without metabolic conversion, and do not seem to act as enzyme co-factors. These features would seem to necessitate the existence of specific recognition molecules, i.e. receptors, which mediate and amplify the 53

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hormonal signal. Because of the precision of the recognition process-the ability to discriminate for example between the auxin-active 2,s-dichlorobenzoic acid and the inactive 2,4-dichloro analogue-it seems most likely that the recognition site forms part of a protein molecule. Research on animal hormone receptors has met with considerable success and has been characterized by a reasonably orderly progression from initial observations on hormone binding, through independent confirmation, extension or modification of the particular system, followed in some cases by correlation of receptor binding with some metabolic response. In contrast, the limited literature on receptors for plant hormones is strewn with preliminary reports or abstracts describing systems which have undergone no further development. Nevertheless, there are now (1976) good grounds for believing that we may at last be escaping from a lengthy “lag-phase” and that the next few years will see real and exciting advances in a neglected area of research, crucial to an understanding of the molecular action of plant hormones. A significant stimulus was provided by the publications of Hertel and co-workers on binding of auxins (Hertel et al., 1972) and an auxin transport inhibitor (Lembi et aZ., 1971) to particulate preparations from corn coleoptiles. This has proved to be a convenient and reproducible system, whose further elaboration will be described in detail. In addition, several initial reports have recently appeared describing plant hormone binding to soluble macromolecular systems and it is hoped that these promising developments will be confirmed and extended.

11. SITES OF HORMONE ACTION A. EFFECTS ON MACROMOLECULAR SYNTHESIS

There is abundant evidence that any sustained response to a plant hormone requires synthesis of new RNA and protein, and that the hormones can elicit not only quantitative changes, but selective qualitative changes in the pattern of enzyme protein synthesis. The extensive literature in this area has been reviewed elsewhere (e.g. Key, 1969; Galston and Davies, 1969; Davies, 1973) and the present discussion will be restricted to a few examples of hormonally induced alterations in enzyme synthesis. Probably the most completely investigated system of hormonal control is that of gibberellic acid regulation of enzyme synthesis during germination of cereal grains. Gibberellins pass from the embryo to the aleurone cells where they evoke the appearance of a-amylase and other hydrolytic enzymes responsible for mobilization of endosperm reserves. By fingerprinting (Varner and Chandra, 1964) and by density labelling (Filner and Varner, 1967; Jacobsen and Varner, 1967) it has been established unambiguously that gibberellic acid (GA,) causes the de nova synthesis of &-amylaseand a protease in barley aleurone layers. Most eukaryotic messenger FWA (mRNA) molecules contain a polyadenylic acid

RECEPTORS FOR PLANT HORMONES

55

[poly(A)] sequence, and GA3 has been shown to enhance synthesis of poly (A)-RNA in aleurone layers (Jacobsen and Zwar, 1974; Ho and Varner, 1974). Furthermore, it has recently proved possible to programme a cell-free protein synthesis system from wheat embryos with total or poly (A)-containing RNA from GA3-treated aleurones and obtain immunoprecipitable a-amylase (Higgins et aZ., 1976). The time-course for the appearance of mRNA for a-amylase was found to parallel the time-course for rate of enzyme synthesis following exposure of aleurones to GA3. Although the authors are careful to point out alternative possibilities, these findings taken in conjunction with the effects on poly (A)-RNA synthesis and earlier inhibitor data, point very strongly to a transcriptional control mechanism for gibberellin action. The most widely studied effect of auxins is the promotion of cell enlargement, a process that involves loosening of the cell wall. Anabolic and catabolic enzymes of cell-wall polysaccharide metabolism have therefore received close attention. Maclachlan and co-workers have examined in detail the regulation of cellulase activity in decapitated pea epicotyls which undergo lateral swelling in response to apical application of IAA. In this system, IAA induces large increases in cellulase activity and specific activity, which are blocked by inhibitors of RNA and protein synthesis (Fan and Maclachlan, 1966). Cellulase induction can take place independently of cell division (Fan and Maclachlan, 1967) and the specific activities of several other hydrolases are not altered by IAA under the same conditions (Datko and Maclachlan, 1968). Polysomes isolated from IAA-treated, but not from control tissue, were able to synthesize cellulase in vitro even though both preparations were active in protein synthesis (Davies and Maclachlan, 1969). Taken together, these results support the idea that IAA de-represses the gene for cellulase mRNA. A system which probably has no direct relevance to auxin-induced cell enlargement, but which has been particularly well studied in relation to auxin specificity, is the N-acylaspartate synthetase of peas. Freshly excised pea tissues are able to form only very small amounts of the aspartate conjugates of IAA or NAA (1 aaphthylacetic acid). Synthesis of these conjugates is greatly enhanced by pre-treatment of the tissue with auxins, while chemically related but physiologically inactive analogues are without effect (Sudi, 1964). Induction is not dependent on auxin-stimulated growth, nor is it a substrate-related effect, since auxins which are unable to form acylaspartates are effective inducers (Siidi, 1966). The specificity of the system is illustrated (Fig. 1) by a comparison of the inductive effect of the non-substrate auxin 2,4-D(2,4-dichlorophenoxyacetic acid) with that of the chemically related but very weak auxin 2,6-D(2,6-dichlorophenoxyacetic acid). Benzoic acid also forms an aspartate conjugate in pea tissues pre-treated with auxin, but the main conjugation product was shown to be the chromatographically similar benzoylmalic acid (Venis and Stoessl, 1969). Induced synthesis of both aspartate and malate conjugates is abolished by low levels of actinomycin D,

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MICHAEL A. VENIS

puromycin or cycloheximide which inhibit RNA (actinomycin) or protein synthesis in the tissues (Venis, 1964, 1972). However, whereas the induction of acylaspartate synthesis is an absolutely auxin-specific process, benzoylmalate synthesis is induced both by auxins and by physiologically inactive aromatic carboxylic acids (Venis, 1972). The time-courses of 2,441-induced synthesis (Fig. 2A) indicate that benzoylmalate synthetase is induced somewhat more rapidly than acylaspartate synthetase. The optimal concentration for induction of NAA-aspartate synthesis is reached at 0.5 mg/litre 2 , 4 - ~but : a 25-fold higher concentration is required for maximal induction of benzoylmalate (Fig. 2B). Although an induction period of 6-8 h is required to demonstrate enhanced

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conjugation (Fig. 2A), experiments involving the delayed addition of actinomycin D suggest that the mRNAs for both synthetases are formed within 2 h of auxin addition (unpublished results). Pea tissues thus possess an N-acylaspartate synthetase (substrates IAA, NAA, benzoic acid), induced specifically by auxins, and a benzoylmalate synthetase which is induced by numerous aromatic carboxylic acids, including compounds with auxin activity. Further work on these systems, in particular a rigorous demonstration of de novo synthesis, is hindered by the inability to establish cell-free systems for conjugate synthesis. The solution of this problem would provide a valuable first step towards a detailed comparison of the induction mechanisms in an auxin-specific and an auxin non-specific system. B. RAPID EFFECTS

While there is some debate as t o the actual time required for manifestation of hormonal effects on macromolecular synthesis, in general no convincing effects on RNA and protein synthesis are observed until at least 30 min after hormone

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MICHAEL A. VENIS

application. Many hormone responses take place far more rapidly than this however, and with the advent of sensitive continuous recording techniques for growth measurements these rapid effects have received considerable attention in recent years (see Evans, 1974 for a review). Auxins promote cell elongation after a characteristic lag period of only 8-15 min, depending on the tissue, and under certain conditions (e.g. use of IAA methyl ester t o aid penetration) the lag period may be considerably shorter (Rayle et al., 1970). It is generally agreed that these rapid responses must precede any changes in RNA or protein synthesis, that some auxin-induced growth can take place even when protein synthesis is inhibited (e.g. Penny, 1971), but that further protein synthesis is nevertheless necessary for any sustained auxin response (Davies, 1973). It has been known for some time that acidic solutions (pH 3-4) can partially mimic the rapid stimulation of cell elongation by auxin. The simdarities between proton-induced and auxin-induced growth led to the suggestion that auxins activate a plasma membrane ATPase, thereby causing an outward pumping of protons from the cytoplasm t o the cell wall (Hager et al., 1971). The resultant acidification of the wall is considered to increase wall plasticity by stimulating a cell wall hydrolase with an acidic pH optimum (Hager et al., 1971) or by directly breaking acid-labile co-valent (Rayle and Cleland, 1970) or hydrogen (Keegstra et al., 1973) bonds in the wall. The choice between these three possibilities has been discussed by Ray (1974). The “proton pump” hypothesis received additional experimental support following the realization that the cuticle presents a considerable barrier to the passage of protons. Using sections from which the cuticle had been peeled away, optimal proton-induced extension growth was observed at pH 5 (Rayle, 1973) compared with the previously determined pH 3 optimum for sections with intact cuticles. These peeled sections were found to excrete protons in response to auxin treatment, acidification of the incubation medium being detectable after 20-30 min (Cleland, 1973; Rayle, 1973). Other supporting evidence for the proton extrusion hypothesis has been obtained and various objections appear to have been answered satisfactorily (see Cleland and Rayle, 1975; Cleland, 1975). C. EVIDENCE FOR TWO SITES OF AUXIN ACTION

The emphasis in recent years on the early kinetics of auxin-stimulated growth has led to the misconception in some quarters that auxin action must be independent of RNA and protein synthesis, and therefore that any search for nuclear or cytoplasmic receptors which might mediate transcriptional effects is of dubious value. While this may very well be true for the initial, rapid phase of auxin action, abundant evidence has been avdable to show that for longer-term effects on elongation and other processes, macromolecule synthesis is required. P. Penny et al. (1972) showed clearly that if growth rate rather than total growth is plotted against time for individual sections, two phases in the auxin-induced growth response are discernible. For lupin sections, the first

RECEPTORS FOR PLANT HORMONES

59

response starts after a lag period of 14-19 min. and reaches a maximum rate at 29-39 min. The growth rate then falls to a minimum after 40-63 min before rising to a second sustained maximum rate (about equal to the first maximum) at 63-77 min. In the presence of cycloheximide, only the first auxin response is observed (D. Penny et aZ., 1972). The dual nature of the auxin response has been re-emphasized more recently by Vanderhoef and StaM (1975) who showed that the second response of soybean sections could also be eliminated by the cytokinin isopentenyl adenine, while the first response was still evident. The normal dual phase growth rate plot was interpreted in terms of two overlapping responses, commencing at 12 min and 3 5 4 5 min after auxin application. The time relationships of the different phases of the growth curve were generally in good agreement with the data of P. Penny et al. (1972) for lupins. Rayle (1973) had noted that proton-induced growth mimicked only a portion of the auxin response and Vanderhoef and Stahl (1975) demonstrated that the low pH growth rate curve showed only a single peak, apparently corresponding to the first auxin response. From the above discussion it appears likely that the overall growth curve observed following auxin application is the summation of a rapid response, frequently detectable within minutes, which is mimicked by low pH, and a second response which is blocked by cycloheximide and whose timing ( 3 5 4 5 min after auxin treatment) is entirely compatible with a requirement for macromolecular synthesis. The timing of the first response and of other rapid hormone responses (Evans, 1974) is suggestive of an interaction with plasma membrane receptors, while longer-term effects of hormones may reflect transcriptional changes mediated by nuclear or cytoplasmic receptors. One could postulate a single class of multi-functional receptors, but as discussed previously (Venis, 1973) the concept of different classes of receptor which are spatially and functionally distinct is perfectly plausible. Moreover, there is now quite good evidence, to be discussed in the following sections, for the existence of both “soluble” and membrane-bound receptors for plant hormones.

111. THE SEARCH FOR HORMONE RECEPTORS A. MODEL SYSTEMS

Over a period of many years, the possibility that plant hormones alter the permeability or other properties of the cell membrane has been examined in model systems by studying interactions with phospholipid (e.g. Havinga and Veldstra, 1948; Brian and Rideal, 1952) or phospholipid-sterol (e.g. Wood and Paleg, 1972; Kennedy and Harvey, 1972) monolayers or vesicles. Alterations in physical properties of the synthetic membrane systems are undoubtedly observed, but in no case is there a correlation with biological activity, i.e. inactive analogues frequently produce changes identical to those produced by active

60

MICHAEL A. VENIS

compounds. This is hardly surprising in view of the simplicity of the systems. While the interactions observed may be relevant to the penetration properties of the hormone for example, a receptor which recognizes specifically only growthactive molecular configurations must almost certainly be locafed in a protein. B. DIRECT INTERACTION WITH ENZYMES

Activation of a membrane-bound ATPase is a requirement of the proton pump model of auxin action. Such activation has so far been reported from one laboratory only. Kasamo and Yamaki (1974) have claimed an approximately 150% stimulation of a plasma membrane ATPase by the in vitro addition of M IAA (the lowest concentration tested). The stimulated rate was essentially independent of IAA concentration over the range 10-13-10-5 M . Making the most favourable possible assumptions, namely a specific activity for a pure ATPase of 100ymol/mg protein per rnin and a molecular weight of 250 000 daltons (Schwartz et al., 1975) it can be calculated from their data that at 10-1 M IAA, the reaction mixture contains at the very most one molecule of IAA for every 250 ATPase molecules. It is difficult to reconcile this ratio with maximal enzyme activation or indeed with any significant activation whatsoever. No information on auxin specificity is available, apart from some data using crude extracts (Kasamo and Yamaki, 1973) where the effects were very small (e.g. 10% stimulation at 10M NAA) and the only inactive compound tested was tryptamine, which is not a carboxylic acid analogue. Tuli and Moyed (1969) claimed that the reactive intermediate in IAA action is an oxidation product 3-methyleneoxindole (3-MO). By analogy with effects observed in a bacterial system, low concentrations of 3-MO were considered to stimulate growth by desensitizing regulatory enzymes to feedback inhibition, while high concentrations were thought to inhibit growth through reaction with sulphydryl groups. To account for the activity of synthetic auxins which are not subject to the oxindole pathway, it was reported (Moyed and Williamson, 1967) that NAA and 2,4-D inhibit the reduction of 3-MO to the inert 3-methyloxindole. Other laboratories have failed to observe any effects of 3-MO on growth and it has been suggested (Evans and Ray, 1973) that the reported growth stimulation by 3-MO might be due to contamination with IAA, used as the starting material for synthesis. Activation of a partly purified citrate synthase by M IAA has been reported (Sarkissian and Schmalstieg, 1969), together with an IAA-induced change in enzyme conformation as judged by alteration of the elution position from a gel filtration column (Sarkissian, 1970). Other workers have failed to detect any effect of IAA on activity of the enzyme (Zenk and Nissl, 1968; Brock and Fletcher, 1969). Once again, calculations of the sort outlined previously for ATPase suggest that at 10-1 M IAA the reaction mixtures of Sarkissian and Schmalstieg (1 969) contained only one molecule of IAA for every 50 molecules of enzyme.

RECEPTORS FOR PLANT HORMONES

61

Van der Woude et al. (1972) observed a very small (515%) stimulation of glucan synthetase activity when 2,4-D was added t o a plasma membrane fraction from onions. No results with other auxin analogues were reported. A rapid, auxin-specific activation of glucan synthetase was found when pea sections were treated with IAA or other active auxins (Ray, 1973), but contrary to the findings of Van der Woude et al. (1972), no effect was observed with IAA added in vitro. Despite extensive investigations purporting to invoke a role for adenyl cyclase in mediating the action of plant hormones, there is no convincing evidence that either adenyl cyclase or cyclic AMP have been detected in higher plants. This subject has been reviewed in detail by Lin (1974). More recently, several reports have described careful investigations into the problems of adequately purifying cyclic AMP from plant tissues, and the unreliability of supposedly specific assays for cyclic AMP (Bressan et al., 1976, and references therein). The result of these studies has been to reduce the upper estimates of cyclic AMP concentrations in plant tissues to levels which are likely to be physiologically irrelevant. It may be concluded that there has been no adequate demonstration of in vitro enzyme activation by a plant hormone, nor of in vivo regulation of cyclic AMP levels. Control of membrane ATPase activity by auxins may be a reasonable effect to look for, but the possibility exists that activation may not be detectable in a disrupted system (Cleland, 1975). C. "SOLUBLE" (NUCLEAR/CYTOPLASMIC) RECEPTORS

1. Some Early Studies Siege1 and Galston (1953) reported the formation of IAA-protein complexes both in vivo and in vitro using peas. Andreae and Van Ysselstein (1960) could find no evidence for such complex formation in pea tissues and the earlier results were regarded as artefacts of the protein precipitation procedure used. An in vitro association between an oxidation product of IAA and pea RNA was claimed by Kefford et al. (1963), but a later report (Galston et al., 1964) suggested that this again was likely to be a spurious result of the method of precipitation. When pea sections were fed IAA-' 4C, radioactivity was found in association with RNA, mainly the 4s RNA fraction (Bendana et al., 1965). It now appears that the bulk of this labelling results from recycling of the radioactive side-chain into associated polysaccharide (Davies and Galston, 1971).

2. Histones and DNA At a time when removal of histones from DNA was still regarded as a possible mechanism for specific gene derepression, we examined by equilibrium dialysis the binding of auxins to purified histones isolated from peas (Venis, 1968). The binding affinities of active auxins were about double those of inactive analogues and there were differences in the binding characteristics of arginine-rich and

62

MICHAEL A. VENIS

lysine-rich histones. However, the lowest dissociation constant observed was 40 p~ and we were discouraged from pursuing this work further by reports suggesting that histone heterogeneity was very limited, and by the sequencing studies from Bonner's laboratory (e.g. DeLange et al., 1968) which demonstrated the remarkable degree of complementarity between corresponding histone fractions from sources as evolutionarily divergent as calf thymus and pea buds. These findings rendered most improbable any role for histones as specific repressor proteins. Fellenberg has published a series of papers describing alterations in the melting temperature (T,) of chromatin or purified DNA in the presence of auxins, cytokinins or gibberellins (e.g. Fellenberg, 1969; Fellenberg and Schomer, 1975; and references therein). Similar results with DNA were reported by Bamberger (1971). Penner and Early (1972) however, showed that the changes in DNA T, could be ascribed entirely to alteration of the solution pH upon addition of the high concentrations of growth regulators used. Comparable effects could be obtained by adding acetic or hydrochloric acids to the appropriate pH, while at constant pH none of the hormones produced any significant change in T, .

3. Auxin Mediator Proteins Matthysse and Phillips (1969) reported that 2 , 4 - ~could stimulate RNA synthesis by tobacco or soybean nuclei only if 2 , 4 - ~was also present in the nuclear isolation medium. In the absence of 2 , 4 - ~the nuclei seemingly released some factor which, when added back to the nuclei, restored auxin sensitivity. This factor, apparently protein, could be partially purified either from the nuclear lysates or from post-chromatin supernatants of pea buds. In the presence of 2 , 4 - ~the factor stimulated RNA synthesis up to 85% with chromatin as template, but not with purified DNA. It was claimed that this effect was auxin-specific and that the stimulation was m@ntained when saturating levels of E. coli polymerase were added, suggesting that the chromatin template had been derepressed. No figures were given in support of these statements. Factors prepared from different tissues of peas showed somewhat greater activity on chromatin derived from the same tissue (Matthysse, 1970). No further development of this system has been reported, although a purification procedure similar to that of Matthysse and Phillips (1969) has been used in the isolation of a receptor from coconuts, discussed later. Treatment of soybean hypocotyls with 2,4-D at 4-24 h before harvesting causes a large increase in the activity of chromatin-bound RNA polymerase (O'Brien et al., 1968), the increase being due to enhanced activity of the nucleolar enzyme, RNA polymerase I (Hardin and Cherry, 1972). A protein fraction prepared from the cotyledons stimulated polymerase from control, but

RECEPTORS FOR PLANT HORMONES

63

not from 2P-D-treated tissue (Hardin et al., 1970). It was suggested that this protein might mediate the action of the hormone in v i m , so that polymerase from treated tissue, having been already “activated”, would be unresponsive to in vitro addition of the factor. The degree of stimulation by the factor in the cell-free system (32%) was very much lower than that obtained following treatment with 2,4-D in vivo (over 200%). A similar situation exists with the nucleolar RNA polymerase of lentil roots, whose activity is doubled in tissue treated with IAA (Teissere et al., 1973). Fractionation of the non-histone chromosomal proteins yielded four fractions which stimulated activity of the polymerase (Teissere et al., 1975). Two of these were studied in more detail and appeared to be initiation factors. It was claimed that the “level” of one of the factors, y, was doubled in auxin-treated tissue compared with control tissue, but this conclusion is based on a comparison of the activities of the single fractions at the apex of the respective elution peaks. If the total activities under each y peak are compared, the effect of auxin treatment works out a t not more than a 25% stimulation in y activity. It is possible that y could bear some relationship to the soybean factor of Hardin et aE. (1970), although the soybean protein was derived from the high-speed supernatant fraction, rather than the chromosomal pellet.

4. Auxin-Binding Proteins Unlike the systems described in the preceding section, the reports now to be discussed are ones in which actual auxin binding has been examined. Using a procedure broadly similar to that of Matthysse and Phdlips (1969), Biswas and co-workers have outlined the preparation, from nucleoplasm of young coconut endosperm nuclei, of a substance referred t o as auxin acceptor protein (Mondal et al., 1972) or IAA receptor protein, IRP (Biswas et al., 1975). In a completely homologous system, containing nucleoplasmic RNA polymerase, DNA and initiation factor, RNA synthesis was doubled by IRP, provided that IAA (1 p.M) was also present. Most of the enhanced RNA synthesis was the result of increased chain initiation. Hybridization and gel electrophoretic analysis of the reaction products suggested that new kinds of RNA were produced in the stimulated reaction. IRP itself binds to 3H-DNA, as judged by retention of the label on membrane filters, but binding is further enhanced by IAA, at an optimum concentration of 0.1 pM. IRP is apparently a single polypeptide of 100 000 daltons. Equilibrium dialysis data performed at one ligand concentration only have been interpreted as showing that IRP binds IAA and 2,4-0, but not benzoic acid. From a fuller kinetic analysis of IAA binding, the dissociation constant, K D , was estimated as 7.5 pM with 0.5 binding sites per protein molecule. Since the binding protein was thought to be homogeneous, the latter figure was taken to indicate either a requirement for two IRP molecules to bind

64

MICHAEL A. VENlS

one molecule of IAA or, more probably, partial inactivation of IRP during purification. Many questions regarding the coconut receptor remain unanswered, e.g. is the requirement for IAA in IRP-enhanced RNA synthesis an auxin-specific requirement? What is the optimum IAA concentration in this regard? Assuming it is about 1 p~ (the only concentration reported on in RNA synthesis data), how is this reconciled with a KO for IRP-IAA binding of 7.5 p M , since at 1 pM IAA, most of the binding sites would be unoccupied? Furthermore, the binding specificity needs to be examined in greater detail than single-point data permit. Nevertheless, the reported properties of IRP are consistent with its being a true auxin receptor and would make it one of the best characterized systems in existence. It is therefore particularly unfortunate that many of the claims described above have been outlined only in conference proceedings (Biswas et al., 1975) and that at no time have the extraction and purification to homogeneity of IRP been described in detail sufficient to permit independent confirmation. Auxinologists in coconut-growing areas can only hope that these omissions d l be corrected. Dextran-charcoal techniques have been used extensively in the analysis of steroid-receptor interactions and recently two reports have appeared describing their application for IAA binding studies. Likholat et al. (1974) found that IAA added to the homogenization and wash media enhanced RNA synthesis by wheat coleoptile chromatin, but was without effect if added only to the in vitro reaction mixture. This situation is comparable with that found by Matthysse and Phillips (1969) for nuclei. Binding of IAA-I4C by the cytosol fraction was detected after addition of dextran-coated charcoal to remove the unbound auxin and was about five times higher in extracts from 36-h plants than from 72-h plants. RNA synthesis by chromatin from 72-h coleoptiles was enhanced (nearly twofold) by addition of a 72-h membrane fraction pre-incubated with IAA. A membrane fraction from tissues grown for 36 h however was without effect on RNA synthesis by chromatin from 72 h coleoptiles and neither membrane fraction (with or without IAA) stimulated activity of chromatin from 36 h tissues. These and other data were interpreted in terms of a transition of the auxin receptors from a “soluble” t o a membrane-bound state during passage of the cells from a dividing (36 h) to an elongating (72 h) phase. Auxin binding by the cytosol fraction was saturable upon addition of unlabelled IAA, but no attempt to estimate the binding parameters was made. Oostrom et al. (1975) have also used a dextran-charcoal method t o detect high-affinity auxin binding in the cytosol of tobacco pith. Using IAA-3H of high specific activity, binding sites with a K D of lo-’ M were found in extracts from cultured pith explants, whereas extracts derived from freshly excised pith showed only low affinity binding. The concentration of binding sites was extremely low (c. 0.01 pmol/g) suggesting losses or inactivation during extraction. As yet no information on binding specificity is available.

RECEPTORS FOR PLANT HORMONES

65

5. Binding of Gibberellins and Cytokinins Nuclei isolated from dwarf peas in the presence of lo-' MGA, were found to be 50-80% more active in RNA synthesis than control nuclei (John and Varner, 1968). The RNA product also had a higher average molecular weight and a different nearest neighbour analysis. Stimulatory activity declined when GA, was added at successively later stages of the nuclear isolation procedure, again suggesting the possible loss of a factor required to mediate the hormonal effect. No attempts to isolate such a factor have been reported. At present, only one publication describing gibberellin binding has appeared. When epicotyl sections of dwarf pea were supplied with high specific activity GA,-3H for 12 h, fractionation of the 20 000 x g supernatant revealed two peaks of radioactivity, apparently unchanged GA,, in association with a high (HMW) and an intermediate molecular weight (IMW) fraction (Stoddart et al., 1974). Similar experiments with two inactive tritiated analogues showed that whereas pseudoGA, was not bound, 16-keto-GA1 bound to about the same extent as the active C A I . HMW and IMW fractions derived from unlabelled extracts were able to bind GA1-3H in vitro (single point equilibrium dialysis data) and the bound radioactivity was 70% exchangeable with excess unlabelled GA, in one hour. Further information on the binding kinetics and specificity is needed to assess the significance of these results. Matthysse and Abrams (1970) reported the preparation of a cytokinin mediator protein from the sucrose interface region obtained during purification of pea chromatin. In the presence of kinetin or zeatin, the protein stimulated RNA synthesis (10-50%) with E. coli polymerase and either chromatin or homologous (pea) DNA as template. Stimulation was not observed when DNA from other sources was used. The effects of inactive cytokinin analogues were not described and no further reports on this system have appeared. Binding of cytokhins to plant ribosomes was reported by Berridge et al. (1970). Only low affinity binding was detected however, and binding of different cytokinin analogues did not altogether correlate with physiological activity. Ribosomal binding was re-examined by Fox and Erion (1975) using lower cytokinin concentrations than those used in the earlier study. In addition to the low affinity binding they detected a class of high affinity sites for benzyladenine (KO = 0.6 p M ) , at a concentration of one site per ribosome. Rat liver and E. coli ribosomes bound four to six times less cytokinin than ribosomes from wheat germ. The high affinity sites could be removed from the ribosomes by washng with 0.5 M KCI. Material present in the supernatant from this ribosome wash could itself bind cytokinins and the binding moiety appeared to be a protein. Results to be reported (quoted in Fox and Erion, 1975; Fox, personal communication) indicate: (a) there is a correlation between cytokinin activity and ability to compete with benzyladenine for the high affinity sites; and ( b ) the binding protein is also present in the supernatant, can be purified either from the supernatant or from the ribosomal wash, and has a molecular weight of about

66

MICHAEL A. VENIS

65 000 daltons. The fiinction of the binding protein is uncertain, but is not thought to be involved in recognition of cytokinin bases in transfer RNA molecules.

6. Applications of Affinity Chromatography (a) Auxins Shortly after Cuatrecasas et al. (1968) defined clearly the conditions favouring satisfactory application of the affinity chromatography principle, we became interested in designing adsorbents which might be used in purifying proteins with affinity for auxins. Column materials were prepared by coupling the E-L-lysine derivatives of 2 , 4 - ~or IAA to cyanogen bromideactivated agarose. Although the auxin carboxyl group, essential for activity, is blocked in these derivatives, it was hoped that introduction of the lysyl carboxyl group would compensate for this deficiency. IAA-lysine and 2,4-D-lysine do show auxin activity, though we cannot be certain that they are active without hydrolysis. When crude supernatants prepared from pea or maize shoots were passed over such columns, small amounts of protein were retained and could be eluted by various means, typically with 1 M NaCl followed by 2 mh! KOH (Venis, 1971). The fractions obtained were tested for their effects on DNAdependent RNA synthesis, supported by E. coli polymerase (holoenzyme). Fractions eluted by NaCl were invariably inhibitory, while KOH fractions promoted RNA synthesis by 40-200%in different preparations. Stimulation was not dependent on the addition of auxin to the reaction mixture (Table I). Activity of the KOH fraction was unchanged by dialysis, but completely TABLE I Activity o f Affinity-column Fractions Derived from Pea Supernatants on RNA Synthesis (Venis, 1971)

ATP incorporated (pmol)

Fraction added

-2,4-D

Control NaCl KOH KOH, dialysed KOH, frozen-thawed KOH, 6OoC, 5 min KOH, i o o o c , 5 min KOH, minus DNA KOH, minus polymerase ~

~~~

+2,4-~

621 472 965 954 629 686 600 17 0 ~

~~~~~

595 459 895

~~

~~

~~~

~

~~

5 pg of NaC1-eluted protein and 10 pg of KOHeluted protein were used. 2,4-D concentration was 0.05 mg/litre. 1 pg of DNA and 1 polymerase unit per assay. Incubation for 30 min at 37°C.

67

RECEPTORS FOR PLANT HORMONES

destroyed by freezing or by brief heating. These and other characteristics suggested that activity was due t o a protein. The active factor does not support any RNA synthesis in the absence of added polymerase, and very little without DNA (Table I). Thus it is not itself a polynucleotide-forming enzyme, nor does it act by presenting additional template. Other trivial explanations for activity, such as inhibition of ATPase or RNase in the reaction mixture, or nicking of the DNA template were also eliminated. Factors prepared from pea or maize supernatants were active in both homologous and heterologous reactions (Table II), though with maize DNA as template the homologous factor was somewhat more active. A fraction prepared in a similar manner from mouse liver was without effect on RNA synthesis. TABLE I1 Activity o f KOH-eluted Factors from Pea, Maize or Mouse Liver on DNA-dependent R N A Synthesis (Venis, 1971) ATP incorporated (pmol) Template Factor added A. Control

Pea Corn B.

Control Mouse liver

Pea DNA

Maize DNA

1050 1550 1540

1050 1450 1640

934 943

551 571

15 pg (protein) of each factor were used. Two polymerase units and 1 pg of DNA per assay (except 0.5 pg of corn DNA in B). Incubation for 30 min at 37°C.

The stimulatory effect of the pea factor increases with reaction time, since RNA synthesis continues for a longer period in the presence of factor (Fig. 3). Rifampicin, which inhibits chain initiation but not elongation, blocks RNA synthesis almost completely when added at zero time. If rifampicin addition is delayed by 2 min, the control reaction is scarcely affected (indicating that initiation is essentially complete by this time), but factor stimulation is reduced by 40-50%. If the factor is added after 15 min, when control incorporation has virtually ceased, a fresh burst of RNA synthesis occurs. The rifampicin results suggest that in the presence of the pea factor, initiation of RNA chains continues beyond 2 min. The factor may therefore act by permitting initiation at template regions not otherwise transcribed, or by facilitating chain release and reinitiation at the same points. The latter possibility is less likely, since rifampicin added after 5 min is without effect on factor stimulation. Another argument in favour of an effect on chain initiation comes from a comparison of the size of the RNA

68

MICHAEL A. VENIS

product formed in the presence and absence of the pea factor (Fig. 4). Although the factor promoted total RNA synthesis by 50%in this experiment, the average product size was smaller. This result is consistent with enhanced initiation of RNA chains in the presence of the factor, with a consequent ieduction in overall

1500

a l-

a 1000 Q)

0

E a c .-0 e

0 L

0 Q L

0 0

-t

500

2

15

30

Time, min Fig. 3. Characteristics of DNA-dependent RNA synthesis in the presence and absence of pea factor: 15 pg of factor; 0.8 p g of rifampicin; 1 pg of DNA; 2 polymerase units (Venis, 1971).

polymerization rate and hence in average chain size due to a crowding effect on the template (Richardson, 1966). We would like to think that the factor acts by promoting initiation at hormone-specific template regions, but we have no direct evidence on this point. There is some evidence, from effects on retention of labelled DNA on membrane filters, and on DNA renaturation kinetics, that the factor is able to interact with

69

RECEPTORS FOR PLANT HORMONES

DNA. Stimulation of RNA synthesis is observed without addition of auxin (Table I), for which several possible reasons suggest themselves:

1. mere passage through the affinity column may transform the factor to an active configuration in which further contact with auxin is not required. 2. In vivo, auxin may simply permit activity to be expressed by transporting an inherently active regulatory protein from the cytoplasm to the nucleus (Hardin et al., 1970). 3. A regulatory sub-unit may be stripped off the protein during passage through the column, as apparently happens during affinity chromatography of cyclic AMP-dependent protein kinase (Wilchek et al., 1971). Binding of labelled auxins by the active fractions could not be convincingly demonstrated by equilibrium dialysis. Again, it is possible that passage through

c v) 8

3

s 1000500 -

I

L

5

10 15 Fraction No.

2(

Fig. 4. Glycerol gradient centrifugation (5-20% v/v) of the RNA products formed in the presence or absence of pea factor after 5 min and 30 min of RNA synthesis.

70

MICHAEL A. VENIS

the column may alter the configuration of the factor to one in which auxin is not bound, either by removal of a binding subunit or through inactivation of the binding site under the rather harsh (2 mM KOH) conditions of elution. The KOH-eluted fractions are heterogeneous by gel electrophoresis, but attempts at further purification always led to loss of activity. We have since designed adsorbents which we hope will permit elution under milder conditions. However, the large body of literature on affinity chromatography over the last few years has pointed up many of its limitations and would suggest that it is more advantageous to apply the technique at a point where some biological activity, e.g. binding, is already demonstrable, rather than to “fish and hope”. Nevertheless the columns described above have been successfully used in other laboratories. Biswas et al. (1975) have reported that their auxin receptor from coconuts can be isolated by chromatography on the IAA-lysine column, using 1 M KSCN elution after the NaCl step. Rizzo et al. (1976), using the 2,4-D-lysine column and preparation methods identical to those previously outlined (Venis, 1971), have obtained a similar though more active factor from soybean hypocotyls. DNA-dependent RNA synthesis with E. coli polymerase was stimulated two- to seven-fold by the KOH-eluted fraction. Stimulation was also observed, in the presence of 0.1 pM 2 , 4 - ~using , soybean polymerase I, but not with the nucleoplasmic enzyme polymerase 11. The effect (25430%stimulation) was much smaller than that obtained with the bacterial enzyme, perhaps because of the impure nature of the soybean enzyme. It may be that the factor is involved in mediating the previously discussed enhancement of RNA polymerase activity following treatment of soybeans with 2 , 4 - ~(O’Brien et al., 1968). ( b ) Gibberellins and cytokinins Knofel et al. (1975) have described the synthesis of three gibberellin affinity adsorbents, all of which involve substitution of the GA, carboxyl group. No experiments describing the use of these adsorbents were presented, but in view of the importance of a free carboxyl group for activity it would be surprising if they prove practical for receptor isolation. An adsorbent consisting of benzyladenine coupled directly to cyanogen bromide-activated agarose has been used by Takegami and Yoshida (1975) to obtain a cytokinin-binding protein from tobacco leaves. Binding of benzyladenine-14C by the adsorbed fraction (eluted rather drastically with 0.1 N KOH) was about 40-fold higher than the binding observed with crude extracts. A nonequilibrium gel filtration method was used to estimate binding, which was reduced by pre-incubation of the affinity column protein with unlabelled benzyladenine or kinetin. Adenine (very slightly active as a cytokinin) competed to a lesser extent, while the inactive adenosine did not compete. The binding protein appears to consist of a single polypeptide of only 4000 daltons. It is somewhat surprising that benzyladenine linked directly without a spacer arm should act as an affinity adsorbent, but even more surprising that it should

RECEPTORS FOR PLANT HORMONES

71

couple to cyanogen bromide-activated agarose, since coupling normally proceeds through a primary amino group. The reaction apparently takes place at pH 4-7, considerably lower than that required for amine coupling, and there is some evidence that the molecule is linked at the N9 position (Yoshida and Takegami, 1976). It is interesting that a small glycopeptide (6000 daltons) which binds cytokinins (as well as IAA, tryptophan and calcium) has been isolated from the cell wall-membrane matrix of the water mould Achlya (UJohn, 1975). A promising cytokinin adsorbent has been prepared by Hermville and Klambt (1976). Isopentenyl adenosine was attached to epoxy-activated agarose, presumably via a ribosyl hydroxyl group. Small amounts of protein from maize shoot extracts were retained by the column, recovered by substrate elution and examined by gel electrophoresis. The gel patterns were relatively simple, but the affinity column proteins have not yet been examined for biological activity.

D. MEMBRANE-BOUND RECEPTORS

1. Binding of NaphthylphthalamicAcid (NPA) The pioneering studies of Hertel and co-workers have been largely responsible for the rapid progress in investigation of membrane-bound receptors over the last few years. Initially, binding of NPA, a potent synthetic inhibitor of polar auxin transport was examined, since this compound could be prepared tritiated to quite high specific activity. Membrane preparations from maize coleoptiles were fractionated on sucrose gradients and their ability to bind NPA-3H was assayed by a pelleting technique (Lembi et al., 1971). Binding of NPA was correlated with plasma membrane content of the fractions as determined by a specific staining method. The main class of binding sites has a dissociation constant for NPA of 2.2 x M, while a smaller fraction of hgher affinity sites (KO = 1.3 x lo-' M) is also present (Thomson, 1972). The NPA sites are Fpparently distinct from auxin binding sites, since IAA, NAA and 2,4-D did not compete. Neither was competition observed with another transport inhbitor, 2,3,5-triiodobenzoic acid (TIBA), nor with GA3, or abscisic acid. The affinity of NPA for the bulk of the binding sites (KD = 2.2 x M ) is well correlated with the saturation kinetics of its effect on auxin transport (Thomson et al., 1973). Several NF'A derivatives were found to displace NPA-3H from the binding sites, in a manner which was reasonably compatible with their relative activities as transport inhibitors (Thomson and Leopold, 1974). In addition, various morphactins, another group of synthetic auxin transport inhibitors, also displaced NPA-3H. The interaction of one of the morphactins was established as competitive by double reciprocal analysis, with a K j value (2.9 x M) about the same as the KO for NPA. The function of these binding sites, which recognize two groups of unnatural

72

MICHAEL A. VENIS

growth regulators but not any of the known plant hormones tested, remains a mystery. It is possible, as suggested by Thomson (1972), that their presence signifies an as yet unidentified natural hormone.

2. Auxin Binding (a) Properties of the binding sites Using procedures similar to those employed with NPA, it was possible to detect saturable binding of NAA-' 4 C and IAA-, H t o heterogeneous membrane preparations from maize coleoptiles (Hertel et al., 1972). Radioactive NAA in the pellets was displaceable by unlabelled auxins such as IAA and 2 , 4 - ~ but , not by the inactive compound benzoic acid. Abscisic acid, GA, and NPA also failed to interact with the binding sites. The observed levels of saturable binding were considerably less than the amounts bound nonspecifically, even over the wide concentration range studied (generally 0.2-200 pM). Nevertheless, it was possible to make approximate estimates of the dissociation constants, 1-2 pM for NAA and 3 4 p M for IAA. Binding activity was destroyed by heating or by treatment with sodium lauryl sulphate, and could be largely separated from nuclear and mitochondrial markers. Subsequent changes in procedure, in particular prior separation of the membranes from the supernatant by pelleting and resuspension, were reported to give improved binding and a KO for NAA of 0.5 p M (Ray and Hertel, unpublished, quoted in Hertel, 1974). We have re-examined binding of NAA-' 4 C by methods similar to those used by Hertel and co-workers, but confining our attention t o a detailed investigation of binding over a restricted concentration range (normally 0.2-1 p ~ ) As . the concentration of unlabelled NAA is increased over this range, radioactivity in the membrane pellet decreases (Fig. 5a), indicating progressive saturation of the NAA binding sites. Scatchard analysis of the data (Fig. 5b) yields a biphasic plot, suggesting the presence within the membrane preparation of at least two sets of high affinity binding sites for NAA, with dissociation constants of 0.15 p~ (site 1) and 1.6 pM (site 2). IAA-14C also appears to bind to two sets of sites ( K , = 1.7 /1M; K 2 = 5.8 p M ; Batt et al., 1976), whose concentrations are in good agreement with those deduced for NAA (nl ,n2 in Fig. 5b). To examine the binding specificity of each class of sites, we constructed complete binding curves for NAA in the presence or absence of a fixed concentration of the auxin analogue under investigation. An example for IAA interaction is illustrated in Fig. 6, together with double reciprocal plots constructed for the site 1 and site 2 regions of the binding curve. The common ordinate intercepts (Fig. 6b, c) indicate that IAA interacts competitively with NAA at both sets of binding sites. The Ki values for IAA (site 1 = 2.6 p ~ site ; 2 = 7.6 pM, mean values from several experiments) compare favourably with the IAA dissociation constants for site 1 and site 2 obtained from direct binding studies (see above). It is therefore most probable that the competitive interactions studied do indeed represent competition for common classes of binding

2400 2200 K, = 1 . 5 ~ 10-7~

$

n,

=

38p mollg

5

cl

h

2 1800

U

E

y’

m I

5 1600

0 c Q L

1200 I4O0l

\

K,= 16.1 x 1 0 - 7 ~

n2 = 96 p mollg

I

20

I

40

60

p mol boundlg fresh wt. Fig. 5. Kinetics of NAA-14C binding. (a) Pellet radioactivity as a function of NAA concentration. (b) Scatchard analysis of the binding data (Batt et aL, 1976; reproduced with permission). 4

w

Ic) Site 2

3000

+ IAA 0.4

5 5

E

? 4

2000

-

0.2

c

p'

1000

1

I

1

0.2

0.5

1.0

NAA concentration,pM (log scale)

-3

-1

0

1

3

5

h

I

I

I

Free. !dvl

Fig. 6 . Effect of IAA on NAA-14C binding. (a) Experimental binding values in the presence and absence of IAA. (b, c) Double reciprocal plots of the data for site 1 and site 2 respectively (Batt ef aL, 1976; reproduced with permission).

RECEPTORS FOR PLANT HORMONES

75

sites. The results of a large number of similar experiments with a range of auxin analogues indicated that all compounds tested, including inactive analogues such as benzoic acid, were able to compete with NAA for site 1 binding. Site 2 on the other hand exhibited binding specificity compatible with the expected properties of an auxin receptor as defined previously by Hertel et al. (1972), in that only active auxins, anti-auxins or transport inhibitors were able to compete with NAA for the binding sites (Batt et al., 1976). The discrepancies between our results and those of Hertel et al. (1972) can be partly accounted for by our more detailed examination of binding at concentrations below 1 @I NAA. Thls permitted detection of site 1 binding, whereas the earlier results appear to reflect predominantly binding to site 2. In regard to competition by transport inhibitors, Hertel et al. (1 972) observed displacement of NAA-14C only at TIBA concentrations of 100 /.LMor more, and subsequently the discrepancy between these concentrations and those required to inhibit polar transport of IAA (50% inhibition at 2 fiM TIBA) was discussed (Thomson et al., 1973). However, we were able to obtain clear competition for NAA binding at much lower TIBA concentrations (Batt et al., 1976) and the calculated Kivalues (about 2 pM for both sites) were well reconciled with the data of Thomson et al. (1973) for inhibition of auxin transport. We could also demonstrate competition by "A, but the binding affinities were about 100-fold lower than those of the NPA-3H binding sites discussed earlier. These results therefore support the suggestion of Thomson et al. (1973) that TIBA and NPA have different sites of action. TIBA may well inhibit transport by binding to auxin receptor sites, but there is little doubt that the more active inhibitor NPA exerts its effect at distinct sites of higher affinity. The discrete nature of the two sets of NAA binding sites was confirmed by experiments in which we were able to achieve substantial resolution of site 1 and site 2 binding (Batt and Venis, 1976). If the membrane preparation was separated by differential centrifugation, it was found that the 4000-10 000 x g fraction contained a single population of NAA binding sites, with a KO = 1.6 pM (Fig. 7), characteristic of site 2, the auxin-specific site. The 10 000-38000 x g fraction, on the other hand, appeared to contain both sets of binding sites (Fig. 7). The total membrane preparation (4000-38 000 x g) could be fractionated on discontinuous sucrose gradients into two bands. The kinetics of NAA-14C binding to each of these bands (Fig. 8) suggested that each band contains one distinct set of binding sites. The dissociation constants, 0.39 /.IMfor the light band, and 1.2 PM for the heavy band, were in reasonable agreement with those determined previously for site 1 and site 2 respectively in unfractionated membrane preparations. This similarity suggested that substantial resolution of the binding sites had been obtained. If the light and heavy bands do indeed contain site 1 and site 2 respectively, then the binding specificities of each site, as determined in unfractionated preparations, should be retained. Specificity was examined by studying the interactions of IAA, TIBA and the inactive compound

\”\

K , = 2.9 x l O - ’ M

K, = 10.2

~ o - ~ M

0

% K = 15.7 x 10--7M

~~~

.1

0.2

0.5

N A A concn. ph’l (log scale)

1 .o

I

I

10

20

I 30

p mol bound/g fresh wt

Fig. 7. (a) and (b) Kinetics of NAA-14C binding by 4000-10 000 x g and 10 000-38 000 x g membiane fractions. (a) Pellet radioactivity as a function of NAA concentration. (b) Scatchard analysis of the binding data (Batt and Venis, 1976; reproduced with permission).

\O

K = 3.9 x 10p7M n = 24 p mol/g 1000

5

\\

5 E

y.

m

> E

4

a,

E

n

K = 11.6 x 10 'M

600

0

4-

n=

P 0

32 p rnollg

Heavy band

300

0.2

0.5 NAA concn., p M (log scale)

1 .(

0

10

20

p mol boundig fresh wt

Fig. 8 (a) and (b) Kinetics of NAA-14C binding to light and heavy bands obtained by sucrose gradient fractionation of a 4000-38 000 x g membrane preparation. (a) Pellet radioactivity as a function of NAA concentration. (b) Scatchard analysis of the binding data (Batt and Venis, 1976; reproduced with permission).

78

MICHAEL A. VENIS

benzoic acid. From double reciprocal analysis of the binding data (Fig. 9) it could be concluded that IAA and TIBA are able to compete with NAA for the binding sites present in both membrane bands. Benzoic acid, on the other hand, is competitive with NAA for the binding sites in the light band, but does not compete for binding sites in the heavy band. It would seem, therefore, that the binding specificities of the light and heavy bands do indeed correspond to those deduced previously for site 1 and site 2 respectively, in that only physiologically active analogues bind to the heavy fraction, whereas both active and inactive compounds can bind to the light fraction. To obtain further information on the localization of the binding sites, crude membrane preparations were fractionated on multi-step sucrose gradients into five bands (Batt and Venis, 1976). Examination of these bands for enzymic and chemical markers suggested, in conjunction with electron microscopy and auxin binding data, that the auxin-specific binding sites (site 2 ) are located in membrane fractions enriched in plasma membrane. Site 1 appeared to be associated with ER (endoplasmic reticulum) and/or Golgi membranes. Results obtained using higher resolution continuous sucrose gradients suggest that site 1 is most probably located in ER (Hertel, personal communication). What is the function of site 1, which binds both active and inactive auxin analogues? It certainly seems unlikely that a binding site of such high affinity would have been evolved without some physiological value. Perhaps site 1 represents a sort of “pro-receptor”, in the process of maturation to a final auxin-specific form in the plasma membrane. In this connection, recent observations by Ray and Hertel (Hertel, personal communication) concerning the effects of a supernatant factor on binding specificity could be relevant. It appears that this factor (heat-stable, low molecular weight) reduces the binding affinity for NAA about three-fold, but the affinity for inactive auxin analogues is reduced to a very much greater extent. The net result is thus an increased specificity of binding. In our terminology, it is as if site 1 has been made, to some extent, to look like site 2. The question of whether in fact some form of receptor transformation is involved and whether the activity of the supernatant factor reflects a real physiological function are intriguing areas for further study. There is an interesting parallel with the multiple forms of steroid hormone receptors and the presence of a steroid receptor-transforming factor, though this factor is known to be macromolecular (Puca et al., 1972). In addition, alterations in specificity of catecholamine (Lacombe and Hanoune, 1974) and opiate (Wilson et al., 1975) receptors have been reported. Two other reports have appeared describing IAA binding to particulate preparations, one from pea buds (JablonoviE and Nooden, 1974) and one from mung bean hypocotyls (Kasamo and Yamaki, 1976). In their present state of development, neither of these systems looks particularly promising. Saturable relative to non-specific binding is low, no kinetic data have been presented, and information on analogue competition is inadequate for any assessment of binding specificity.

79

RECEPTORS FOR PLANT HORMONES 12)

Light Band

1 4 7

Heavy Band

14r

/f

+lAA5phl

f/

P

m

L // I -2

0.5

I -3

I

I

3

5

-

1 -1

I 1

1

. I 5

3

+ TIBA 4 pM

J'

L

d/

-3

-1

I

1

I

1

1

3

5 1 Free, p.M C4420

Fig. 9. (a)-(c) Competition for NAA-14C binding sites in the light and heavy bands obtained by sucrose gradient fractionation of 4000-38 000 x g membrane preparations. Double reciprocal plots of NAA binding in the presence and absence of (a) IAA, (b) benzoic acid (BA) and (c) TIBA (Batt and Venis, 1976; reproduced with permission).

80

MICHAEL A. VENIS

( b ) Probing the active site. Auxin binding activity in maize coleoptile membranes can be rendered soluble using non-ionic detergents such as Triton (Batt et a l , 1976), but various difficulties are encountered in attempting to purify the solubilized binding entity. One means of circumventing some of these problems would be t o use an auxin affinity label, or active site-directed irreversible inhibitor, i.e. a compound that looks sufficiently like an auxin to have preferential affinity for the receptor site over other potential binding sites, and which bears a chemically reactive function capable of attaching the molecule co-valently to a suitable amino acid residue in the active site environment. The perfect affinity label would react on@ at the active site; in practice this is seldom the case, but with the help of a few tricks the ideal situation can frequently be approached. If the molecule is made radiolabelled, then the receptor can be followed during purification from a mixture of proteins by simply monitoring the radioactive tag. In addition (and a more frequently used aspect of affinity labelling) the method can provide valuable information about the amino acids in the neighbourhood of the active site. A modification of the besic principle is that of photoaffinity labelling, in which the reactive species is generated in situ photolytically, following binding of an appropriate precursor. Aryl azides (generating nitrenes) have frequently been used for this purpose, and an azido auxin analogue was suggested in discussions by Dr A. J. Trewavas (Department of Botany, Edinburgh University) several years ago. One disadvantage is the very high reactivity of the nitrene radical, which increases the probability of non-specific labelling. We therefore preferred to investigate initially the potential of amino auxin analogues, from which diazonium salts can be readily prepared. Azo coupling should be more restricted than nitrene labelling, but if the compounds proved unsatisfactory, then they could be converted readily to the azido derivatives. The diazo salt of 2-chloro-4-aminophenoxyacetic acid (CAPA) was the first compound we examined as an affinity label. (The synthesis and biological activity of the corresponding 4-azido analogue have recently been described by Leonard et al. (1975), but without any affinity labelling data.) The reactivity of the non-radioactive compound was investigated by incubating maize coleoptile membranes with the diazonium salt at 25”C, normally for 15 min, then diluting out into cold buffer and collecting the membranes by centrifugation. Binding of NAA-l 4C is then compared with that of control membranes treated similarly but without the diazonium salt. It is found that diazo-CAPA does inhibit subsequent binding of NAA-l 4C, but only if the coupling reaction is carried out at pH 8-9 (Fig. 10). If the compound is acting as a genuine affinity label, i.e. coupling co-valently with a reactive residue in the neighbourhood of the receptor site, then prior addition of an auxin should impede the coupling of CAPA to the receptor. After separation from the reaction mixture by centrifugation and resuspension in buffer, the “protected” membranes should retain greater auxin binding activity

81

RECEPTORS FOR PLANT HORMONES

than unprotected membranes. Unfortunately, the conditions required for coupling of diazo-CAF'A (pH 8 or above, Fig. 10) are exceedingly unfavourable for auxin binding (pH optimum 5.5, Batt and Venis, 1976). It was therefore not surprising to find that auxin pretreatment at pH 8 affords no significant protection. The compound may indeed be acting as an affinity label, but it is not possible to obtain supporting evidence by ligand protection. Investigation of other amino auxin analogues revealed that reaction of membranes with diazo-Chloramben (2,5,-dichloro-3-aminobenzoicacid) at pH 70 -

1

0

6

0

7

8

9

PH Fig. 10. Effect of reaction pH on inhibition, by diazonium salts of CAPA or Chloramben (150 PM) of NAA-14C binding by maize coleoptile membranes.

values as low as 6 (25"C, 15 min) resulted in substantial inhibition of their auxin binding capacity (Fig. 10). Inhibition is largely independent of reaction pH over the range pH 6 to 9, in contrast with the behaviour of diazo-CAPA. Figure 11 illustrates the concentration dependence of diazo-Chloramben inhibition at pH 7 and 25°C; reaction at 0°C reduces inhibition about fourfold. Experiments of this type are difficult to perform at pH <6 because incubation of the membranes at 25°C under these conditions results in a marked reduction in control binding activity. Nevertheless, pH 6 is sufficiently close to the pH optimum for auxin binding (5.5) to permit investigation of auxin protection against diazoChioramben reaction. Addition of 25 p M NAA immediately prior t o the diazonium salt (150 pM) does indeed appear to reduce substantially the extent

82

MICHAEL A. VENIS

of coupling at both site 1 and site 2, as judged by subsequent ability to bind NAA-14C (Table 111). Benzoic acid, on the other hand, protects only site 1, which is in accordance with the site binding specificity observed for this inactive compound (Batt et al., 1976).

30

75

150

300

[Diazo-Chloramben] pM Fig. 11. Inhibition of NAA-14C binding to maize coleoptile membranes after treatment with different concentrations of diazo-chloramben at pH 7. Reaction temperature 25°C except as noted.

From the above results, from the known variation of auxin binding with pH and from consideration of structure-activity rules, several tentative conclusions can be drawn regarding the environment of the receptor sites:

1. The presence of a free anionic function, normally carboxyl, is one of the essential structural requirements for auxin activity, suggesting binding of the carboxyl anion to a positively charged amino acid group in the receptor site. Binding of NAA is maximal at pH 5.5 and falls to very low values at pH 4 or pH 7 (Batt and Venis, 1976). The imidazolyl= NH' of histidine (pK, = 6.0) is the only candidate carboxyl-binding group whose ionization properties are compatible with loss of binding activity over the

83

RECEPTORS FOR PLANT HORMONES

range pH 6-7. Reduction of binding on the acid side of the pH optimum can be attributed to protonation of the carboxyl anion (pK,NAA = 4.3). 2. A fractional positive charge 0.55 nm distant from the -COO' is also regarded as an essential feature of active auxin molecules (Porter and Thimann, 1965). This requirement most probably implies electrostatic bonding to an anionic residue in the binding site located at the appropriate distance from the carboxyl-binding imidazolyl function. At pH 5.5 the choice of negatively charged non-terminal polypeptide groups is restricted to the p- or y-carboxyl of aspartate or glutamate respectively (pK, 4.0). Protonation of this carboxyl would also contribute towards the fall-off in auxin binding at low pH. 3. The pH dependence of diazo-CAPA inhibition (Fig. 10) is consistent with an azo coupling controlled by a functional group of an amino acid with a high pK, i.e. tyrosine or lysine. 4. The effectiveness of diazo-Chloramben at pH 6 (Fig. 10) suggests that under these conditions, where tyrosine and lysine would not be expected to react, coupling to a histidine residue is involved. From Dreiding models it appears improbable that a Chloramben molecule can both azo couple via the 3-amino group and bind through its carboxyl group to the same histidine residue. This would imply either the presence of a second histidine near the recsptor site, or azo coupling to the carboxyl-binding histidine, in which case the Chloramben carboxyl would not be suitably positioned for electrostatic bonding. The use of general group-modifying reagents is also being investigated, starting with sulphydryl reagents. Results to date implicate a cysteine residue in TABLE 111 Affinity Labelling with Diazo-Chloramben in the Presence or Absence o f N A A or BA (Benzoic Acid)

Inhibition of subsequent auxin binding (%) ~~

-

Site 1 Site 2

NAA

72.1 50.6

+NAA

-BA

+BA

25.4 13.4

54.5 39.1

29.0 35.7

Membranes were resuspended at pH 6 and reacted, in the presence or absence of 25 fiM NAA or BA, with 150 p M diazoChloramben (25"C, 15 min). Control membranes received diazotization mixture without Chloramben iNAA or BA. The reaction mixtures were then diluted into two volumes of ice-cold buffer and centrifuged (80000 x g , 30 min). Membranes were resuspended at pH 5.5 and saturable auxin binding determined using 0.2 p M NAA-14C f 0.2 pM or 0.8 pM cold NAA. Site 1 and site 2 binding were estimated from the differences in radioactivity bound between 0.2-0.4 rM NAA and 0.4-1.0 pM NAA respectively. Inhibition values were calculated relative to the appropriate controls (+ NAA or BA during the 25°C pre-incubation). The protective effects of NAA and BA were determined in separate experiments.

84

MICHAEL A. VENIS

the vicinity of the binding site (inhibition by p-hydroxy-mercuribenzoate, PHMB, reversible by thiols). Table IV summarizes current speculation on some of the amino acid residues which may be present at or near the auxin binding sites in maize coleoptile membranes. Because modification by diazoChloramben at pH 6 may be more specific than modification by diazo-CAPA at higher pH, and because auxin protection can be demonstrated at pH 6 , Chloramben has been selected for tritiation in preference to CAPA. Recently it has proved possible to separate the binding entity from the membranes in a soluble form without recourse to detergent TABLE IV Possible Amino Acid Environment of Auxin Receptor Sites Observation 1. pH dependence of auxin binding

2. Diazo-Chloramben inhibition at pH 6 3. Diazo-CAPA inhibition at pH 8-9 4. PHMB inhibition

Inference Histidine (-COO‘-binding) Aspartate/glutamate (6 +-binding) Histidine Tyrosine/lysine Cysteine

treatment and to obtain small-scale purification of 100-200-fold in two steps. It is hoped that co-valent attachment of tritiated Chloramben by azo-coupling to membrane-bound or to solubilized receptors will prove a further aid in receptor purification and in investigation of the active site environment.

IV. CONCLUDING REMARKS The status of plant hormone receptor research is still embryonic and most of the available information relates to auxins. No directly relevant reports on abscisic acid have appeared, though we do have preliminary evidence for high affinity binding of tritiated abscisic acid to partly purified membranes from bean leaves (Clapham, et al. , unpublished observations). There are probably functionally distinct ‘‘soluble’’ and membrane-bound receptors for auxins, but characterization and comparison of such receptors from the same tissue will be needed to decide on this point. Hardin et al. (1972) have reported stimulation of soybean FWA polymerase by a factor that is released from plasma membrane fractions by 2,4-D. They suggest that auxin binds to a membrane receptor and causes its release and transfer to the nucleus, where it modifies RNA polymerase activity. T h ~ s constitutes a one receptor hypothesis to unify slow and fast auxin responses. The nature of the released factor has not been characterized in any way, but there is no reason to believe that it is related to the soluble soybean factor described earlier from the same laboratory (Hardin et al., 1970). Nor does it seem to resemble the soybean factor obtained by affinity chromatography

RECEPTORS FOR PLANT HORMONES

85

(Rizzo e t al., 1976), since the latter is active on a different soybean polymerase. Substantially more evidence wdl be necessary to lend support to the hypothesis of Hardin et ul. (1972). At present the only repsit that is in any way comparable is that by Likholat et al. (1974), discussed in Section III.C(4). The only system reported in which actual binding of a hormone to a receptor has been observed and correlated with some form of response (alteration in RNA polymerase activity) is the auxin receptor protein from immature coconuts studied by Biswas and co-workers (Mondal et ul., 1972; Biswas et al., 1975). Unfortunately the nature of the source material makes independent evaluation of this receptor difficult. Nevertheless, publication of adequate experimental details might facilitate the search for comparable receptors in systems more amenable to wider study. In terms of convenience and reproducibility, the membrane-bound auxin receptor system of maize coleoptiles, first studied by Hertel and colleagues, affords great promise for further detailed investigation. The receptor concentration is high (about 100 pmol/g tissue), the first steps in purifying the solubilized receptor have been taken, and we already have inferential information on some of the amino acids likely to surround the binding site. With larger quantities of purified receptor, it may well prove possible to map the active site of a plant hormone receptor for the first time. It will also be intriguing to discover whether the binding sites of the purified receptor exhibit negative co-operativity. We have tended to confine our attention to the kinetics of high affinity binding at auxin concentrations not exceeding 1 p M , but binding continues to saturate gradually with concentration for more than two further orders of magnitude without ever reaching a true plateau. This behaviour could be caused by a large number of additional, low affinity sites in the membranes, but might alternatively reflect negative co-operativity of the binding sites already studied, i.e. a situation in which binding of one auxin molecule reduces the binding affinity for subsequent molecules. Such a mechanism would permit the plant to continue responding to auxin concentrations far in excess of those which would otherwise have saturated the receptors. Since physiological response-dose curves for auxins and indeed for other plant hormones frequently span more than four orders of magnitude in concentration, negative co-operativity of binding sites may prove to be a general feature of plant hormone receptors.

REFERENCES Andreae, W. A. and Van Ysselstein, M. W. (1960). Plant Physiol. 35, 225-232. Bamberger, E. S . (197 1). Phytochernistry 10, 957-966. Batt, S. and Venis, M. A. (1976). Planta 130, 15-21. Batt, S., Wilkins, M. B. and Venis, M. A. (1976). Planta 130, 7-13. Bendana, F. E., Galston, A.. W., Kaur-Sawhney, R. and Penny, P. (1965). Plant Physiol. 40, 977-983.

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MICHAEL A. VENIS

Berridge, M. V., Ralph, R. K. and Letham, D. S. (1 970). Biochem. J. 119, 75-84. Biswas, B. B., Ganguly, A., Das, A. and Roy, P. (1975). In “Regulation of Growth and Differentiated Function in Eukaryote Cells” (G. P. Talwar, Ed.), 461-477. Raven Press, New York. Bressan, R. A., Ross, C. W. and Vandepeute, J. (1976). Plant Physiol. 57, 29-37. Brian, R. C. and Rideal, E. K. (1952). Biochim. biophys. Acta 9, 1-18. Brock, B. L. W. and Fletcher, R. A. (1969). Nature, Lond. 224, 184-185. Cleland, R. (1973).Proc. natn. Acad. Sci. U.S.A. 70, 3092-3093. Cleland, R. E. (1975). Planta 127, 233-242. Cleland, R. E. and Rayle, D. L. (1975). Plant Physiol. 55, 547-549. Cuatrecasas, P., Wilchek, M. and Anfinsen, C. B. (1968). Proc. natn. Acad. Sci. U.S.A. 61, 636-643. Datko, A. H. and Maclachlan, G. A. (1968). Plant PhysioZ. 43, 735-742. Davies, E. and Maclachlan, G. A. (1969). Archs. Biochem. Biophys. 129, 58 1-587. Davies, P. J. (1973). Bot. Rev. 39, 139-171. Davies, P. J. and Galston, A. W. (1971). Plant Physiol. 47, 435-441. DeLange, R. J., Fambrough, D. M., Smith, E. L. and Bonner, J. (1968). J. BioZ. Chem. 243,5906-5913. Evans, M. L. (1974). Ann. Rev. Plant Physiol, 25, 195-224. Evans, M. L. and Ray, P. M. (1973). Plant Physiol. 52, 186-189. Fan, D. F. and Machlachlan, G. A. (1966). Can. J. Bot. 44, 1025-1034. Fan, D. F. and Machlachlan, G. A. (1 967). PZant Physiol. 42, 1 1 14-1 122. Fellenberg, G. (1969). Z. Pfl.Physiol. 60, 457-466. Fellenberg, G. and Schomer, U. (1975). Z. Pfl.PhysioZ. 75,449-456. Filner, P. and Varner, J. E. (1967). Proc. natn. Acad. Sci. U.S.A. 58, 1520-1526. Fox, J. E. and Erion, J. L. (1975). Biochem. biophys. Res. Commun. 64, 694-700. Galston, A. W. and Davies, P. J. (1969). Science, N. Y. 163, 1288-1297. Galston, A. W., Jackson, P., Kaur-Sawhney, R., Kefford, N. P. and Meudt, W. J. (1 964). In “Rkgulateurs Naturels de la Croissance VBgBtale”, 25 1-264. CNRS, Paris. Hager, A., Menzel, H. and Krauss, A. (1971). Planta 100,47-75. Hardin, J. W. and Cherry, J. H. (1972). Biochem. biophys. Res. Commun. 48, 299-3 06. Hardin, J. W., O’Brien, T. J. and Cherry, J. H. (1970). Biochim. biophys. Acta. 224, 667-670. Hardin, J. W., Cherry, J. H., MorrB, D. J. and Lembi, C. A. (1972). Proc. natn. Acad. S C ~U.S.A. . 69, 3146-3150. Havinga, E. and Veldstra, H. (1948). Rec. trav. chim. 67, 855-863. Hermville, E. and Klambt, D. (1976). Submitted t o Biochem. biophys. Res.

Commun. Hertel, R. (1974). In “Membrane Transport in Plants” (U. Zimmerman and J. Dainty, Eds), 457-461. Springer-Verlag, Berlin. Hertel, R., Thomson, K. S. and Russo, V. E. k (1972). Pluntu 107, 325-340. Higgins, T. J. V., Zwar, J. A. and Jacobsen, J. V. (1976). Nature, Lond. 260, 166-169. Ho, D. T.-H and Varner, J. E. (1974). Proc. natn. Acad. Sci. U.S.A. 71, 4783-4786. Jablonovi;, M. and Nooden, L. D. (1 974). Plant Cell Physiol. 15,687-692. Jacobsen, J. V. and Varner, J. E. (1 967). Plant Physiol. 42, 1596-1 600.

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87

Jacobsen, J. V. and Zwar, J. A. (1974). Proc. natn. Acad. Sci. U.S.A. 71, 3290-3293. John, M. M. and Varner, J. E. (1968). Proc. nutn. Acad. Sci. U.S.A. 59, 269-276. Kasamo, K. and Yamaki, T. (1973). Sci. Pup. gen. Educ. Univ. Tokyo 23, 131-138. Kasamo, K. and Yamaki, T. (1974). In “Plant Growth Substances 1973”, 699-707. Hirokawa, Tokyo. Kasamo, K. and Yamaki, T. (1976). Plant Cell Physiol. 17, 149-164. Keegstra, K., Talmadge, K. W., Bauer, W. D. and Albersheim, P. (1973). Plant Physiol. 51, 188-196. Kefford, N. P., Kaur-Sawhney, R. and Galston, A. W. (1963). Acta chem. scand. 17, S313-318. Kennedy, C. D. and Harvey, J. M. (1972). Pestic. Sci. 3, 715-727. Key, J. L. (1 969). Ann. Rev. PZant Physiol. 20, 449-474. Knofel, H. D., Miiller, P., Kramell, R. and Sembdner, G. (1975). FEBS Lett. 60, 39-41. Lacombe, M. L. and Hanoune, J. (1 974). Biochem. biophys. Res. Commun. 58, 667-673. LCJohn, H. B. (1975). Can. J. Biochem. 53, 768-778. Lembi, C. A., MorrB, D. J., Thomson, K.-St. and Hertel, R. (1971). Planta 99, 37-45. Leonard, N. J., Greenfield, J. C., Schmitz, R. Y . and Skoog, F. (1975). Plant Physiol. 55, 1057-1061. Likholat, T. V., Pospelov, V. A., Morozova, T. M. and Salganik, R. I. (1974). Soviet Plant Physiol. 21, 779-784. Lin, P. P.-C. (1 974). In “Advances in Cyclic Nucleotide Research” (P. Greengard and G. A. Robison, Eds), Vol. 4,439-46 1. Raven Press, New York. Matthysse, A. G. (1 970). Biochim. biophys. Acta 199, 5 19-521. Matthysse, A. G. and Abrams, M. (1970). Biochim. biophys. Acta. 199, 51 1-518. Matthysse, A. G. and Phillips, C. (1969). Proc. natn. Acad. Sci. U.S.A. 63, 897-903. Mondal, H., Mandel, R. K. and Biswas, B. B. (1972). Nature, New Biol. 240, 111-113. Moyed, H. S . and Williamson, V. (1967). J. Biol. Chem. 242, 1075-1077. O’Brien, T. J., Jarvis, B. C., Cherry, J. H. and Hanson, J. B. (1968). Biochim. biophys. Acta. 169, 35-43. Oostrom, H., Van Loop&-Detmers, M. A. and Libbenga, K. R. (1975). FEBS Lett. 59, 194-197. Penner, D. and Early, R. W. (1972). Phytochemistry 11, 3135-3138. Penny, P. (1 97 1). Plant Physiol. 48, 720-723. Penny, P., Penny, D., Marshall, D. and Heyes, J. K. (1972). J. Expl. Bot. 23, 23-26. Penny, D., Penny, P., Monro, J. and Bailey, R. W. (1972). In “Plant. Growth Substances 1970” (D. J. Cars, Ed.), 52-6 1. Springer-Verlag, Berlin. Porter, W. L. and Thimann, K. V. (1 965). Phytochemistry 4, 229;243. F’uca, G. A., Nola, E., Sica, V. and Bresciani, F. (1972). Biochemistry 11, 4157-4165. Ray, P. M. (1973). Plant Physiol. 51, 601-608. Ray, P. M. (1 974). In “The Chemistry and Biochemistry of Plant Hormones” (V. C. Runeckles, E. Sondheimer and D. C. Walton, Eds). Academic Press, New York.

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Rayle, D. L. (1973). Planta 114, 63-73. Rayle, D. L. and Cleland. R. (1970). PZunt Physiol. 46, 250-253. Rayle, D. L., Evans, M. L. and Hertel, R. (1970). Proc. natn. Acad. Sci. U.S.A. 65, 184-191. Richardson, J. P. (1966). J. molec. Biol. 21, 115-127. Rizzo, P. J., Pedersen, K. and Cherry, J. H. (1976). Submitted to Biochem. biophys. Res. Commun. Sarkissian, I. V. (1970). Biochem. biophys. Res. Commun. 40, 1385-1390. Sarkissian, I. V. and Schmalstieg, F. C. (1 969). Naturwissenschaft 56, 284. Schwartz, A., Lindenmayer, G. E. and Allen, J. C. (1975). Pharmac. Rev. 27, 3-1 34. Siegel, S. M. and Galston, A. W. (1953). Proc. natn. Acad. Sci. U.S.A. 39, 111 1-1 118. Stoddart, J., Breidenbach, W., Nadeau, R. and Rappaport, W. (1 974). Proc. natn. Acad. Sci. U.S.A. 71, 3255-3259. Siidi, J. ( 1 964). Nature, Lond. 201, 1009-1 0 10. Siidi, J. (1 966). New Phytol. 65, 9-21. Takegami, R. and Yoshida, K. (1 975). Biochem. biophys. Res. Commun. 67, 7 8 2-7 89. Teissere, M., Penon, P. and Ricard, J. (1973). FEBS Lett. 30, 65-70. Teissere, M., Penon, P., Van Huystee, R. B., Azou, Y . , and Ricard, J. (1975). Biochim. biophys. Acta. 402, 391-402. Thomson, K. S. (1972). In “Hormonal Regulation in Plant Growth and Development” (H. Kaldeway and Y. Vardar, Eds), 83-88. Verlag-Chemie, Weinheim. Thomson, K. S. and Leopold, A. C. (1974). Planta 115, 259-270. Thomson, K. S., Hertel, R. Muller, S. and Tavares, J. E. (1973). Planta 109, 337-352. Tuli, V. and Moyed, H. S. (1969). J. Biol. Chem. 244,4916-4920. Vanderhoef, L. N. and Stahl, C. A. (1975). Proc. natn. Acad. Sci. U.S.A. 72, 1822-1825. Van der Woude, W. J., Lembi, C. A. and MorrB, D. J. (1972). Biochem. biophys. Res. Commun. 46, 245-253. Varner, J. E. and Chandra, G. R. (1964). Proc. natn. Acad. Sci. U.S.A. 52, 100-1 06. Venis, M. A. (1 964). Nature, Lond. 202, 900-901. Venis, M. A. (1968). In “Biochemistry and Physiology of Plant Growth Substances” (F. Wightman and G. Setterfield, Eds), 761-775. Runge Press, Ottawa. Venis, M. A. (1971). Proc. natn. Acad. Sci. U.S.A. 68, 1824-1827. Venis, M. A. (1 972). Plant Physiol. 49, 24-27. Venis, M. A. (1973). Curr. Adv. Plant. Sci. 2, 21-28. Venis, M. A. and Stoessl, A. (1969). Biochem. biophys. Res. Cornmun. 36, 54-56. Wilchek, M., Salomon, Y., Lowe, M. and Selinger, Z. (1971). Biochern. biophys. Res. Cornmun. 45, 1177-1 184. Wilson, H. A., Pasternak, G. W. and Snyder, S. H. (1975). Nature, Lond. 253, 448-4 50. Wood, A. and Paleg, L. G. (1972). Plant Physiol. 50, 103-108. Yoshida, K. and Takegami, T. (1 976). Submitted to J. Biochem. (Tokyo). Zenk, M. H. and Nissl, D. (1968). Naturwissenschaft 55, 84-85.

Plant Cell Wall Synthesis“ DAVID G. ROBINSON

Pfanzenphysiologixhes Institut der Universitat Untere Karspiile 2. 0.34 Gottingen. Federal Republic of Germany I. I1.

Int-roduction . . . . . . . . . . . . . . . . . Structural Considerations . . . . . . . . . . . . . A Non-Cellulosic Components . . . . . . . . . . B. Cellulose . . . . . . . . . . . . . . . . 111. The Electron Microscopy of Cell Wall Formation . . . . . A . Sitesof Synthesis . . . . . . . . . . . . . . B . The Orientation of Cellulose . . . . . . . . . . IV . Cell Fractionation Studies . . . . . . . . . . . . . A. Preparation of Cell Fractions and Their Identification . . B . Analysis of Fractions from Pulse-Chase Experiments . . C. Transport of Synthesized Materials . . . . . . . . V . In Vitro Synthesis . . . . . . . . . . . . . . . A . Higher Plant Cellulose . . . . . . . . . . . . B. Bacterial Cellulose . . . . . . . . . . . . . Chitin . . . . . . . . . . . . . . . . . C. D . Non-Cellulosic Materials . . . . . . . . . . . Lipid Intermediates . . . . . . . . . . . . . E. VI . Conclusion . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

.

. . . . .

. . . . .

. .

.

. .

.

.

.

.

89 91 91 96 99 99 105 111 11 1 11 8 125 136 136 138 138 139 140 142 143 143

I . INTRODUCTION “Well you know. the best plant cell for Golgi extraction is really liver because it doesn’t have a cell wall.” D . J . M o d 1st Meeting International Association of Plant Physiology. Wiirzburg. 1974. Only rarely do plant cells in nature exist without a wall and even for those which do. such as certain unicellular algae. the naked protoplast is a transitory stage

* Literature survey completed 15th April 1976. 89

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DAVID G. ROBINSON

associated with reproduction. The plant cell wall is an externally lying product of the protoplast. As a consequence, all wall formation includes not only the synthesis of complex macromolecules, but also the transport of the precursors of these components to their sites of deposition and their eventual incorporation into the wall. The extent to which the synthesis, transport and incorporation of cell wall components may be related spatially and in time to one another is only now being unravelled. One may investigate the synthesis of the plant cell wall with three approaches: 1. An in vivo method in which the production of the wall is followed in situ.

This may be followed using the electron microscope, for example, in the development of a wall between daughter cells following mitosis, or in the generation of a wall around naked protoplasts. 2. In in vitro methods in which particular subcellular fractions from homogenized material are provided with a radioactive precursor whose incorporation into a cell wall component may be followed. 3. A combination of the in vivo and in vitro methods in which whole cells/tissue may be fed with a radioactive precursor and then homogenized allowing the nature of the intermediates and their location within the cell to be found.

The first approach is essentially a static one and can at best only provide knowledge of where a particular heavy metal-staining pattern is to be seen at any particular time; one usually cannot say exactly what biological structure is being stained nor where it came from or is going to in the cell. Although the coupling of autoradiography to the pulse-chase technique (Northcote and Pickett-Heaps, 1966) has introduced a more dynamic facet t o electron microscopy, the resolution, i.e. the assignment of silver grains to a particular cell component, is not fine enough to allow accurate pinpointing of the radioactive source. Methods (2) and (3) involve homogenizing the material under investigation which introduces a somewhat ironical aspect of t h s field of research. If one is interested in retaining the structural integrity of cell organelles, whether for indentification and/or for purity purposes, one should employ the gentlest methods of homogenization: sufficient only, for example, to rupture the plasmalemma. More destructive procedures introduce artefacts but unfortunately the presence of a resilient cell wall often necessitates the use of such methods. Even in animal cells where often a few turns of a Potter homogenizer releases the cell contents, the need for purity of intracellular fractions is only achieved at the expense of a low percentage recovery of these fractions (Jamieson and Palade, 1967a). There exists, then, the paradox that, to various degrees, the wall itself may be a barrier to studies of its synthesis. Research into the structure and synthesis of individual cell wall components has increased in recent years and there is an urgent need t o bring together

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structural (electron microscopical) and functional (chemical) information. Such is the object of this review although a number of prominent secondary components, most notably lignins, will be excluded. 11. STRUCTURAL CONSIDERATIONS

As is well known, cells produced by division in root or shoot meristems of higher plants are subject to a period of elongation, during which time the deposited cell wall is known as a primary wall, followed by a period of differentiation when no further elongation takes place and a thick secondary wall is deposited. The terms primary and secondary are not to be used universally however: most higher plant seed cells, e.g. endosperm and aleurone cells, do not “grow” in the above sense, and many algae which possess thick cellulosic walls, reminiscent of secondary walls of higher plants, can nevertheless increase in length and girth (Preston, 1974). A. NON-CELLULOSIC COMPONENTS

It is also well known that the non-protein, polysaccharide portion of the cell wall has for many years been subdivided largely on the arbitrary criterion of solubility of certain components in boiling water, EDTA or ‘dilute acid (the pectins) followed by concentrated, cold alkali (the hemicelluloses), the residue after such treatments being the a-cellulose. These treatments however can modify the chemical composition of the cell wall: dilute acid hydrolyzes the methyl and acetyl groups of uronic acids and sugars respectively (Talmadge e l al., 1973); alkali has a similar action and also effects the P-elimination of glycosidic bonds to serine in cell wall protein (Lamport, 1973) and the transelimination of polyuronides into uronic acids (Neukom and Deuel, 1958). Therefore, although it has been known for some time that pectins regularly consist of an acid (a-1,4 linked galacturonic acid) fraction and a neutral (galactans, arabinans, arabinogalactans) fraction (Aspinall, 1970) and that hemicelluloses consist of xylans, mannans, glucomannans, arabinogalactans and arabinoxylans in varying proportions (Whistler and Richards, 1970), the relationships between these wall components and the cellulose and protein of the cell wall is lost when conventional extractions are carried out. Recognition of this and the use, instead, of specific polysaccharidases has recently enabled Albersheim and his co-workers to make a significant contribution t o the field of plant cell wall structure (Albersheim et al., 1973; Bauer et al., 1973; Keegstra e l al., 1973; Talmadge e l al., 1973; Valent and Albersheim, 1974; Albersheim, 1975). As experimental material Albersheim’s group have used suspension cultured cells of sycamore callus because they are easily cultivated, possess only primary walls and release into the growth medium some polysaccharides closely resembling those of the wall. As shown in Fig. 1 cell walls isolated from these cultures were subjected to sequential degradation with a polygalacturonase, a p-1,4

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endoglucanase (cellulase) and finally with pronase. By gel filtration/Sephadex chromatography the neutral polymers could be separated from acidic polymers for each extract. The total cell wall and these various fractions were then subjected t o a painstaking structural analysis involving the methylation of all free hydroxyl groups, hydrolysis of the polymers followed by reduction and acetylation of the constituent sugars, producing volatile derivatives which could be identified by gas chromatography, (see Albersheim et al., 1973; and Albersheim, 1975 for details of the chemistry involved here). Essentially where an hydroxyl group is absent the sugar is linked to another sugar and therefore from such an analysis one can determine the percentage of each linkage “species” and thus possible structures for the polysaccharide may be deduced. UNTREATED CELL WALLS

v

Polygalacturonase

Soluble “PECTIC POLYMERS”

Residue

p-1,L Endoglucanase

h

Soluble “HEMICELLULOSES ”

Residue

Pronase

Residue Fig. 1. Enzymatic fractionation of suspension-cultured sycamore callus cells as employed by Albersheim’s group.

The main component released by endopolygalacturonase was found t o be an acidic polymer of a-1,4 galacturonic acid containing after every 8 uronic residues a 1,2 linked rhamnosyl residue, causing the polymer to assume a “kinked” shape. Also released by this enzyme were several neutral polymers: a 1,3 linked galactan with arabinose residues frequently attached at the 6 carbon of the galactose, a 1,4 linked galactan and a 2,5 linked arabinan. From the endoglucanase treatment of the wall and also from the polysaccharides secreted into

PLANT CELL WALL SYNTHESIS

93

the medium (here it must be noted that these polysaccharides are representative of the total wall; according to Stoddart and Northcote (1967) they are rich in glucose and xylose, poor in arabinose and possess little galacturonic acid) a neutral xyloglucan polymer was obtained. Pronase extraction of walls pretreated with polygalacturonase and endoglucanase released protein and a further amount of carbohydrate. Linkage analysis of the acidic components of this digest revealed a close similarity to the polysaccharides released by polygalacturonase; the neutral portion contained a hydroxproline-rich protein and arabinose. Cell wall hydroxyproline arabinoside glycoproteins are not just characteristic of sycamore cells (Heath and Northcote, 1971) but are now known to be very widespread in the plant kingdom (Lamport and Miller, 1971). Table I shows the proportions of these various polymers in the non-cellulosic portion of the sycamore wall. In good agreement with the total amounts of rhamnose and galacturonic acid in the cell wall is the determined amount of rhamnogalacturonan. However, the proportion of xyloglucan is clearly too high for the corresponding levels of xylose and glucose as given by Talmadge et al. (1973); not only is the ratio of xylose to glucose in xyloglucan polymer about 0.75 as compared to 2.04 in the total non-cellulosic hydrolysates but earlier measurements by Lamport (1965) show a much higher level of glucose to xylose in the non-cellulosic fracticrls. TABLE I Composition of the Primary Wall o f Suspension Cultured Sycamore Callus Cells Fraction and proportion in walla

Non-cellulosic polysaccharides (66%)

a-Cellulose

Protein (10%)

% sugara amino acid'^ in each fraction Rhamnose Galacturonic Acid Arabin ose Galactose Fucose Xylose . Glucose Glucose

4.9 21.2 33.2 20.2 2.1 12.1 5.9 100c

% polymer composition in wall carbohydrutesa Kharnnogalac turonan 3,6 linked Arabinogalactan 1,4 linked Galactan + 2,5 linked Arabinan Arabinose oligosaccharides (attached t o wall protein) Xyloglucan

24 3

21 14 32

Hyp. 20.6; Ser. 10.62; Lys. 8.7; Glu. 1.34; Asp. 6.7; Pro. 6.44; Gly. 5.76; Val. 5.54; Ala. 5.2; Leu. 4.96; Thre. 4.0; Ileu. 2.94; @Ala.2.71; Arg. 2.39; His. 2.37; Tyr. 1.69; Cys. 1.13; Met. SO7 1.13

From Talmadge el al. (1973). b From Lamport (1965) Depending on the severity of the extraction small amounts of other sugars, particularly mannose may be present. a

C

,

94

DAVID G. ROBINSON

Although by these enzyme treatments Albersheim and his group have only succeeded in removing about 60% of the non-cellulosic polysaccharides of the cell wall they have concluded on the basis of the following evidence that the individual polysaccharide and glycoprotein fragments are in fact attached to one another and to the cellulose of the wall; their reasoning is as follows: 1. Evidence for connection between xyloglucan (XG) and cellulose: (a) Isolated XG binds to Whatman cellulose powder (up to 90% binding having been demonstrated) which can be partially released by treatment with the endoglucanase. ( b ) Polygalacturonase pre-treated walls can release XG after treatment with 8 M Urea or 0.5 N NaOH suggesting the existence of non-covalent linkages between the XG and cellulose. 2. Evidence for connection between XG and other non-cellulosic polysaccharides: (a) Presence of small amounts of XG in neutral sugar rich fractions released by polygalacturonase. (b) Release of further neutral sugar-rich pectic polysaccharides from polygalacturonase pre-treated walls with 0.5 N NaOH and the cochromatography of these fragments with XG released by endoglucanase. 3. Evidence for connection between non-cellulosic polysaccharides and wall protein. (a) Linkage analysis of a hydroxyproline-rich glycoprotein isolated and purified from the growth medium of the sycamore cells revealed a 1,3 llnked galactan bearing occasional C 6 linked arabinobe residues as side chains, i.e. similar t o that found in extracts of cell walls made with polygalacturonase. (b) The existence of a terminal rhamnose on this arabinogalactan, suggesting a relationship with the rhamnogalacturonan polymer. (c) The existence of galacto-serine glycopeptides (Lamport, 1969) which are relatively stable to the 0-elimination of serine to alanine, suggesting a polymer rather than a single sugar attachment here (Lamport, 1973). (d) Attachment of other polysaccharides to the hydroxyproline arabinosides seems unlikely in view of the fact that such arabinosides may be obtained with concentrations of alkali which do not break glycosidic bonds.

Figure 2 shows in diagrammatic form the molecular models postulated by Albersheim’s group. In their first published model (Keegstra et al., 1973) the non-cellulosic polysaccharides were linked to the wall proteins; in a later article (Albersheim, 1975) the protein has been omitted, “since the structural role of the protein has not been established”. While the evidence for the linkages in

PLANT CELL WALL SYNTHESIS

95

these models is tentative, the linkage analysis patterns for the non-cellulosic polysaccharides in sycamore d o not appear to be unique. Rhamnogalacturonans are often present in pectins ( e g Rees and Wight, 1969); Wilder and Albersheim (1973) have reported an almost identical xyloglucan from the walls of suspension cultured kidney bean cells; Labavitch and Ray (1974a, b) and Jacobs and Ray (1975) have shown that auxin and low pH apparently stimulate the conversion of wall-bound xyloglucan to a soluble form in pea epicotyl cells, and Kooiman (1960, 1961) has demonstrated the presence of xyloglucan in the cotyledon cell walls of several plants. On the other hand the primary walls of

CFLLULOSE 5LUCAN

ALACTAN

[

WALL F’ROTEIN

\

RHAMNOGALACT~URONAN

LJ

Fig. 2. Suggested schemes for the structure of the primary wall of sycamore callus cells. Models (not to scale) redrawn from the work of Keegstra et 01. (1973) and Albersheim (1975). Postulated linkages: 0 covalent bonding; = hydrogen bonding.

monocotyledon cells seem to have a considerably different composition (Burke el al., 1974) with no xyloglucan, smaller amounts of uronic acids, a cell wall protein containing very little hydroxyproline and with the principal noncellulosic polysaccharide a 1,4 linked xylan with single arabinosyl residues as side chains. The work of Albersheim’s group is important not only for the results themselves-indeed much was already known of the linkages involved in non-cellulosic polysaccharides (see for example the reviews by Timell, 1964, 1965; Aspinall, 1970, 1973;and Whistler and Richards, 1970)-but also for their successful employment of new and powerful methods.

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DAVID G . ROBINSON

B. CELLULOSE Cellulose is the most abundant biological macromolecule and was already extensively investigated both chemically and crystallographically before the Second World War. Methods for the demonstration of cellulose may be divided into two types: 1. Chemical. Here the methods employed are usually hydrolytic, e.g. by acids or enzymes; by solubility and the measurement of degree of polymerization. 2. Physical. Includes X-ray diffraction; infra-red spectroscopy, electron microscopy and polarization microscopy. A basic difference between these two groups is exactly what is demonstrated after their successful application. With the first, only the type and degree of linkage, i.e. the polymeric nature, is determined; with the second, the association of polymer chains, i.e. the crystallinity is demonstrated. I These are very simple points which should not need to be stated; nevertheless the facts that cellulose always exists in a crystalline microfibrillar form and that no single &1,4 glucan chain cellulosic precursors have been demonstrated in plants, do not seem to have deterred many workers from claiming the synthesis of cellulose or the location of a “cellulose synthetase” in a particular preparation, purely on the basis of chemical methods. The dangers in so doing become apparent when one asks the question: are there in the cell wall any p-1,4 linked glucans which are not cellulose. As separate chains the answer is no, but as segments of another molecule the answer is most definitely yes. The best-known examples are the cereal glucans and lichenin (Clarke and Stone, 1963), but from the above section on the cell wall non-cellulosic components we now know that the xyloglucan to be found in many plant cell walls also possesses a p-1,4 glucan backbone (Fig. 3). Using undefined cell fractions from Avena coleoptiles it has been qhown by Ordin and Hall (1967, 1968) that mixed /3-1,4; p-1,3 glucms may be synthesized in vitro using UDP-D-glucose as the donor. Furthermore the 0-1,4 glucan synthetase in Pisum epicotyl tissue shown to be localized in Golgi dictyosomes by Ray et al. (1969) has now been shown by Ray (1975) and Villemez and Hinman (1975) t o work in concert with a UDP-xylose transferase in the production of cell wall xyloglucan. The care that Ray et al. (1969) took in avoiding the words cellulose synthetase in their paper is, in retrospect, a commendable example of the caution to be adopted in this field. Such caution has not been demonstrated by other authors however: for example, owing to a lack of appreciation of the distinction between the in vitro synthesis of p-1,4 linked glucan and the in vivo incorporation of radioactivity into cellulose, Brown and co-workers (Brown et al., 1973; Herth et al., 1975) have cited the paper of Ray et al. (1969) as an example of Golgi-based cellulose synthesis and have even

97

PLANT CELL WALL SYNTHESIS

alluded to a “controversy” between this and another paper from Ray’s laboratory which claims cellulose synthesis at the plasmalemma (Robinson and Ray, 1973). Although there is a large amount of evidence (see later) for the participation of nucleotide sugar-dependent 0-glucan synthetases in cellulose synthesis, it must be emphasized that the demonstration of &1,4 glucan synthetases cannot be automatically assumed to be the demonstration of cellulose synthesis. This is particularly so when the only existing X-ray diffraction evidence (Robinson and Preston, 1972b) for such in vitro products indicates short oligosaccharides and, 1. CEREAL GLUCAN 4Glul-3

Glu 1-

4 Glu1-4

Gh1-3

Glul-

XYl

Xyl

CGlul-4

Glul-4

Glul-3

Glul-

2. TAMARINDUS A M YLOID Gal 1

-4

Gal 1

. Gal 1

G l ~ l + 4 G l ~ l - 4 GIU 1-

3. SYCAMORE XYLOGLUCAN

-

Xyl

XYl

6

6

i

L Glul-4Glu

1

1

-

4GIul-

i

6

L Glul-

1

t

6

X’II

1

6

4 G l ~ l - 4 G l u 1-24 Glul-

Fig. 3. Occurrence of p-1,4 glucan in non-cellulosic polysaccharides.

furthermore when the planis used for the in vitro analysis have large amounts of non-cellulosic glucan in their walls, e.g. some cereal plants (Buchala and Meier, 1973). Information from X-ray diffraction on cellulose has been with us for over forty years. The crystal structure of cellulose first inferred from X-ray studies some forty years ago (Meyer and Misch, 1937; see Preston, 1974 for detailed information) remained largely unchallenged until recently. Important modifications now require that for some plants the cellulose crystallite is so long and well ordered that unit cells larger than the typical 5-chain Meyer and Misch model are necessary to index all the available reflections. Thus Nieduszyuski and Atkins, (1970) have demonstrated an 8-chain unit cell for Cladophora and Valonia cellulose. Furthermore the antiparallel arrangement of the glucan chains in the Meyer-Misch model, a feature of particular importance when considering the

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synthesis of cellulose, can now be replaced by a parallel arrangement (Gardner and Blackwell, 1974) in which chains in the 020 plane are hydrogen bonded together giving cellulose a sheet-like structure. Despite these recent advances, the basic X-ray diagram for cellulose is constant throughout the plant kingdom and therefore can be used in a histological sense for the detection of cellulose. The morphological unit of cellulose, the microfibril, must be interpreted with care. It is a preparation artefact just as are many other cell structures revealed by the electron microscope. The transmission electron microscope depends primarily on the distribution of heavy metals such as osmium, lead and uranium to produce contrast. But, electron microscopical stains, because they are aqueous, cannot penetrate the cellulose crystal (Heyn, 1966; O’Brien, 1972); the X-ray diagram for cellulose also remaining the same in dry or we1 conditions (Preston et al., 1948). Therefore the cellulose crystallite will always be negatively stained. Only rarely is it possible to section cell walls and see clearly the relationship between the non-stained cellulose and its surroundings (see for example Figs 12 and 14). Such a concentration of heavy metal ions around the crystallite must indicate a binding. What is then responsible for this stain accumulation? When cell walls are fractionated by conventional acid-alkali methods to remove pectin and hemi-cellulosic materials, the residue, called a-cellulose, seldom consists of glucose alone on hydrolysis (Dennis and Preston, 1961; Preston, 1962). Among the other sugars detected in smaller amounts in such hydrolysates are xylose and mannose. It now appears that a considerable amount of protein may also occur in this fraction. Heath and Northcote (1971) have reported that over 90% of the cell wall protein is in the a-cellulose fraction in sycamore cells. Furthermore Herth et al. (1972) have shown that the alkalipurified, scale a-cellulose of Pleurochiysis scherffelii contains up to 30%protein which is covalently bonded to the polysaccharide, and Brown et al. (1973) have demonstrated the existence of protein in a-cellulose fractions in a number of higher plants. The network structure of non-cellulosic polysaccharides and protein as shown in Albersheim’s models are either removed or altered in the preparation of “microfibrils” for electron microscopy. Even when these substances are not removed, water is, and this may have the effect of concentrating the noncellulosic materials around the cellulose crystallite. The concept of the cellulose microfibril is therefore in my opinion a somewhat arbitrary one which tends to exaggerate the status of the microfibril in the cell wall. I do not of course deny the existence of microfibrils, but the only safe measurement of them is the size of the cellulose crystallite they contain. This is normally only possible with negatively stained, isolated, microfibrils or by suitable X-ray diffraction methods (see Preston, 1974, pp. 144-146). From the latter technique the cellulose crystallite tends to be at least 4 nm in its smallest axis, but these values are for material from highly crystalline sources. Other objects which have been studied by negative staining have produced low values in the region of 1-2 nm (Franke

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and Ermen, 1969). Caution is to be recommended before accepting these values on account of the difficulties with the method itself (see Preston, 1971) but also because such small values can only represent 1 or 2 unit cells. 111. THE ELECTRON MICROSCOPY OF CELL WALL FORMATION A. SITES OF SYNTHESIS

The Golgi apparatus and plasmalemma are implicated in the synthesis of cell wall components on ultrastructural grounds. Occasionally, as in the cases of Pleurochiysis scherffelii (Brown et al., 1973) and Chiysochromulina chiton (Manton, 1967; Allen and Northcote, 1975), the site of synthesis and the secretion of cell wall components can be demonstrated directly. In these and other Chrysophycean algae, the “cell wall” contains highly ornamented cellulose scales which develop in the Golgi cisternae, are transported in secretory vesicles and extruded through the plasmalemma. In many other cases electron microscopical evidence for the participation of the Golgi apparatus has been reported, but mainly by inference, and as such has been heavily criticized (O’Brien, 1972). . The root cap, particularly of Zea Mays (Fig. 4), which secretes a polysaccharide slime (MorrC, et al., 1967) has often been sectioned. The root cap cells

Fig. 4. Section through root cap cell of Zea mays. Typical for this tissue are the large hypertrophied dictyosome cisternae and vesicles. x16 500. (Courtesy H. H. Mollenhauer and D. J. Morr6.)

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contain large numbers of Golgi dictyosomes almost all of which possess inflated (hypertrophied) cisternae and occasionally large vesicles may be seen fusing with the plasmalemma. The presence of polysaccharide in these cisternae has been demonstrated by Pickett-Heaps (1967a, 1968) using the periodic acid-silver hexamine (PASH) method; and Northcote and Pickett-Heaps (1966) using autoradiography, coupled with the pulse chase technique, have introduced a dynamic aspect to these observations. Using wheat root cap cells these authors have demonstrated that radioactivity, lying approximately over the Golgi after a 10 min feeding of 3H-glucose (“pulse”) is, after a subsequent period in non-radioactive glucose (“chase”), to be seen lying over the cell wall. Autoradiography has also been applied to the developing secondary walls in sycamore vascular tissue (Wooding, 1968) with the conclusion that xylans are deposited into the wall from Golgi dictyosomes. In agreement with the lack of a cell wall, animal cells divide apparently without the need of Golgi dictyosomes (Szollosi, 1970; Bluemink, 1970). On the other hand plant cytokinesis is invariably accompanied by the building of a new cell wall between the daughter protopiasts, whether achieved by centrifugal cell plate formation as in land plants (Ledbetter and Porter, 1970) or by a centripetal furrow as in some algae (Pickett-Heaps, 1975; Robinson et al., 1976a for examples). Although a large number of papers have been written on ultra-structural aspects of cytokinesis in plant cells, few have employed cytochemical or autoradiographic methods. Discrepancies exist; Pickett-Heaps (1967b) and Dauwalder et al. (1969) have shown that radioactivity may be located above cell plates in both epidermal and meristematic root cells, but only in the epidermal cells could radioactivity over Golgi dictyosomes be located. A similar result was obtained using the PASH method (Pickett-Heaps, 1968) with the Golgi dictyosomes of the inner meristematic cells of the root giving no positive reaction. Since the Golgi dictyosomes are, after the nucleus and mitochondrion,‘the most ubiquitous of organelles, it is of interest to compare the respective functions which have been ascribed to this organelle in animal and plant cells. Table I1 summarises the many different materials which are claimed to either originate in, or to be transported through, the Golgi apparatus. Protein, lipid and carbohydrate have been demonstrated in various combinations and proportions as secreted structural and non-rtructural substances. Nevertheless whether as non-structural or structural components, the Golgi secretions from plant cells appear t o be predominantly polysaccharide in nature in contrast t o those of animal cells which tend to be mostly protein. Most of the work in animal cell biology referred to in Table 11, has been carried out with the autoradiographic technique and the variety of results undoubtedly reflects the large number of animal cell types. On the other hand the application of pulsechase autoradiography to wall deposition in elongating cells in plants has not been carried out and the cytological evidence for the participation of the Golgi apparatus in wall formation falls heavily upon

TABLE I1 Overview of'the Various Secretory Functions of the Golgi Apparatus Cell or organ f y p e

Substance secreted

Reference

Non-structural components Animals:

Adrenal medullary cell Blood plasma cell Brunner's gland cell Intestinal goblet cell

I

Intestinal epithelial cell Liver cell Milk cell of the mammary gland Neurons Pancreatic acinar cell Thyroid follicle cell Plants:

Digestive gland cells from Dionea and Mercurialis Root hair cells Trapping-slinie from secretory cells of insectivorous plants Vacuolaria virescens Glaucocystis iiostochinearum Enferomorpha zoospores

Catecholomine I m munoglo b ulin (protein) Mucus substances Mucigen (enzyme and mucus substances) Chylomicra (lipid)

Kirshner and Kirshner (1971) Uhr (1970) Friend (1965) Neutra and Leblond (1966a and b) Friedman and Cardell ( 1 972)

Milk proteins, glycoproteins and lactose Acetylcholine Z y m o z e n (proteases, lipases, carbohydrases, nucleases) Thyroglobulin (Protein and polysaccharide) Enzymes

Keenan ef al. (1975) Smith ( 1 9 7 I ) Jamieson and Palade (1967a, b; 1968a, b) Whur e f a1 (1969) Schwab e f al. (1969); Figier (1969) Mom&e f al. (1967)

Slime (Glycoprotein? + polysaccharide) Polysaccharide

Schnepf (1 969)

Water

Schnepf and Koch (1966a, b)

Adhesive (glycoprotein)

Callow and Evans (1973)

Structural components Animals.

Plants:

Chondroblasts Cornea cells Odontoblasts Oocytes Spermatocytes Chrysophyceae Cells of higher plants and algae

Koilagen. ctiondroifin suiphate Keratin sulphate, hyaluroriir acid Protein of the Zona pellucida Glycolipids, glycoproteins and enzymes of the Acrosome Scales (some out of cellulose) Non-cellulosic wall materials

Weinstock and Leblond (1974) Meyer (1969) Beams and Kessel ( I 969) S u s i e t a l . (1971) Allen and Northcote (1975) See this review for references

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observations made with the root tip where the situation is often unclear due to the production of slime. It is now generally accepted that the synthesis of cellulose takes place at or near the plasmalemma. Even though precursors may be formed within the cell the assembly of the microfibril takes place at the cell surface. Electron microscopical evidence supporting this contention has been mainly obtained from algae, possessing large parallel oriented microfibrils. The first clear results came from the large filamentous green alga Chaetomorphu melugonium (Frei and Preston, 1961). Cells which were plasmolysed with mannitol before fixation showed on their innermost lamellae (after stripping of the lamellae and heavy metal shadowing) elongated groups of granular material. In an oft-published picture of this work (Preston, 1962, Fig. 16; 1964, Fig. 8; 1966, Fig. 18; Preston and Goodman, 1968, Fig. 10) one may see individual microfibrils apparently emanating from these areas. From such observations, and in recognition of the alga’s need to synthesize alternating lamellae of parallel arrays of microfibrils, Preston presented verbally in 1963 his now famous enzyme-particle-complex model for the synthesis of cellulose microfibrils (see Fig. 5). Only minutes later Frey-Wyssling communicated the first results obtained with the then newly developed, freeze-etch technique which supported Preston’s “hypothesomes” (see Preston, 1964 for this interesting exchange). These results were from yeast (Moor and Muhlethaler, 1963) and have been repeated many times in various laboratories (e.g. Fig. 6). Depicted at the plasmalemma, were areas of close-packed particles some of which showed clear attachment to the fibrils in the wall. Since that time the freeze-etch technique has been applied to numerous plant cells with only limited degrees of success as far as validating Preston’s hypothesomes. Barnett and Preston (1970) published very atypical freeze-etch pictures purporting to show stacks of organized particles at the surface of naked zoospores of the alga CZudophoru, A later investigation from the same laboratory (Robinson and Preston, 1971) could not confirni these observations but tentatively drew attention to short fibrillar bodies attached to some of the plasmalemma particles in zoospores of Chuetomorpha. A problem with zoospores of these two algae is that the first-formed wall layer consists of randomly oriented microfibrils (Nicolai, 1957) and the parallel orientation develops later as the zoospore settles and grows, thus a geometric relationship as suggested from Preston’s model may only be obtainable with later stages when regularly alternating lamellae are to be seen in the wall. Such stages have not been investigated. An alga which does not present these difficulties is Oocystis upiculafu (solitaria): from the beginning of wall development the microfibrils are oriented parallel to one another and a change in microfibril direction is immediately undertaken with the second lamella (see Figs 11 and 12). The wall consists of two sets of helically wound microfibnls and a helically wound arrangement of “granule bands”, reflecting one of the two microfibril directions, is also t o be seen at the plasmalemma (Robinson qnd Preston, 1972a) (Fig. 7).

6

5

7 Fig. 5. Preston’s model for the synthesis and orientation of cellulose microfibrils. Each sphere represents an enzyme complex approx. 50 nm diameter. Fig. 6. Freeze-fracture micrograph of the yeast plasmalemma. A paracrystalline array of particles, some attached to wall microfibrils are here characteristic. x45 000. Fig. 7. Freezeetch micrograph of the plasmalemma of Oocystis solifaria ( - a p i c u h z ) . In this organism particles (8.5 nm diameter) are arranged in bands (80- 100 nm wide; up to 5 Mm long) which, like the cell wall microfibrils, are wound helically on the cell surface. x33 000.

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Unfortunately in none of these cells have connections to microfibrils been seen and this is also true for higher plant cells. Long files of particles on the plasmalemma in root tip cells have been shown by Northcote and Lewis (1968) and Branton and Deamer (1972) but these seem to be an exception. Until recently, the only clear example for the attachment of microfibrils to plasmalemma particles has been yeast and here the microfibrils are not cellulosic but rather glucomannan. Staehelin and Pickett-Heaps (1975) have shown highly organized areas of particles and fibrils emanating from them, for the unicellular green alga Scenedesmus pannonicus. They prefer, however to interpret these fibrils as having more to do with the existence of a protective layer of sporopollenin than with cellulose biosynthesis. Using another Scenedesmus sp. (S. obliquus) I have also observed such particles, but on the basis of micrographs as in Fig. 8 I would prefer the interpretation that they do represent centres of fibril synthesis. Support for plasmalemma particles has also come from thin sectioning (Roland, 1967; Robards, 1969; Murmanis, 1971) but it is purely a matter of opinion whether these particles are the particles seen in freeze-etched prep-

Fig. 8. Freeze-fracture micrograph of Scenedesmus obliquus. Revealed are four daughter cells in a mother cell, two of which show 11-nm particles and microfibrils emanating from them. x27 500.

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arations. If plasmalemma particles are involved in the synthesis of cellulose then it is disturbing that they have been so infrequently and imperfectly demonstrated with the freeze-etch method. On the other hand the first successful in vitro synthesis of a chitin microfibril (Ruiz-Herrera et al., 1975) (see Fig. 30) has been achieved with a fraction containing small particles 35-100 nm diameter, which are in fact in the size range for Preston’s original “hypothesomes”. B. THE ORIENTATION OF CELLULOSE

The orientation of cellulose microfibrils within the cell wall may range from random to parallel. The primary wall at inception usually possesses randomly oriented microfibrils but when the cells begin to elongate, their innermost, newly formed, microfibnls tend to become more parallel, oriented at 90” to the growth axis (Roelofsen, 1965). Very parallel microfibrils, the directions of which alternate in different layers, are present in secondary walls but are not just a characteristic of non-elongating cells, for example the algae Cladophora and Chaetomorpha (Preston, 1974) which possess alternating lamellae of highly parallel microfibrils, still increase in length. Ever since the discovery by Ledbetter and Porter (1963) of microtubules immediately below the surface of the plasmalemma, the literature has become full of papers either supporting or contradicting this observation. As O’Brien (1972) points out, the situation as regards a similar orientation for‘cortical microtubules and microfibrils in the primary wall is particularly unclear. With secondary walls and especially primary xylem tracheary elements, the agreement between microfibril and microtubule orientation is consistent in many publications (Hepler and Newcomb, 1964; Cronshaw and Bouck, 1965; Esau et al., 1966; Cronshaw, 1967). Figure 9 shows diagrammatically this relationship. Another, more recent, example for agreement in orientation between microfibril and microtubule is in the lorica-forming alga Porteriochromonas stipitata (Schnepf et al., 1975) (Fig. 10). These authors claim-although in my opinion the micrographs are not tremendously clear-that over each microtubule lies a microfibril. Further evidence for a relationship between microfibril and microtubule orientation in these cells lies in the application of colchicine. This drug, which is known t o bind to the 6 s tubulin sub-units (Wilson, 1975) leading to the disassembly of microtubules, has been shown in numerous experiments on xylem secondary wall elements to create a “smeared” secondary wall with no thickenings, and of course no microtubules (F’ickett-Heaps, 1 9 6 7 ~Hepler ; and Fosket, 1971; Torrey et al., 1971). Similarly Schnepf et al. (1975) have shown that, in the absence of microtubules, a stunted lorica with more randomly oriented microfibrils is formed with Porteriochromonas. Together with I. Grimm and H. Sachs, such colchicine experiments have been recently repeated in my laboratory but with a much better experimental system, namely the alga Oocystis solitaria (Robinson et al., 1976b; Robinson and White, 1972; Sachs et al., 1977; Grimm et al., 1977). O’Brien in his 1972 review

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mentions that all previous work on microtubule/microfibril orientation is subject to the caveat that the direction of microfibril orientation is represented as electron dense fibrils in sections and this may not be the true direction. This critique and the further one that colchicine affects the gross distribution of the wall rather than microfibril orientation itself is also overcome with this alga.

9

10 Fig. 9. Diagrammatic representation of microfibril and microtubule orientations in xylem cells with secondary wall thickenings. (Pl-plasmalemma; PW-primary wall; SWsecondary wall; T-tonoplast; Mf-microfibrils; Mt-microtubules). (Courtesy H. Sachs.) Fig. 10. Cell and lorica of Porteriochromonas stipitata after negative staining. Thin microfibriIs (approx. 3.5 nm diameter) lie over microtubules both of which are helically arranged around the cell axis x30 000. (Courtesy of W. Herth and E. Schnepf.)

As shown in Figs 11 and 12 the directions of the two microfibril orientations are without any shadow of a doubt clearly recognizable. In a mature wall of -1 pm thick, the microfibrils bear a superficial resemblance to microtubules in their staining, revealing a central unstained core (the cellulose crystallite of about 7 nm diameter, see above). Microtubules subjacent to the plasmalemma are to be seen before the wall is developed in naked autospores, during the development of the amorphous “outer layer”, a feature of many green algae and possibly containing sporopollenin (Atkinson et al., 1972), and during the development of the cellulose wall itself. When microtubules are transversely sectioned in the latter phase, the first layer of microfibrils on the outer surface of the plasmalemma are also transversely sectioned. The number of microfib-rils per microtubule in such sections is usually 2-3. These cells divide into 8, 16 or 32 autospores and have a developmental cycle

PLANT CELL WALL SYNTHESIS

107

11

12A

126

1X

12D Figs 11 and 12. Normal cells of Oocystis solifunu. Fig. 11 depicts a tangential section through the cell wall revealing the typical crossed microfibrillar structure. x19 500. Fig. 12 A, B, C and D show the presence of microtubules subjacent to the plasmalemma at all stages in wall development. A: before wall formation (x56 000); B: during formation of the “outer layer” (x40 000); C & D: during formation of the wall itself (C x60 000; D x75 000).

13

14 Figs 13 and 14. Cells of Oocystis solifaria after treatment with 10-2 M colchicine. Fig. 13. Tangential section of the cell wall. x17 000. Fig. 14.Transverse section of the cell wall. Note the lack of orientation alternation for the cellulose microfibrils. x44 000.

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of at least 10 days so that a synchronous culture has not been achieved. However with continuous inspection, cultures having at least 80% mother cells can be obtained. When such cultures are supplied with colchicine* some of the newly formed autospores die but a large proportion continue to grow and, in so doing, do not develop the typical ellipsoid shape for normal cells. Sections of vegetative cells (Figs 13 and 14) reveal a startling effect. Instead of complete random orientation as is to be expected from all previous results, we see that the microfibrils still develop parallel t o one another, but the regular alternating pattern of microfibril layers, typical of normal cells, is lost. Within any one

Fig. 15. Section through the cell wall of Oocystis solitaria after recovery from colchicine treatment. The outer microfibril layers show the typical lack of alternating orientation for colchicined cells, and the inner, developed after the colchicine has been washed out, show the normal microfibril orientation once again. x77 000.

section all microfibrils are not transversely sectioned, large sectors of the wall also being occupied with many longitudinally sectioned microfibrils. It is at this rrlbment difficult to give a description of the total wall archtecture after such treatment. Cells treated with colchicine for 24 h and then frequently washed with fresh growth medium over a period of 48 h show a recovery and the normal wall pattern, is again obtained. Figure 15 shows a mixed wall with normal and colchicine-induced microfibril orientations from a recovered cell.

* The concentration used here was 10-2 M. This is a very high concentration compared to the required amounts for microtubule disorganization in animal cells (10-6-10-7 M; Borisy and Taylor, 1967) but within the range already used for algae (Wanka, 1968; Marchant and Pickett-Heaps, 1974; Schnepf et al., 1975) and higher plants (Pickett-Heaps, 1967~).

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In all previous experiments with colchicine there has never been any estimate of how much cellulose synthesis has been affected, although the impression gained is always that synthesis is not affected. In Oocystis the same proportion of cellulose, 4876, (expressed as total cell carbohydrate) has been observed for normal and colchicine treated cultures. These observations, give considerable support to the participation of microtubules in microfibril orientation and also provide an excellent parallel to the studies of Green (1963), who, before the discovery of microtubules, was able to demonstrate a reversible change in the wall texture of NitelZa upon colchicine addition and removal. Preston (1974), when discussing microtubules and microfibrils, ignores experiments with colchicine. In a recent paper (Nelmes et el., 1973), contend that the orientation of microfibrils through microtubules “. . . has now been generally abandoned in favor of the ordered-granule hypothesis. . . .” as put forward by (Preston, 1964). In the light of the present results this would seem to have been a premature conclusion. Preston’s original cellulose biosynthesis model (see last section) was created out of the need to present a unified concept for the synthesis, parallelity and change of direction for microfibrils. However it is quite clear, even from Preston’s earlier experiments with plasmolysis (Frei and Preston, 1961), that when the plasmalemma is disturbed, parallel orientation is lost but microfibril synthesis nevertheless continues. Preston’s model certainly explains how many of the structural features, e.g. interweaving microfibrils from one layer to the next, may be accomplished, but gives no indication of how such synthetic complexes may be organized or controlled by the cell. Since it is now recognized that microtubules can not only control cell form, e.g. in Ochromonas (Bouck and Brown, 1973; Brown and Bouck, 1973) and in the colony-building algae Hydrodictyon and Pediastmm (Marchant and Pickett-Heaps, 1974), but also may influence the plasmalemma topography in animal cells (Oliver, 1975 ; Shields, 1975), they are clearly one of the best candidates for the control of microfibril orientation. In any event microtubule control of microfibril orientation is not irreconcilable with Preston’s model as has already been suggested by Heath (1974). With respect to the control of orientation through microtubules there are three prominent questions t o be answered: 1. How d o the microtubules transfer their influence to the plasmalemma?

Although evidence for the existence of direct positively stained connections between microtubules is seldom seen (see for example Franke, 1971; Allen, 1975; Schnepf et al., 1975), the oft forgotten original observation of Ledbetter and Porter (1963) that each positively stained microtubule is surrounded by a “zone or layer of material from which all major particulate or membranous elements of the cytoplasm are excluded” (Ledbetter and Porter, 1970, p. 28) is

PLANT CELL WALL SYNTHESIS

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an indication that contact may exist despite no visibly stained bridges. That a non-staining outer zone of the microtubules, at least 8 nm thick does exist has recently been shown by Behnke (1975) using cationic stains w h c h suggest the presence of glycoprotein. Inspection of my own and many published micrographs of cortical microtubules does leave one the impression that there is a non-staining material between the microtubules and the plasmalemma. 2. If microtubules are always running in one direction how can they induce changes in microfibril direction through YO"? One has here two choices: either to accept that it is so, which makes the synthesis-orientation mechanism rather unusual or to postulate that prior to each microfibril direction there is a change in microtubule direction as well. With the current knowledge of the assembly and disassembly of microtubules (Soifer, 1975) this is not too unreasonable a postulate, particularly considering that for example in Oocystis at the present we have occasionally found longitudinally sectioned microfibrils directly opposite transversely sectioned microtubules.

3. How does one explain the lack of coincidence between microtubule and microfibril direction in so many cells? Schnepf (1976) points out that those examples where no coincidence between microfibril and microtubule direction exists are always from elongating cells. Now if Roelofsen's multinet hypothesis (Roelofsen and Houwink, 1953; Roelofsen, 1965), whereby the initial transverse microfibril orientation with respect t o the major cell axis is passively altered to a longitudinal direction through the wall, is applicable to such cells then the great majority of microfibrils will no longer coincide in orientation with the microtubules. Ledbetter and Porter (1 963) demonstrated a transverse microtubule direction in cylindrical root cells and Green (1963) a similar transverse orientation for the first formed microfibrils in Nitella, albeit by indirect shadowing methods. If there is a passive reorientation of microfibrils in such cells, and if the microfibrils in the walls are so difficult to demonstrate in section as O'Brien (1972) suggests, then one must be careful in assuming that a coincidence in microfibril/ microtubule does not exist.

IV. CELL FRACTIONATION STUDIES A. PREPARATION OF CELL FRACTIONS AND THEIR IDENTIFICATION

In order to make the electron microscopical evidence for the participation of certain cell organelles in cell wall synthesis less circumstantial and more convincing, one must obtain enriched fractions of these organelles, which

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invariably involves some form of density gradient centrifugation. An important problem in density gradient analysis of cell fractions is the identification of the various vesicle-containing bands. When homogenized, the plasmalemma, endoplasmic reticulnm (rough and smooth) and tonoplast are all broken down into smaller pieces which, in the homogenate, tend spontaneously to form small vesicles. These “artificial” vesicles therefore exist side by side in the homogenate with the naturally occurring secretory vesicles. The correct allocation of radioactive precursor incorporation to one vesicle type and not to another is of profound importance, since it can determine whether or not the endoplasmic reticulum is the initial site for the formation of wall polymers, as it is in the animal cell for the formation of proteins and glycoproteins. The earliest attempts at isolation of plant Golgi bodies (Morre‘ and Mollenhauer, 1964; MorrB et al., 1965) established two prerequisites for good isolation, namely the use of homogenization methods with minimum shear and the maintenance (stabilization) of the cisternal stack by the inclusion of glutaraldehyde in the homogenization medium. Gentle of homogenization has also been emphasized in the work of Ray et al. (1969) who have shown that the distribution profile of Golgi-bound 0-glucan synthetase in density gradients is considerably altered when harsh mortar-grinding, instead of hand-chopping with a razor blade, is used. Similar gentleness may be obtained with a mortar and pestle but the grinding periods must be very short (<30 sec (Robinson and Ray, 1977; Brett and Northcote, 1975)). Discontinuous density gradient centrifugation for the isolation of plant Golgi bodies was also introduced by Morre et al. (1965) but despite the warning of de Duve (1971) that “it is a very dangerous procedure, in that it creates the illusion of a clear-cut separation”, it has been adopted by other workers (Mertz and Nordin, 197 1 ; Bowles and Northcote, 1972,1974; Wright and Bowles, 1974; Brett and Northcote, 1975). Such a centrifugation method may produce a relatively clean Golgi preparation, but the vesicle fractions are arbitrarily separated. For example, Bowles and Northcote (1972) using maize roots separate a “mixosomal” fraction from a “mitochondria1 and smooth membrane” fraction yet both fractions, as EM pictures illustrate, contain rough endoplasmic reticulum and smooth vesicles to various degrees. In a later paper (Bowles and Northcote, 1974) these two fractions change their designaticn and are referred to “rough endoplasmic reticulum” and “smooth membrane” fractions. This is particularly disturbing when Morr6 (1970), who used the same technique, (albeit with onion stems) reports his microsomal fraction to be “heterogeneous and contained smooth vesicles, fragments of endoplasmic reticulum and ribosomes”. Only with continuous linear density gradients is it possible to distinguish the various vesicle types from one another and the dictyosomes, but even this cannot be done with one simple isopycnic centrifugation. Since such a centrifugation involves the separation of particles according to density, then the separation of secretory vesicles, whose density (particularly those which have just been released from the cisternae) is probably the same as that of the Golgi

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PLANT CELL WALL SYNTHESIS

dictyosomes, is not possible. When intact dictyosome stacks, through the use of glutaraldehyde, are present in the homogenate, it may be possible to separate dictyosomes from all vesicles, including secretory vesicles, on the basis of their size and mass, i.e. on the basis of their sedimentation coefficient. Such particle separations (rate-zonal centrifugation) have been known for many years (Anderson, 1955) but have never gained popularity in plant biochemistry. A combination of rate-zonal and isopycnic-zonal centrifugations for non-glutaraldehyde stabilized Pisum epicotyl tissue has already been introduced by Ray et a1 (1969) and this has now been successfully adapted for glutaraldehyde-stabilized homogenates from which Golgi bodies and all vesicle types may be delineated from one another (Ray et al., 1976; Robinson et al., 1976). The principle of this method is the subjection of a glutaraldehyde-stabilized homogeaate t o an initial rate-zonal centrifugation of short duration and low power. Figure 16 shows comparatively the results obtained when no glutaraldehyde is present in the homogenization medium. From in vivo labelled tissue.

I

50

I

A

Distance sedimented lmmt

m

I

6

Fig. 16. Effect of the presence of glutaraldehyde in the homogenizing medium on the rate zonal sedimentation characteristics of pea epicotyl organelles. Compared are the distributions of (i) in vivo non lipid incorporation from 3H-glucose ( 0 - 0 , dpm x 10-3); (ii)in vitro UDPG-glucan synthetase ( 0 - 0 , dpm x 10-2); (iii)in vitro cytochrome C-oxidase (A-AAEE, 550 nm min-1). @ without glutaraldehyde; @ with 0.3% glutaraldehyde in the homogenization medium. (N.B. For details of assay conditions see Ray et al. (19761.)

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DAVID C. ROBINSON

essentially two peaks are to be found in the gradient, the lowermost corresponding to a sharp peak for in vitro measured p-glucan synthetase activity. If the portions of the gradient containing these two peaks are then transferred to separate linear sucrose gradients and centrifuged until isopycnic conditions are reached, the peaks for both in vivo incorporation and 0-glucan synthetase activity are seen to occur at the same density ( - 1 . 1 4 g ~ m - ~ ) . Electron microscopy of these fractions is depicted in Figs 17 and 18. The lowermost of the incorporation/synthetase peaks in the rate-zonal separation (“fraction 11”) is without doubt a Golgi-dictyosonie-enriched fraction with a high degree of purity and structural integrity. The uppermost fraction (“fraction I”) is a heterogeneous fraction of vesicles equivalent to a conventional microsomal fraction of differential centrifugation; it contains both smooth vesicles and vesicles bearing ribosomes. The pellet, which is always found in the rate-zonal separations, is also heterogeneous and contains principally mitochondria and etioplasts together with nuclei and some dictyosome aggregates. Good peak resolution in such rate-zonal separations is dependent upon the type of homogenization and the amount of glutaraldehyde present in the homogenization medium as well as centrifugation conditions. For a given pulse of radioactive glucose the size of the Golgi peak may be lowered or increased depending on whether mortar-grinding or hand-chopping with a razor blade are used. The increased peak height of the vesicle fraction in relation to that of the Golgi fraction upon mortar-grinding is undoubtedly due to the disruption of some dictyosomes which, as smaller elements, will travel slower under these separation conditions. Best peak resolution in the Pisum epicotyl system was always obtained with 0.5% glutaraldehyde in the homogenization medium. Greater concentrations of glutaraldehyde produce clumping and a concomitant increase in the size of the rate-zonal pellet. Concentrations of 0.3% though still allowing good separation of dictyosomes from vesicles, also permits a significant amount of enzyme activity to be retained. Seemingly unaffected by the presence or absence of glutaraldehyde, is the position of mitochondria as denoted by the distribution of cytochrome C-oxidase in Fig. 16. The question as to which organelle(s) is responsible for bearing the radioactivity in the vesicle fraction has also been elucidated by Ray etal. (1976). This involved the comparison of the dktribution of marker enzymes and compounds in the vesicle fraction prepared from homogenates in the presence of high (3 mM) or low (0.1 mM) magnesium concentrations. It has been known for some time that the retention of ribosomes on rough endoplasmic reticulum (ER) in fractionation experiments is magnesium-dependent (Sabatini et al., 1966; Lord Fig. 17. Section through the vesicle fraction (“Fraction I”) of pea epicotyl homogenates as prepared after the Ray et al. (1976) method. x12 000. Fig. 18. Section through the dictyosome fraction (“Fraction 11’’). Intact Gola dictyosome stacks are predominant in this fraction. x18 000.

PLANT CELL WALL SYNTHESIS

7%

115

116

DAVID G. ROBINSON

et al., 1973; Adelman et al., 1973). At high Mg2+ concentrations ribosomes tend to remain on the ER thereby providing a higher isopycnic density than ribosome free ER. The results of such an experiment are shown in Fig. 19A and B. As expected the distribution of the ER marker enzyme NADH-cytochrome C-reductase (Lord et al., 1973; Beaufay et al., 1974) moves from a density of -1.10 g cm-3 to -1.17 g cm-3 in high Mg2+ gradients. Similarly the RNA content (i.e. ribosome position) instead of being distributed throughout the gradient also moves to a peak in the region of 1.17 g cmP3. This effect is to be seen with homogenates, with or without glutaraldehyde but is more

1.1 0

1.1 L

1.1 8

Density g . c m -3 Fig. 19. Effect of MgZ+ concentration on the isopycnic distribution of vesicle fraction parameters. The distributions of (i) in vivo non-lipid incorporation from 3H-glucose ( 0 - 0 , dpm x 10-3); (ii) specific NPA binding ( 0 - 0 ) ; (iii) RNA (X----X, mg ml-l x 1.2); (iv)NADH: cytochrome C-reductase (A----A, AE 550 nm min-1) are compared for vesicle fractions of pea epicotyls from homogenates made in the presence of 0.1 mM MgC12 (@) or 3 mM MgCl, (@). This effect of Mg2+ on the distribution of the ER is to be seen for homogenates f glutaraldehyde, but is most obvious in the absence of glutaraldehyde (from Ray et al. (1976).

PLANT CELL WALL SYNTHESIS

117

marked in the absence of the fixative. Although the plasmalemma, as indicated by the position of the naphthylphthalamic acid (NPA) marker (tembi er aZ., 1971; Thom e t al., 1976) moves a little to higher densities with a higher MgZf concentration, no effect whatsoever is observed on the distribution of the component responsible for carrying the radioactivity in the vesicle fraction. Since the peak for radioactivity is clearly not rough ER or plasmalemma it must, by deduction, refer t o secretory vesicles (smooth ER being almost non-existent in this tissue and tonoplast vesicles being unlikely candidates). ‘Thus, despite the presence of other vesicles, Ray et al. (1976) have felt it justifiable to designate this heterogeneous fraction as a secretory vesicle fraction. That the procedures devised by Ray e t al. (1969, 1976) for dictyosome isolation are adaptable to systems other than Pisum epicotyl tissue, is seen in the recent work of Gardiner and Chrispeels (1975). Using carrot phloem parenchyma tissue, these authors have reversed the procedure described above, using an initial isopycnic centrifugation followed by a rate-zonal separation, and have also achieved a relatively pure Golgi-dictyosome fraction. Corroborative evidence for the movement of the ER vesicles in response to a change in Mg2+ concentration for Pisum epicotyls comes from Shore and MacLachlan (1975) who have also shown no effect on 0-glucan synthetase distributions. However some inconsistencies with the results of Ray e t al. (1976) are to be seen. In the isopycnic gradients of Shore and MacLachlan pea epicotyl plasmalemma bands at 1.13 g c n P 3 . This is based upon the PTA-chromic acid stain (Roland et al., 1972) whose specificity is now questionable since “mature” secretory vesicles have also been shown to stain with this reagent (Frantz e t al., 1973). Furthermore the available literature on plant cell plasmalemma isolation (Hardin e t al., 1972: Hodges e t al., 1972; Shiberi e t al., 1973; Strobe1 and Hess, 1974; Patni et al., 1974; Van Der Woude e t al., 1974; Leonard and Van Der Shore and Woude, 1976) provides values between 1.16 and 1.18 MacLachlan do in fact show a large peak for 0-glucan synthetase activity a t a density of 1 .I8 g ~ r n - from ~ 2-day-old decapitated epicotyls but only coniniented that this fraction was mainly composed t o mitochondria. Since this synthetase activity was obtained a t high UDPG coricentrations it would seem possible that it reflects the position of the plasmalemma as has been shown by Van Der Woude e t al. (1974) for onion stem. Particularly interesting in Shore and Macbchlan’s results is that they have observed a large increase in P-glucan synthetase activity after the epicotyl segments had been treated with auxin. The peak for this activity coincided with that of NADH-cytochrome C-reductase but did not move upon changes in magnesium concentration as did the latter, indicating its location in a “smooth ER vesicle”. Unfortunately, the density of this component at 1 .lo-1.1 1 g cmP3 is much lower than that found b y Ray et al. for the corresponding vesicle fraction P-glucan synthetase in their experiments. In searching for an explanation for this difference, attention should be drawn to the fact that whereas Ray et al. used an

118

DAVID G. ROBINSON

overlay of 16% sucrose on a 20-42.5% linear sucrose gradient (16 ml) for their isopycnic zonal separations, Shore and MacLachlan used a 12% sucrose overlay on a 25-55% (10 ml) linear gradient. Thus for the same amount of material (from 50-100 epicotyl segments) not only are the gradients of Shore and MacLachlan more heavily loaded but they approach discontinuous conditions which are known to produce artefacts (Anderson, 1955). These doubts are confirmed by Paul1 and Jones (1976) with Zea root tips. They have shown that when a homogenate of Zea root tips in 17% sucrose, is added to a linear gradient of 30-50% sucrose, a very sharp peak of phosphorylcholine transferase, an ER marker (Lord et al., 1973), is obtained at the boundary of the overlay and the gradient, i.e. at a density of 1.13 g c m P 3 . Material incubated with 3H-fucose also provided a sharp peak at this density under these centrifugation conditions and the usual conclusion would be that ER incorporates fucose. When, however, the homogenate is added to a linear 20-50% gradient, the boundary conditions no longer existed and, instead of two coincidental peaks at 1.13 g cm-3, several non-coincidental peaks for both the ER marker enzyme and the incorporated fucose label were to be found between densities of 1.12 and 1.25 g cmP3. B. ANALYSIS OF FRACTIONS FROM PULSE-CHASE EXPERIMENTS

The purpose of this section is essentially an attempt at answering two questions: 1. In which organelles are cell wall precursors synthesized? 2. To what extent d o the precursors resemble cell wall components? 1. Cell Wall Proteins Two observations suggest that the hydroxyproline of the cell wall protein is formed post-translationally. Firstly, it is well recognized that a pool of hydroxyproline residues does not exist in plant cells (Pollard and Steward, 1959) and secondly ribosome-associated polypeptides isolated from carrot tissue after a 7 min exposure to 4C-proline contain n o radioactive hydroxyproline (Sadava and Chrispeels, 1970). Since such experiments have usually involved conditions in which ribosomes are not tightly bound to the ER, i.e. low Mg2+ concentrations in the homogenizing medium, it is not possible to say how much of the synthesis of the hydroxyproline-containingpolypeptide, occurs in free ribosomes and how much in ER-attached ribosomes. Using the fractionation technique of MorrC et al. (1967), Dashek (1970) who presented I4C-proline for 15 min to sycamore callus cells, showed radioactive hydroxyproiine to be present in three fractions: a fraction rich in Golgi dictyosomes and smooth membranes, a microsomal fraction containing both rough ER and smooth membrane components and a ribosomal fraction which also contained some rough ER. More recently Gardiner and Chrispeels (1975) have been able to show more conclusively that for carrot root discs pulsed for

PLANT CELL WALL SYNTHESIS

119

20 min with 14C-proline, most of the radioactive hydroxyproline resides in the dictyosome fraction. Not commented upon by these authors, is that a substantial shoulder to this peak exists. This lies higher in the rate zonal gradient and is also in the same position as the peak for the ER marker enzyme NADH-cytochrome C-reductase. However, since this shoulder lies in a mixed vesicle fraction, and the analysis of such a fraction as described for Pisum epicotyl tissue by Ray et al. (1976) has not been carried out, it is not possible to say whether the hydroxyproline is carried in ER vesicles or in secretory vesicles. Confirming the involvement of the Golgi in synthesis of cell wall protein is the further observation of Gardiner and Chrispeels that coincident with the dictyosome hydroxyproline incorporation peak, is a peak for the activity of UDP-arabinose arabinosyl transferase. Since Brysk and Chrispeels (1 972) have already shown that a particle-bound high molecular weight glycoprotein (MW 200 000; bearing hydroxyproline and serine which can be extracted from a 1000-48 000 g differential centrifugation fraction from carrot tissue), contains arabinose as the principal sugar, it is likely that the arabinose residues are attached to the cell wall glycoprotein within the Golgi apparatus. Since this glycoprotein does not contain galactose, it has been suggested by Sadava and Chrispeels (1973) that the serine-o-galactose linkage, i.e. and connection to the matrix polysaccharides, is formed within the cell wall itself.

2. Polysaccharides Work over the last 5 years with enriched cell fractions has confirmed the polysaccharide-bearing nature of the Golgi dictyosomes. First to demonstrate this from in vivo labelled material were Harris and Northcote (1971). After feeding pea roots ''C-glucose for 40 min an extracted dictyosome fraction upon acid hydrolysis yielded over 80% non-glucose residues with galactose, galacturonic acid and arabinose as principal labelled sugars. Bowles and Northcote (1972) have also shown for Zea mays roots incubated for 2 h in ''C-glucose that less than 10% of the radioactive sugar residues from a dictyosome fraction hydrolysate are glucose (Table 111). With this tissue, however, one has the complication of the production of large quantities of extracellular non-cell-wall polysaccharides (MorrC et al. , 1967) whose principal constituents are galactose and fucose (Harris and Northcote, 1970). Although both Harris and Northcote (1970) and Paull and Jones (1975) showed that only the cap area of the Zea root is responsible for the production of the fucose-rich extracellular slime, these two reports differ on whether fucose-containing polysaccharides exist outside of this region. Whereas Harris and Northcote claim that no such polymers exist, Paull and Jones show that fucose-containing polysaccharides are present in the cell walls of root cap cells (representing about 18% of the total, i.e. slime plus wall, secreted fucose polysaccharides) and in cells higher along the root. Furthermore the wall

TABLE 111 % S u g a r Radiocomposition of Cell and Cell Wall Fractions after In Vivo Labelling with 14C-Glucose A. Zea ma,ys root tissue; a: root cap tissue; b: older tissue’ Microsomes Smooth membranes + Mitochondria a b a b

Galacturonic acid Glucuronic acid Galactose Glucose Mannose Arabinosc Xylose

Fucose Ribose + Khamnose

11.2 1.6 31.4 7.3 5.0 22.4 16.4 4.5

5.2 1.2 21.2 8.5 3.4 20.4 38.8 1.1 -

.-

10.2 5.6 26.0 7.3

8.6 4.6 37.4 6.4 5.3 16.3 13.4 8.1

-

14.7 32.8 2.8

-

Golgi

Cell wall

Slime

a

b

a

b

11.6 4.7 34.1 6.8 1.8 14.7 18.5 6.2 1.7

10.3 1.6 21.5 3.95 1 .o 19.8 41.4 0.2

7.8 1.3 17.4 38.3 2.5 12.5 13.5 4.9 1.2

2.7 0.8 6.9 62.5 1.8 8.8 15.6 0.4 0.7

-

10.1 1.8 23.9 4.0 2.1 13.8 15.0 28.3

B. Pirum sativum epicotyl tissue2

Galacturonic acid Galactose Glucose Mannose Arabinose Xylosc a 1 rom

Vesicles

Golgi

Cell wall non-cellulosic polysaccarides

18.6 28.33 30.12 4.07 12.62 6.02

16.0 24.53 22.68 8.82 19.15 8.65

22.6 39.94 18.19 0.23 12.15 6.89

Bowlcs and Northcote (1972) (material pulsed for 2 h). (1976) (cell organellc values are after 5 min pulse and 10 min chase; cell wall values after a 5 min pulse and 40 min chase).

b From Robincon ef al.

-

PLANT CELL WALL SYNTHESIS

121

fraction containing the water-soluble fucose polysaccharides after incubations in H-fucose was the hemicellulose fraction, thus eliminating the possibility of slime contamination in these experiments. Bowles and Northcote (1974) analysed the composition of the cell wall and cell organelles after varying incubation periods in ‘‘C-glucose and noted that radiocomposition values were constant after 30-min incubation. Although not specifically mentioned from which cells the fractionations were made, a good agreement exists between the root cap cell wall values of the Bowles and Northcote (1972) and the “cell wall” values of the Bowles and Northcote (1974) papers. On the other hand a comparison of dictyosome and microsome fractions as reported in these two papers, indicates that the material used in the 1974 paper is from either whole roots or older root tissue. Such discrepancies are unfortunate and the 1974 paper of Bowles and Northcote cannot therefore be used for a radiocomposition comparison between organelles and cell wall. Lookkg at the data of Bowles and Northcote one sees an agreement in radiocomposition values between the cell organelles and the extracellular slime for all sugars except fucose, which is present in fourfold higher concentration in the slime. Since these were apparently equilibrium conditions and the actual amount of fucose incorporated into the cell wall is considerably smaller than that in the slime (c. 6000 cpm versus 43 000 cpm-my calculations) one must postulate an non-organelle source for this additional fucose incorporation. A similar conclusion has also been reached by Robinson ef al. (1976) to account for the discrepancy between levels of galactose in the isolated cell organelles and that in the non-cellulosic cell wall polysaccharides in Pea epicotyl tissue (Table 111). Also interesting in this system is the substantial amount of mannose (12%) in dictyosome hydrolysates which is not to be found in the matrix polysaccharides, but in the a-cellulose fraction (up to 670). Thus a simple comparison of radiocomposition between cell wall fractions and cell organelles can be somewhat misleading, although it is clear that polysaccharides are contained in dictyosomes and other cell organelles. When sugars are detected in a hydrolysate it is usually accepted that a polysaccharide was previously present, which of course is not necessarily true. Outside of hydrolysis two further lines of evidence prove conclusively that dictyo, somes and certain other organelles bear cell wall polysaccharides. The first of these involves the examination of solubilized organelle fractions by gel filtration. Jilka et al. (1972) have demonstrated that all cell fractions obtained from discontinuous sucrose gradients by the technique of Morri et al. (1965) contain high molecular weight material. Both a hot 0.05 M KOH extract treated with pronase and a hemicellulase-pectinase extract from the dictyosomes release a significant proportion of polysaccharide eluting in the void volume of Sephadex (3-75. A similar distribution for hot water-soluble material has also been observed in the pea epicotyl system with Sephadex G-100 (Robinson, unpublished observations).

122

DAVID G. ROBINSON

Better than Sephadex for gel chromatography of polysaccharides are the Bio-gels and they have recently been used by Bowles and Northcote (1976) for an examination of the polysaccharides in the roots of Zea. These authors have found water-soluble polysaccharides with a MW greater than 40 000 in both “dictyosome” and “rough E R ’ fractions. Water-insoluble material (20% and 70% of total incorporated radioactivity for dictyosomes and rough ER respectively) could be rendered soluble with pronase and produced fucose-containing polymers having a MW greater than 4000 from the dictyosome and rough ER fractions and smaller non-fucose-containing polymers from the rough ER fraction. The second line of evidence involves the application of polysaccharidases on the various cell organelle and cell wall fractions. With the pea epicotyl system

Fig. 20. Radiochromatographic scans of p-1,2 glucanase hydrolysates of cell wall pectin (EDTA wall extract) and cell organelle fractions (dictyosomes and vesicles) from pea epicotyl segments. Segments (usually 75 per sample) were incubated in approx. 100 pCi U-14C-glucose for 1 h , and then fractionated. Fractions were incubated with the enzyme (1 mg/ml) a t pH 4,5 for 5-6h and the soluble material then chromatographed against known oligosaccharide standards (adapted from Ray et al., 1976).

PLANT CELL WALL SYNTHESIS

123

Ray et al. (1976) have used the following endo-glucanases: p-1,2 glucanase from Penicillium verruculosum; /3-1,3 glucanase from Rhizopus arrhizus; and 0-1,4 glucanase from Streptomyces QMB814 sp. From each, a separate and distinctive partial hydrolysis pattern was obtained (Figs 20, 21, 22) which is similar for all cell organelle fractions. For p-1,2 and p-1,3 glucanases these characteristic patterns are also t o be seen for the cell wall non-cellulosic polysaccharides (EDTA cell wall extract). When the individual oligosaccharides from Clabelled organelle and 3H-cell wall digests are mixed they run together, further indicating their similarity. Although these enzyme preparations are supposed to be specific (Reese and Mandels, 1959, 1963; Reese et al., 1959, 1961) the oligosaccharides and monosaccharides released by these enzymes were certainly not restricted to glucose. For example the disaccharide in the p-l,2 glucanase digests contained xylose and glucose and the trisaccharide typical of p-1,3 digests yielded glucose, galactose, xylose and arabinose upon acid hydrolysis indicating the presence of more than one oligosaccharide in this peak. Important from the point of view of the detection of intracellular cellulosic precursors, is the action of &1,4 glucanase (cellulase). Unlike Shore and

Fig. 21. As for Fig. 20 but with p-1,3 glucanase.

124

DAVID G. ROBINSON

MacLachlan (1975) small amounts of a glucose disaccharide running in the same position as cellobiose were obtained by Ray et al. (1976) for both dictyosome and vesicle fractions. 4C-labelled samples of this disaccharide mixed with -> _ IQOOO d Dm-

+,

0 ~

3

\

i*_

+_

-~ L,

i

r

CB

.

LZ

GLC

PLANT CELL WALL SYNTHESIS

125

3H-samples of cell wall cellobiose again run in the same position upon rechromatography. The fact that a similar small cellobiose peak from the non-cellulosic portion of the wall may also be detected, together with the in vitro xyloglucan synthesis discussed above, page 96) means that the cellobiose released from the organelles cannot be unequivocally regarded as a precursor of cellulose. Due t o the different action of cellulase on the total cell wall as compared with purified cellulose (Fig. 22) Ray et al. investigated the possibility that organelle values for cellobiose were lowered by the presence of large amounts of non-cellulosic polysacchaddes. However organelles pretreated with 0.1 N H2S04 and the acid extracts themselves did not produce higher levels of cellobiose upon cellulase digestion. The likelihood that cellulosic precursors are to be found in these organelles is thereby further reduced. Engels (1974) and Engels and Kreger (1974) have claimed the presence of cellulose in vesicles isolated by discontinuous gradients from Petunia hybn’da pollen tubes. Although the presence of polysaccharide in pollen tube vesicles has been previously demonstrated (Van Der Woude e t al., 1974) this is the first time that X-ray diffraction has been applied to relatively pure organelle fractions. Alkali-resistant cell wall and vesicle fractions which yield principally glucose on hydrolysis produce a similar X-ray diagram. This diagram however only resembles that of the cotton cellulose standard and the innermost diffraction rings of cellulose (6.01 and 5.35 are completely absent. Upon treatment with 20% NaOH the cellulose of cotton changes to the crystalline I1 form and the diagrams of the Petunia vesicle and wall also change in a similar way. Engels (1974) notes that since no fibrillar material was detected in the vesicles by electron microscopy, the cellulose was “not to be detected in the vesicles in the form of a crystalline structure”. Apart from the questionable nature of his X-ray diagrams, Engels’ interpretation is considerably weakened by the report from Herth et al. (1974) that the cell wall of Lilium Longiflorurn pollen tubes contains only small amounts of cellulose, but large amounts alkali-insoluble 0-1,3 glucan.

a

a)

C. TRANSPORT OF SYNTHESIZED MATERIALS

1. Kinetics In the preceding section we have considered the evidence that the Golgi bodies and fractions containing rough ER, contain protein and polysaccharides to a certain extent resembling those found in the cell wall. We may now ask a further two questions: 1. Where does the synthesis first take place, in the ER or in the Golgi dict yo som es? 2. At what rate are the synthesized materials transported to the cell wall?

To determine whether the initial steps in cell wall synthesis are carried out in the ER or in dictyosomes we have, unfortunately, only two sources of

126

DAVID G . ROBINSON

information: the work of Bowles and Northcote (1974, 1976) on Zea roots and that of Robinson et al. (1976) on pea epicotyl tissue. In a time-course of incorporation study Bowles and Northcote (1974) have shown that after 2 min incubation in 14C-glucose only the fraction containing rough ER and the cell wall are labelled. After a 5-min incubation the dictyosomes become labelled but have here, and for all subsequent pulse periods, always less than 10% of the radioactivity in the ER fraction. Since the amount and composition of the radioactivity in the cell fractions was seen to be constant after 30 min incubation in 14C-glucose, these authors considered that the increase in radioactivity in the cell wall in a further 15-min 4C-glucose incubation, represented the rate of incorporation of exported materials from the organelles within the cell. From this radioactivity increase a value (in pmole/min) was calculated for the rate of addition of materials to the cell wall. Similarly from the saturation levels of radioactivity in the cell organelles, actual values for their sugar composition were obtained. From the composition of the extracellular slime and the assumption that fucose-containing polysaccharides do not occur elsewhere, Bowles and Northcote then determined what proportions of the exported material were slime and true wall material. Corresponding values for the slime and non-slime components in the cell organelles were also calculated. They reached the conclusion that, in order to explain the calculated rates of increase, the slime components would have to pass through the dictyosomes in 20 s and that if this material was replenished by the ER then the ER content would turn over every 7 min. In contrast to the values for slime transport the passage of cell wall materials through the dictyosomes is 2.5 min. To explain this difference the authors allude t o certain cases where dictyosomes possessing different functions may co-exist in the cell; alternatively different cisternae in a dictyosome may carry different products (Dauwalder and Whaley, 1973; Fraser and Gunning, 1973). Lending support to their contention that the ER is the initial site of polysaccharide synthesis are the results of Bowles and Northcote (1976) where a large proportion of non-fucose polysaccharides of low MW are formed in the ER and are subsequently elaborated in the dictyosomes. The results of Bowles and Northcote should not be accepted without criticism. It has already been pointed out (p. 112) that the purity of their rough ER fraction is questionable, moreover, in the absence of gradient analyses employing marker enzymes and changes in Mg2+ levels, one must regard the designation here, of rough ER as the first site of synthesis, as sub judice. It is possible, for example, that the components in their rough ER fraction responsible for the initial uptake of radioactivity, are dictyosome fragments, which have been disrupted during the homogenization and later are collected in another portion of the gradient. Certainly the fact that mortar grinding was used, makes this possibility even more likely. Secondly the legitimacy of using, as a basis for the separation of slime and non-slime components, the fact that only slime con-

127

PLANT CELL WALL SYNTHESIS

tains fucose, is highly questionable in the light of the findings of Paul1 and Jones (1975) (see p. 119). Furthermore, since the slime is produced only by root cap cells, and the radiocomposition of the dictyosomes here give xylose values of 1876, whereas those from older tissue have 42% xylose, then xylose should also be taken into account in the calculations since the xylose content of the walls is the same for root cap and older tissue cells (Bowles and Northcote, 1972). The pertinent results of Bowles and Northcote have been obtained using a “mixed cell population” (Bowles and Northcote, 1974, p. 142) and I feel that the simplicity of their calculations does not match the complexity of the system. It would be informative to carry out the& experiments on root tips and older tissues separately. The complication of slime production is not present in the pea epicotyl system. Robinson et al. (1976) have followed the passage of labelled precursors through the two main classes of organelles over short periods. In contrast to the results of Bowles and Northcote (1974) the initial uptake was into the dictyosome fraction (Fig. 23). Even after 5-min incubations in 14C-glucose the level of dictyosome-associated radioactivity was higher than that for the vesicle fraction. Only after an 8-min pulse was the radioactivity in the vesicle fraction comparable in amount to that o f the dictyosome fraction. In experiments in which an 8-min isotope pulse is followed by incubation with non-labelled substrate (Fig. 23), the radioactivity in the dictyosome fraction declined continuously. The vesicle fraction however showed a considerable

PULSE-

CHASElminl

+

Fig. 23. Non-lipid 14C-incorporation from glucose in vivo for short-term pulse-chased pea epicotyl segments. Abraded epicotyl segments (2 parallel samples of 100 segments per time point) were pretreated for 2 h in 1 7 p M IAA then incubated in U-14C-glucose ( 5 pCi) and followed by a chase-out in the presence of 50 mM glucose. At the times indicated samples were homogenized and dictyosomes (.-I and vesicles ( 0 - 0 ) separated.

128

DAVID G . ROBINSON

increase in radioactivity during the first 5 min of chase-out before declining, a feature to be expected when radioactivity is transferred from the dictyosomes to secretory vesicles. Confirming the transport pathway as soluble pool -+ dictyosomes -+ secretory vesicle -+ cell wall are the values for the changing isotope transient during these short-term pulse-chase experiments. Values for the ratio of glucose t o non-glucose radioactivity are, for the cell wall non-cellulosic polymers, the vesicles and dictyosomes after a 5 min pulse 1.89, 1.75 and 0.64 respectively which drop to 0.64, 0.59 and 0.43 at the end of a subsequent 10-min chase-out. This is fully in keeping with the idea that radioglucose must be metabolized in soluble pools to other sugars, which takes a finite time, and during this period polysaccharide precursors will be taken up into the dictyosomes. Therefore the earliest transported materials (i.e. those arriving in the cell wall first) will contain a higher radioglucose content, the proportion of which will progressively fall during the chase-out. In agreement with the calculations of Bowles and Northcote (1974) on the displacement times for wall polysaccharides in the dictyosomes, Robinson et al. (1976) have also arrived at a similar value (3 min) and have further estimated the displacement time for the vesicle fraction as 3.5 min. Superficially these results are at variance with the findings for the kinetics of secretion in animal cells (Jamieson and Palade, 1967a; Glaumann et aZ., 1975; Redman et al., 1975) where the microsornal fraction (i.e. that containing much rough ER) precedes the dictyosome fraction in uptake of radioactive precursors. However, since in nearly all cases the secreted material in animal cells is protein or glycoprotein (see Table 11) an initial ER uptake and synthesis is to be expected. The kinetics for the uptake of polysacchande precursors and transfer of polysaccharides of Robinson et al. (1976) do not, however, preclude the transfer of membrane or other materials from ER to dictyosomes, as occurs in animal cells. Most glycoproteins secreted by animal cells do in fact show the uptake of sugar moieties into the dictyosomes rather than the ER (Neutra and Leblond, 1966a, b ; Sturgess et al., 1973).

2. Physiology The physiology of Golgi secretion in animal cells includes papers on zymogen synthesis and secretion in guinea pig pancreatic cells (Jamieson and Palade, 1968a, b, 1971) and it is somewhat surprising that experiments of this type have only recently been attempted upon plant cells (Robinson and Ray, 1973; Robinson, 1976; Robinson and Ray, 1977). It was shown for pancreatic acinar slices that cycloheximide at concentrations of 5 x M inhibited protein synthesis by more than 90% within 1.5 min of addition. When presented to the tissue after a 3-min pulse with 3H-leucine, secretory proteins synthesized at the ER were detected 37 min later, in the zymogen granule fraction. The amount of transport from ER through the Golgi complex to zymogen granules was estimated at 80% of that for non-

129

PLANT CELL WALL SYNTHESIS

cycloheximide treated controls. Thus protein synthesis is not a prerequisite for intracellular transport in this system. Using this observation Jamieson and Palade (1968b) investigated the effects of various inhibitors on the transport process between ER and zymogen granule in the absence of protein synthesis. To accomplish this, material was labelled for short periods as before and the particular inhibitor to be investigated was added to the cycloheximide chase-out medium. In these experiments transport of protein was (a) not affected by glycolytic inhibitors, e.g. iodoacetate, fluoride; ( b ) blocked by respiratory inhibitors e.g. KCN, antimycin A; (c) blocked by inhibitors of oxidative phosphorylation e.g. Oligomycin, Dinitrophenol, and (d) the blockage achieved with KCN was reversible in a subsequent period of incubation in the absence of the inhibitor. Jamieson and Palade (197 1) demonstrated that the further transport of protein in the acinar cell, i.e. the release of the zymogen granule to the cell exterior, was also dependent upon respiration. The pea epicotyl proves very convenient for similar experiments. When segments are incubated for 5 min with ‘‘C-glucose and then transferred to non-labelled glucose for 5 min in the presence of 1 mM KCN, there is an increase in radioactivity in both dictyosome and vesicle fractions (Table IV). During TABLE IV Effect of Cyanide on the Chase-out o f Pisum Epicotyl Segmentsa Fraction

Re-treatment

Chase-out Time (min)

(+/- 2-deoxyglucose) 5

0

20

dPm

Cell organelles (Total) Cell wall No n-cellulo sic Polysaccharides Cell wall Cellulose ~

-2-DG +2-DG -2-DG +2-DG

2480 26444 1556 3744

6029 43377 2426 3989

3393 41666 41 15 4133

-2-DG +2-DG

4699 8500

9877 18777

10100 17444

~~

~

~

~

a Abraded epicotyl segments (3 parallel samples of 30 for -2-DG, 2 parallel samples of 75 for +2-DG) were pretreated for 2 h in 17 p M IAA 50 mM 2-deoxyglucose, then

*

incubated in [U-14C]-D-glucose (2.1 pCi for -2-DG samples; 35 pCi for + 2-DG samples) for 5 min. Samples were then chased for various periods in 50 mM glucose + 1 mM KCN before analysis of radioactivity distribution.

longer “chase” periods with non-labelled glucose + KCN there is still a loss of radioactivity from the dictyosome fraction; if however the epicotyl segments are pre-treated with 2-deoxyglucose (2-DG) there is no loss of label from the dictyosomes during “chase” periods of more than 5 min. Complete and immediate inhibition of incorporation of glucose into the non-cellulosic portion of the cell wall was never achieved with 2-DG.

130

DAVID G . ROBINSON

Ray (1973b) also showed that pea epicotyl segments pretreated with 50mM 2-DG took up less externally supplied 14C-glucose and the ratio of incorporation into the cell wall : cell organelles was reduced from c. 4 : 1 to c. 1 : 1. The effects of 2-DG on carbohydrate metabolism are many (Webb, 1966) but most important for cell wall studies are the observations that deoxyhexose/ pentose nucleotides are formed (Fischer and Weidemann, 1964; Farka3 et al., 1968; Keppler et al., 1970; Zemek et al., 1971) which apparently are not utilized in cell wall formation (Megnet, 1965; Johnson, 1968; Farkaz et al., 1970; Biely et al., 1971), and that cellular ATP levels are reduced (McComb and Yushok, 1964). Thus a pre-treatment with 2-DG causes a reduction in the pool sizes of wall polysaccharide precursors and makes the cells more susceptible to inhbitors such as KCN. The inhibition of 4 C - g l u ~ incorporation ~~e into the non-cellulosic portion of the cell walls of epicotyls pretreated with 2-DG, when chased in the presence of KCN, takes place very rapidly, but the inhibition of incorporation into cellulose takes effect more slowly (Table IV). Furthermore KCN recovery experiments with 2-DG pretreated material reveal an almost “stoichiometric” transfer of radioactivity from cell organelles to cell wall non-cellulosic polysaccharides (Table V). Due to the continual addition from soluble precursor pools to the dictyosomes during a chase-out, such “stoichiometry” is not usually detectable when measurements of loss of radioactivity from dictyosomes and gain in cell wall are compared. Ray (1973b) showed that over a 1-h incubation period, concentrations of cycloheximide as high as 2 x M have no effect on the incorporation of radioactive precursors into the cell walls of pea epicotyls. Thus, although not yet investigated by the analysis of radioactivity from isolated organelles it can, I think, be concluded that in this tissue also, transport of polysaccharides does not require protein synthesis. Electron microscopy of pea epicotyl tissue prepared during the 2-DG and KCN addition and removal experiments described above has been carried out (Robinson, 1976; Robinson and Ray, 1977). After 2-DG pre-treatment the Golgi do not appear altered to any degree (Fig. 24) but after 20 min in KCN they adopt a beaker form (Fig. 25). Such a morphology for Golgi has been know for many years (Schnepf, 1963; Whaley et al., 1964; MorrC and Mollenhauer, 1964) but has always been demonstrated after several hours in KCN or a CO-containing atmosphere. It is remarkable that during such a 20-min KCN chase when no transfer of radioactivity, and presumably therefore no vesicle release occurs, membrane synthesis still continues. Calculations from measurements of many dictyosomes showed that the average surface area of the cisternae following exposure to KCN (assuming their circular shape) is four times that of dictyosomes before the addition of KCN (cf. Figs 24 and 25 which are of the same magnification). Removal of the KCN results in a complete recovery of morphology as well as function. Thus we now have experimental proof for the

TABLE V Reversibility o/ Cyanide Inhibition in Relutiori t o Transfer betw~reriOrganelles and Cell Wall Fractions" ~~

Fraction

Cell organelles (Total) Cell wall Non-cellulosic Polysaccharides Cell wall Cellulose

Pre-treatment (+/- 2-deoxyglucose)

A: dprn after KCN chase

B: dpm 15 min after KCN removal

A A and B; Changes in radioactivity as a result of KCN removal

-2-DG +2-DG -2-DC; +2- DG

6029 36000 2426 4644

5250 20711 5062 21000

-779 -15223 +2636 +16356

-2-DG +2-DG

9877 8655

11475 10155

+1598 + I 500

a Treatments same as for Table IV up to and including the radioactivc pulse. Thcn either A: 5 niin 50 mM glucose + 1 mM KCN or B: 5 min 50 mM glucose + 1 m M KCN plus 15 min 50 r n M glucose without KCN.

132

DAVID G. ROBINSON

suggestion of Whaley et aE. (1964) that vesiculation and membrane synthesis are separable processes. That the dictyosomes possess considerable membrane synthetic capacity is supported by other independent observations: (a) in the onion shoot a dictyosome fraction possessed the highest incorporation of C-acetate and 4C-choline (MorrC, 1970; Montague and Ray, 1975); ( b ) the in vitro measured activity for lecithin biosynthesis was highest in the dictyosomal fraction of onion shoots (MorrC et al., 1970). Taken together with the knowledge that, in pea epicotyl

24

25 Fig. 24. Normal dictyosomes from pea epicotyl epidermal cells (in situ). x30 000. Fig. 25. Beaker-shaped dictyosomes formed after KCN treatment of pea epicotyl segments. Treatment of the segments was exactly as in the legend to Table IV. Samples for electron microscopy were taken after 15 min in 1 mM KCN chase-out medium. x30 000.

tissue and other tissues (Schnepf, 1969) no forming or muturing face to the dictyosome is to be seen and no smooth ER is present (Shore and MacLachlan, 1975), such observations throw doubt on the universality of the so-called “endomembrane concept” (Grove et al., 1968; Franke et al., 1971; Morri and Van Der Woude, 1974; MorrC and Mollenhauer, 1974) where nuclear envelope, ER, Golgi, secretory vesicles and plasmalemma are held to exist as a functional continuum. Occasional connections (see MorrC et al., 1971 for examples)

PLANT CELL WALL SYNTHESIS

133

between these components support this theory which embodies the concept of membrane flow from nuclear envelope to the plasmalemma. This concept fits conveniently for animal cells where protein synthesis may occur on rough ER ribosomes, but for some plant cells were the principal dictyosome product is polysaccharide a membrane flow beginning at the ER may not be necessary. Of course difficulty exists with the synthesis and transport of hydroxyproline wall protein, but as yet no good evidence exists (see pp. 118-119 for the ER as the site of synthesis. Mollenhauer and MorrC (1976) have reported that the fungal derivative cytochalasin B (CB) inhibits the migration of secretory vesicles in root cap cells of Zea (see Fig. 26 and compare with Fig. 4). A similar feature has also been recorded by Robinson and Ray (1976) though the effect is considerably easier to appreciate in Zea where the dictyosome cisternae tend t o hypertrophy and the secretory vesicles are large and conspicuous. Corroborating the ultra-structural evidence are cell fractionation data (Ray et al., 1976). As mentioned before (p. 127) radioactivity in the vesicle fraction from pea epicotyl segments rises during the first 5 min of chase-out after a short pulse. For segments pre-treated with cytochalasin B (10 pg/ml) this post-pulse

Fig. 26. Zeu mays root cap cell after 2-h treatment with 100 p g ml-1 Cytochalasiii B. The cytoplasm is seen to be packed with secretory vesicles (cf. Fig. 4). x16 500. (Courtesy H. H. Mollenhauer and D. J. MorrB.)

134

DAVID G . ROBINSON

increase in radioactivity in the vesicle fraction is particularly large, concomitant with a reduced incorporation into the cell wall. CB is well known as an inhibitor of cytoplasmic streaming (Wessells et al., 1971) and the retardation of transport through the vesicles is supported by observations of its effect on the growth of the pollen tube (Franke et al., 1972; Mascarenhas and LaFountain, 1972) whose growth is dependent upon vesicle transport to the growing tip (Rosen et al., 1964). However the effects of CB are numerous (Hepler and Palevitz, 1974) and with regard to its effect on protein secretion often conflicting, for example CB inhibits a-amylase secretion from rat parotid gland tissue (Butcher and Goldman, 1972) but not from barley aleurone layers (Chrispeels, 1972). Other effects of CB which do not involve microfilaments or streaming (Copeland 1974). could be invoked to explain the action of CB in Zea andPisum. Since low turgor, induced by external concentrations of mannitol, has been known for some time to severely inhibit cell wall synthesis (Bayley and Setterfield, 1958; Ordin, 1960; Ray, 1962; Cleland, 1967) one might also expect the functioning of the intracellular secretory pathway to be altered. Robinson and Cummins (1977) have studied the effects of plasmolysis on the secretion of cell wall materials in pea epicotyls. Although 100% inhibition of incorporation of label into the cell wall itself occurs when cells are chased in the presence of 0.45 M mannitol (Fig. 27) the loss of materials from the dictyosomes and the

TOTAL CELL PARTICLES

?

I

1

5

- MANNI TOL 1

1

20

35

1

CHASE rnin

Fig. 27. Reversibility of plasmolysis inhibition of cell wall incorporation in pea epicotyl segments. Abraded epicotyl segments (75 per sample) were pre-treated with 0.45 M mannitol -t 17 p M IAA for 30 min at RT; then incubated in 0.45 M mannitol containing U-14C-glucose (30 NCi) for 5 min. Samples were then chased with 0.40 M mannitol + 0.05 M glucose and then with glucose alone (from Robinson and Cummins, 1977).

135

PLANT CELL WALL SYNTHESIS

vesicles proceeds as for untreated epicotyls. During this plasmolysis chase-out, however, polysaccharides, detectable as an ethanol-insoluble fraction from homogenates after cell wall and organelles have been centrifuged out, accumulate without being incorporated into the cell wall and may even be detected in the chase-out medium. Removal of the mannitol results in a drop in the levels of the accumulated soluble polysaccharide and a corresponding renewed incorporation into the cell wall some 20 min after the original radioactive incubation had ended. Thus synthesis and transport of at least non-cellulosic wall polymers must be regarded as separate processes to their incorporation into the wall. In fact the independence of wall material secretion from turgor is implicit in studies of polymer secretion during wall regeneration in higher plant protoplasts which are suspended in high concentrations of mannitol or sorbitol (Cocking, 1972; Hanke and Northcote, 1974), in the observations of Kiermayer (1965) on plasmolysis in the desmid Micrasterias and of Schroter and Sievers (1971) on Tradescantria root hairs under low turgor. One can clearly imagine why, if plasmalemma-cell wall relationships are important, the turgid condition is necessary for cellulose synthesis which appears to take place at the plasmalemma, but it is not so obvious for non-cellulosic polysaccharides whlch, as Ray (1967) has demonstrated, are incorporated throughout the body of the wall. Certainly the knowledge that the incorporation of polysaccharides into the cell wall, during the reversal of plasmolysis inhibition in pea epicotyls, is inhibited by KCN, rules out the simple physical effect of bringing the plasmalemma once again in contact with the wall. A summary of what I think can now be said concerning the physiology of the secretion in animal cells as against that in pea epicotyls is shown in Fig. 28. From the dictyosomes to the release of the products outside the plasmalemma the two systems are comparable in their energy dependence. However, one must not forget the difference in rates of secretion in the two systems. Passage of protein from ER to a mature zymogen granule in the pancreas takes 60-90 min (Castle e t al.,

-,.. .....

RESPIRATORY INHIBITORS (KCN etc) 2- DEOXYGLUCOSE CYTOCHALASIN B PLASMOLVSIS / LOW TURGOR

-.--

4

ER VESICLE

-

DICTYOSOMES

SECRETORY VESICLES

RAWALEMMA

t I I SUGAR-/ POLYSACCHARIDE PRECURSOR POOLS

SECRETION IN PlSUM EPICOTYL SYSTEM

9s>

PRODUCT

SECRETION IN ANIMAL SYSTEMS

Fig. 28. Diagrammatic represencation of the similarities and differences between secretion in animal cells and that of the pea epicotyl system.

136

DAVID G. ROBINSON

1972) and the passage through the Golgi complex takes 10-20 min. On the other hand secretion in plants is apparently much quicker: the entire process taking only 7 min in pea epicotyl tissue.

V. IN VIITRO SYNTHESIS The most favoured glycosyl donors for cell wall polysaccharide synthesis are the sugar nucleotides which possess the highest negative free energy of hydrolysis (-7.5 K cal mole-’) of all monosaccharide donors (Leloir et al., 1960). The enzymes responsible for the production of cellulose and chitin precursors (Fig. 29) are well known (Ginsburg, 1958; Hall and Ordin, 1967; Abdul-Baki and Ray, 1971 for higher plants; Leloir and Cardini, 1953; Glaser and Brown, 1955; ,ATP

ADPr,

----_--__--_ -- - - - - - - - - - _ _ Y‘

ATP

Glucose

ADP

UTP

2

Glc-6-@

Glc-l-@-

N-Acetylglucosarnme----f GlcNAc-6-@Hexokina=

~

l

G l C N A C - I @ h Phosphornulase

-

G

l

UOP-Fyro~s~rylase

lu c

UDP-GlcNAc

Cellulose Chlltn

“Polysaccharide synlhetase”

Fig. 29. Biochemical pathway for the formation of cell wall microfibril intermediates.

McMurrough et al., 1971 for fungi) and occur free in the cytoplasm. Cell-free systems from fungi and bacteria, usually consisting of fractions obtained by heterogeneous differential centrifugation, which are capable of transferring the glucose moiety from the nucleotide sugar to a “polysaccharide” were first employed by Glaser and Brown, (1957, chitin) and Glaser (1958, cellulose). A. HIGHER PLANT CELLULOSE

In the years since 1958 numerous in vitro glycosyl transfers have been achieved with cell-free systems from higher plants (Barber et al., 1964; Brummond and Gibbons, 1965; Ordin and Hall, 1967; Ray et al., 1969; Van Der Woude et al., 1974; Shore and MacLachlan, 1975). Apparently important in these cell-free systems in determining what type of glucan is formed and therefore whether or not “cellulose” has been synthesized, is the concentration of the substrate, but the literature is somewhat conflicting. Ordin and Hall (1968) first demonstrated that, with an extract from oat coleoptiles, 4 PM UDPG produced a material which on treatment with cellulase yielded mainly cellobiose, but at 1 mhf mainly laminaribiose. Tsai and Hassid (1971) confirmed these findings and were further able to separate two enzymes: one responsible for p-1,3 linkages and the other for p-1,4. More recently Van Der Woude et al. (1974) using onion stems have shown that, although at high UDPG concentrations (1 mM) the principal products were p-l,3 glucans for both Golgi

PLANT CELL WALL SYNTHESIS

137

and plasmalemma fractions, the proportion of 0-1,4 glucan synthesized in the latter fraction had increased by a factor 10-fold greater than that for the Golgi ) On the other hand when compared to the values at low (1 p ~ concentrations. Ray (1973a) using a heterogeneous fraction from pea epicotyls and Shore and MacLachlan (1975) using various organelle fractions from pea were not able to detect any difference in reaction product at low (1-6 p M ) or high (0.5-0.6 mM) substrate concentrations. Whereas only 10% or less of the product could be solubilized with /3-1,3 endoglucanase, cellulase produced only cellobiose, the product therefore being apparently a /3-1,4 glucan. However despite the fact that in none of these papers have X-ray diffraction methods been used, and in only one (Villemez, 1974) is there evidence that the polymers formed are large (8% > 1.2 x lo6 MW; 92% > 28 000 MW by gel permeation chromatography), the synthesis of cellulose in vivo is almost certainly achieved through the participation of nucleotide sugars. UDPG has been isolated from many tissues actively forming a cell wall (Gregoire et al., 1965; Elnagy and Nordin, 1966) and sometimes in high concentrations (30 pM-Smith and Stone, 1973). Furthermore, with detached cotton fibres during secondary wall development, a very high incorporation of radioactivity from UDPG into cell wall cellulose has been achieved (Franz and Meier, 1969; Delmer el al., 1974). With the cotton fibre system which is essentially analogous to the pea epicotyl section, product analysis is once again proving troublesome. Heiniger et al. (1975) showed that the product from UDPG, which is the preferred substrate for the synthesis of alkali insoluble material in secondary walls is predominantly /3-1,3 linked. Abdul-Baki and Ray (1971) and Shore and MacLachlan (1975) have noted however that the incorporation rate for these cell-free systems is “only a minute fraction of the rate of wall glucan synthesis in vivo”. A possible reason for this discrepancy and also why no successful cellulose microfibril synthesis from higher plants has yet been achieved, may lie in the degree of disruption of the tissue. The high rates of incorporation with cotton fibres are questionably in vitro. Delmer et al. (1974) have considered the possibility that the fibre when removed from the ovule, repairs quickly its dainhged end and, due to the different end products received from GDPG and UDPG, may not absorb these compounds into its cytoplasm. This implies that the enzymes responsible for the incorporation are located at or outside of the plasmalemma and are only partially recovered in the normal in vitro systems. Shore and MacLachlan (1975) have substantiated this: 1-mm sections of pea epicotyl tissue are over twenty times better at incorporating radioactivity from UDPG into alkali insoluble glucan than the total 500-130 000 x g membrane fraction, furthermore when separated walls and membrane fractions are recombined higher incorporations are also achieved. Moreover Shore et al. (1975) now claim that the activities measured in tissue sections fall in the range of those for cellulose synthesis in vivo .

138

DAVID G . ROBINSON B. BACTERIAL CELLULOSE

Hestrin and Schrainni (1954) showed that nonviable lyophilized preparations of Acetobacter xylinum could still generate cellulose from glucose. The participation of nucleotide sugars, e.g. UDPG, has often been demonstrated (Glaser, 1958; Ben-Hayyim and Ohad, 1965) but only recently has it been possible with cell envelope fractions from this bacterium to produce cellulose from glucose 1- or 6-phosphates (Cooper and Manley, 1975b). Based on the loss of cellulose synthesizing capacity after trypsin treatment of lysed cell preparations, Dennis and Colvin (1965) suggested the importance of the plasmalemma in the synthesis of cellulose in A . xylinum. This has now been extended by Cooper and Manley (1975b) to include the cell wall itself, although the centrifugation fractions, as the authors themselves admit, are somewhat heterogeneous. Cooper and Manley (1975a) have also shown that 1 mM EDTA treatment which disrupts the cell wall, but not the plasmalemma, releases glucose-1-phosphate, UDPG and various nucleotides into the medium, but not glucose-6-phosphate which is apparently retained within the cytoplasm. These authors have therefore suggested that a pool of UDPG and glucose-1phosphate exists between plasmalemma and cell wall, namely in the “periplasm”. Interestingly, calculations of the pool size of UDPG in the periplasm are also in agreement with the substrate levels (4-5 mM) required for cellulose synthesis by the cell envelope in vitro. Recent work from Colvin’s laboratory on the nature of the material synthesized in vitro from UDPG with A . xyZinum extracts, shows that the radioactivity taken up by these extracts bands a t about 45% sucrose in isopycnic centrifugations (Kjosbakken and Colvin, 1973). Since cellulose has a density of 1.5-1.6 g c m P 3 it appears that microfibrillar cellulose is not formed in such incubations. Further work by King and Colvin (1976) has shown that a hot borate fraction from the in vitro UDPG incubation contains a /3-1,4 glucan which is protein-free, non-dialysable, and based upon gel-chromatography has a MW of at least 30 000. When isolated and added back to the in vitro system, radioactivity from this glucan is then transferred to an alkali-insoluble fraction (cellulose) suggesting that intermediate polymers of glucose are formed in the pathway to the cellulose microfibril. C. CHITIN

The successful synthesis of chitin micro fibrils by a cell-free system, confirmed by electron microscopy and X-ray diffraction data, has been achieved using the yeast stage of Mucor rouxii, (Ruiz-Herrera and Bartnicki-Garcia, 1974; BartnickiGarcia e t al., 1975; Ruiz-Herrera et al., 1975). Using the yeast stage of Mucor rouxii, enzyme complexes in particle form, 35-100 nm in diameter, which mediate this synthesis have also been isolated. Arising from these particles, after incubation in the presence of the nucleotide sugar UDP-n-acetyl glucosamine (UDP-GlcNAc) and the activator n-acetylglucosamine (McMurrough et al.,

139

PLANT CELL WALL SYNTHESIS

1971), are one, sometimes two, microfibrils 12-15 nm thick and up t o 2 pm long (Fig. 30). There are superficially three types of chitin synthetase: one which is bound to the cell walls, one which is bound to a mixed membrane fraction (sedimenting between 2000 and 54 000 x g) and one which occurs free in the supernatant of the 54 000 x g fraction. Interesting, and of great significance for workers in the field of cellulose synthesis is the recognition of an inactive form (the “zymogenic state”-not to be confused with zymogen in the pancreatic

Fig. 30. An in virro-synthesized chitin microfibril from a particulatc preparation from

Mucor rouxii. x71 000. (Courtesy Bartnicki-Garcia.)

acinar cell) which can be transformed, through proteolysis with acid protease or trypsin, to an active state (Cabib and Farkas, 197 1). Incubation of cell extracts with UDP-GlcNAc and GlcNAc also brings about a solubilization of the chitin synthetase which is bound to the 54 000 x g fraction. D. NON-CELLULOSIC hlATERIALS

Participation of nucleotide sugars in the biosynthesis of pectins and hemicelluloses is well substantiated. The interrelationships of the various nucleotide sugar precursors and their origin from UDP-glucose is shown in Fig. 31. The enzymes responsible for the interconversions have been documented; dehydrogenase and decarboxylase by Strominger and Mapson (1957); epimerases by

1

dehydrogenase

epimerase

UDPGal

1

decarboxylase

epimerase

UDPGalU

1

epirnerase

UDP-Ara

Fig. 31. Sugar nucelotide interconversions leading to non-cellulosic cell wall polysaccharide precursors.

140

DAVID G. ROBINSON

Feingold et al. (1960). Cell-free systems containing heterogeneous particle fractions have shown the transfer of

1. galacturonic acid to a - l , 4 polygalacturonic acid (Villemez et al., 1965) 2. xylose to &1,4 xylan (Odzuck and Kauss, 1966) 3. galactose to p-1,4 galactan (McNab et al., 1968; Panayotatos and Villemez, 1973) 4. mannose to /3-1,4 mannan (Heller and Villemez, 1972) 5. arabinose to arabinan (Odzuck and Kauss, 1972) from the appropriate nucleotide sugar These studies have also shown the post-polymerization methylation of pectic and hemicellulosic uronic acid residues from S-adenosyl-L-methionine (Kauss and Hassid, 1967; Kauss, 1969a). Experiments by Kauss et al. (1969) have further indicated that, at least for methylated polygalacturonic acid (M-PGA), a compartmentalization of the product from the cytoplasm exists. When soluble pectin methyl esterase was added to particle-bound in vitro formed M-PGA, hydrolysis was only achieved after the membranes were denatured with detergents such as Triton X-100 or dodecyl-sulphate. It follows that polysaccharide synthesis probably occurs within membrane vesicles, substantiating the in vivo results already discussed. Both Villemez (1974) and Kauss (1974) have drawn attention to several important problems with these in vitro systems. First of all it would appear that in many cases no polymerization has occurred but rather only a transfer of, at most, a few sugar monomers. Secondly, the particulate preparations themselves contain a certain level of the enzymes necessary for the formation and interconversion of the nucleotide sugars (Feingold et al., 1960). As a result, when a nucleotide sugar is presented to such a preparation a heterogeneous polysaccharide often results. For example, the transfer of glucose from GDPglucose to a polymer originally called cellulose by Barber el al. (1964) with mung bean hypocotyl extracts is now known to be a glucomannan (Heller and Villemez, 1972; Villemez, 1974). Further important observations have recently been made by Villemez (1 977) concerning the nature of the in vitro xyloglucan produced by pea epicotyl extracts. When the purified xyloglucan is hydrolysed for total sugars, and not just those which are radioactive, then many sugars other than xylose and glucose are found. This seems to indicate the capacity to build entire crosslinked units of the wall within the cell membrane systems. E. LIPID INTERMEDIATES

Ever since Colvin (1959) showed that an ethanol extract of A . xylinum could, together with the particulate enzyme, give rise to rnicrofibrils, considerable evidence has become available implicating lipid intermediates between nucleotide sugar and acceptor molecule. Kauss (1974) has briefly reviewed the

PLANT CELL WALL SYNTHESIS

141

literature pertinent to lipopolysaccharide synthesis in bacterial, yeast and mammalian cells. Apparently all of the glycolipid intermediates are polyprenol mono- or di- phosphates:

r3

H- [CH2 - =CH-CH2]

-Ow@

where n is at least 11. Transfer of the sugar from the nucleotide may be accomplished with (e.g. in bacterial wall peptidoglycan (Strominger et al., 1972)) or without (e.g. in Micrococcus (Lennarz and Scher, 1972)) an accompanying phosphate group. In a similar way small oligosaccharides can be built attached t o the lipid, which are then transferred to the acceptor. For the synthesis of cellulose in A . xylinurn the following information is now available: using cell-free extracts Kjosbakkeri and Colvin (1973) have extracted the radioactivity incorporated from UDPG with chloroform : methanol (2 : 1). The labelled lipids were retained on a DEAE cellulose column but could be eluted with ammonium acetate and were very acid labile. After acid hydrolysis glucose and cellobiose were the main sugar components found. Using frozen and thawed EDTA-pre-treated cells as the “enzyme” Garcia et al. (1974) followed the incorporation of 4C from UDPG into a butanol extract. Analysis of the extract enabled the isolation of lipid diphosphate &-glucose, lipid diphosphate a-cellobiose and lipid monophosphate /I-galactose. From P3 labelling experiments it became clear that a-glucose-1-phosphate was being transferred. From both studies the solubility and chromatographic properties of these lipid intermediates suggested a polyprenol nature. For higher plants cotton has been investigated by Forsee and Elbein (1973) and Delmer el al. (1974). Both groups detected steryl glucosides and an acid labile glucosylated polyprenoid phosphate. Using pea roots Brett and Northcote (1975) extracted the in vitro UDPG product with butanol and phenol. The butanol extract was acid labile releasing sugars which were also released after phosphodiesterase treatment, indicating the presence of a phosphate ester bond. The lipids in this extract were again a steryl glucoside and a glucosylated polyprenoid phosphate. From the results of a Smith degradation, the sugar portion appeared to be about 4 units long, 3 of which were /3-1,3 linked. A phenol extract also gave a glycosylated polyprenoid phosphate with 1 1 sugars, again with a larger proportion of 8-1,3 linkages. A mannosyl polyprenoid phosphate from mung bean extracts, after incubation with GDP-mannose, is also known (Kauss? 1969b; Storm and Hassid, 1972). Several points emphasizing the importance of these glycolipids as intermediates in the production of cell wall polysaccharides are: 1 . Their formation is extremely rapid in the in v i m incubation experiments. 2. Their pool sizes are very small.

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3. The reaction nucleotide-sugar -tlipid + nucleotide + lipid-sugar is reversible indicating that the lipid sugar must also be a good donor for sugar units, and of course the advantages of having a hydrophobic carrie’r particularly with respect to synthesis at the plasmalemma are great. VI. CONCLUSION In comparing what is known about the secretion of plant cell wall components to that of digestive enzymes from the pancreatic acinar cell O’Brien (1972) wrote: “We are a long way from having this kind of information . . . in any plant cell.” I believe that, in the intervening years, this information has become available: circumstantial electron microscopical evidence, for the participation of Golgi dictyosomes and other membrane compartments in the synthesis and secretion of cell wall components, is now heavily supported by the appropriate cell fractionation data. However, there are many problems still to be solved. The techniques employed by Albersheim and his co-workers must now be applied to the various isolated cell fractions to see just how closely the intracellular wall precursors resemble the wall polymers themselves. In this respect the biologist must make way for the chemist. But the biologist still has a fruitful field before him. All of the work described here has been accomplished with heterogeneous cell populations and clearly much more definitive results could be obtained from homogeneous material. Some algae offer this possibility. The question of the relationship of wall synthesis to growth is a topic on its own and encompasses not only the promotive effects of hormones but also the negative effects of polysaccharidases which are active at the same time as the synthetase enzymes. Again some algae show this feature particularly well. There are still many points which need to be investigated with cell fractions. If there does not appear to be a contribution from the ER to the dictyosomes in Rsum, and if this is true for some other plant tissues, then how exactly are cell wall proteins and enzymes such as peroxidases, not to mention the various synthetases, incorporated into the dictyosomes? Do the polyribosomes described by Mollenhauer and MorrC (1974), as associated with dictyosomes, serve this function? Clearly this aspect of pea epicotyl cells is in contradiction to the endomembrane concept of MorrC and Mollenhauer (1974). But this concept is based on biochemical information from animal sourceb, and, as we have seen, secretion in some plant cells is much faster than in animal systems. If the major secreted material is polysaccharide it seems rather uneconomical for two vesiculation steps (ER + dictyosome; dictyosome plasmalemma) t o be used in plants. With the lack of sufficient data from other plant cells one must question the ubiquity of the “endomembrane concept” for all plant cells. On the other hand a comparison with animal secretory systems may be useful. Many of these systems can be induced t o secrete by application of chemicals, such as --f

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choliner ic drugs (pilicarpine, nicotine) and sometimes adrenergic drugs (adenyl cyclasefi%f& vesicle production and fusion also take place in plant secretion processes, and since many of these drugs are of plant origin, it would be interesting to investigate their effects on plant cells. If the success of Bartnicki-Garcia’s group is a good indicator, then the in vitro synthesis of cellulose microfibrils from plant sources cannot be too far off. Whether glucose/cellobiose units are added serially to the ends of a microfibril as suggested on ultrastructural grounds (Preston, 1964) and thermodynamic grounds (Stockman, 1972) or, from pre-formed glucan chains as suggested by the recent results from Colvin’s laboratory on Acetobacter xyZinum, remains a problem for investigation. Whether or not cellulose can be synthesized outside of the plasmalemma as suggested by the high levels of wall-bound chitin synthetase, is yet another problem. The mechanism of orientation of cellulose microfibrils clearly lies at the plasmalemma and the potential of organisms like Oocystis are great in studies on this problem. Although different microfibril orientation paiterns and the loss of microtubules may both be achieved with the application of colchicine, the results available t o us at present do not allow one to say categorically that the two effects are directly related. In the same vein, even if paracrystalline arrays of plasmalemma particles, revealed by freeze-etching, do mirror microfibril orientation, they cannot, in the absence of microfibril attachments, be regarded as categorical proof for cellulose synthesising complexes.

ACKNOWLEDGEMENTS

I would like to express my appreciation to Professors R. D. Preston and P. M. Ray from whom I have learnt much. My thanks also go to the various members of the Pflanzenphysiologisches Institut Gottingen, in particular my technician Frl. I. Hammerl, who have helped me in various capacities. The National Science Foundation ( U S A ) , the Deutsche Forschungsgemeinschaft and the Land Niedersachsen are gratefully acknowledged for the financial support of various experiments reported here.

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The Evolution of Vascular Land Plants in Relation to Supracellular Transport Processes

J . A . RAVEN Depurtment of Biological Sciences. University of Diindee. Dundee DD1 4HN. Scotland

Introduction . . . . . . . . . . . . . . . . . . The Progenitors of Vascular Land Plants . . . . . . . . . The Structure of Early Vascular Plants . . . . . . . . . . The Xylem and Liquid-phase Water Transport . . . . . . . A. The Transpirational Flux of Water . . . . . . . . . The Xylem as a Low-resistance Pathway for Mass Flow of B. Water . . . . . . . . . . . . . . . . . . C. The Significance of Lignification . . . . . . . . . V . Transport in t h e Gas Phase . . . . . . . . . . . . . The Robleni of H 2 0 Loss as a Concommitant of PhotoA. synthetic C 0 2 Fixation: Poikilohydry . . . . . . . . Homoiohydry in Vascular Land Plants and the lntercellular B. Space-cuticle-stomata Complex . . . . . . . . . VI . Transport of Dissolved Solutes . . . . . . . . . . . . A. Xylem . . . . . . . . . . . . . . . . . . B. Phloem . . . . . . . . . . . . . . . . . . C. Syniplast and Apoplast . . . . . . . . . . . . . D. Excretion . . . . . . . . . . . . . . . . . VII . The Evolution of Vascular Land Plants; a n Hypothesis . . . . VIII . Appendix A: Secondary Plant Products in Relation t o Vascular Plant Evolution, with Particular Reference to Lignification . . . IX . Appendix B: Role of Intercellular Gas Spaces in Respiratory Gas Exchange of Vascular Land Plants . . . . . . . . . . . X . Summary and Conclusions . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

I. I1. 111. IV .

153

154 155 162 170 170 172 177 181 181 182 192 192 193 197 198 199

206 207 210 211 211

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J. A. RAVEN

I. INTRODUCTION This article sets out to explore some aspects of the significance of transport processes in the evolution of vascular land plants from their putative ancestors, the green algae. The vascular land plants have, in addition to the transport processes at the cell level which are common to all organisms (and specifically to vacuolate plant cells with a cell wall: see Raven, 1976a), important transport processes at the supracellular level which involve complex anatomical features. These transport processes may be divided into three main groups. One is the apoplast, which will be used here (cf. Salisbury and Ross, 1969) to include all water-filled spaces within the plant body but outside living cells. It thus includes cell walls, water-filled intercellular spaces (schizogenous spaces, i.e. produced by separation of cell walls) and water-filled dead cells, produced by specific developmental processes. Of this latter category, the lysigenous spaces, the most significant from the viewpoint of this article is the xylem. The apoplast corresponds t o the free space of Briggs and Robertson (1957). In subterranean organs it is continuous with the soil water. Its functional continuity as far as the passage of water and water-soluble solutes within the plant is concerned may be interrupted by the endodermis (Slatyer, 1967). A second structurally defined transport system is the symplast. This consists of the living cytoplasm of individual cells linked by plasmodesmatal connections through cell walls. It is separated from the apoplast by the plasmalemma, and from the cell vacuoles by the tonoplast. The phloem may be regarded as a specialized development of the symplast, just as the xylem is a specialized kind of apoplast. The symplast includes almost all of the living cells in the plant, although some cells (e.g. stomata1 guard cells) may be excluded from it (Raschke, 1975). The third transport system to be considered is the gas-filled continuum of intercellular spaces. T h s is generally a schizogenous system present throughout the plant with the exception of meristems and vascular bundles. It communicates with the atmosphere in aerial parts of the plant via the lenticels of woody tissue and the variable-resistance stomata of herbaceous tissue. These three transport systems have been described and discussed by Crafts (1961), Crisp (1963), Crafts and Crisp (1971), Clarkson (1974), Peel (1974), Robards (1975), Raven (1976a), Euchli (1976)and Spanswick (1976). The extent to which these transport systems are found in the green algal ancestors of vascular plants, and in the earliest known fossil vascular plants themselves, is discussed as a preliminary to the consideration of the significance of these transport systems in extant vascular plants and the selective pressures involved in their evolution. Finally, a possible evolutionary sequence for these transport-related characteristics of vascular land plants, based on selective pressures related to the terrestrial environment, is presented. Another very significant physiological factor in vascular land plants in comparison with their algal ancestors is related to energetics. Thus the vascular land plant has extra energy demands related to the synthesis, maintenance and

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operation of the supracellular transport systems not found in the algae. Further, they have much more non-green tissue per unit of photosynthetic tissue than do the algae; this has important repercussions for the direct use of photosynthetically produced co-factors for processes other than CO, fixation. These aspects of the energetics of vascular land plants are discussed by Raven (1976b, c). 11. THE PROGENITORS OF VASCULAR LAND PLANTS

A considerable body of cell structural and chemical evidence suggests that the vascular land plants (and bryophytes) evolved from the Chlorophyta, i.e. the green algae defined in a broad sense (Fritsch, 1916, 1935, 1945a, b; Scagel et al., 1965; Klein and Cronquist, 1967; Clowes and Juniper, 1968; Stewart, 1974). These similarities may be considered under three main headings. First there are a number of well known characters which serve to distinguish the land plants and green algae from other groups of algae; these are mainly related to the photosynthetic apparatus. The chloroplast pigments of land plants (chlorophylls a t b, and the carotenoids, 0-carotene, lutein, violoxanthin, antheroxanthin, neoxanthin and zeaxanthin) are also found in most green algae (the major exceptions being certain marine coenocytes); this specific combination of pigments is not found in other algae. Chloroplast ultrastructure, particularly the tendency to form grana (or in the green algae, at least the formation of thylakoid stacks containing at least three thylakoids) is not found in other algal groups. Further, the occurrence of starch (a 1,4-glucan) solely within plastids is another character found only in land plants and green algae. A second category of similarities between green algae and land plants is perhaps less widely appreciated, 'and relates to the synthesis of such important land plant components as lignin and sporopollenin. Some green algae can produce sporopollenin (or some very similar component: see Section VB), and the enzymic complement of green algae is more closely related t o the lignin-synthesizing reaction pathway of vascular plants than is that of other algal groups (Section VIII). Again, we find green algal characters associating them specifically with the land plants. Finally there are a number of characteristics of land plants which are also found in most green algae, but which are present in some other classes of algae as well. Two such features are the presence of cellulose and hydroxyproline-rich protein (Gotelli and Cleland, 1968; Lamport and Miller, 1971; Northcote, 1972; Mackie and Preston, 1974) in the cell wall, and flagella characteristics (Manton, 1965; Duckett and Racey, 1975). These similarities between the green algae and the land plants strongly suggests that the green algae gave rise to the vascular land plants and bryophytes. Recent evidence has focused attention on one particular group within the green algae as the ancestors of the land plants. This is a group which contains the Charales and a number of members of the class Rasinophyceae. The evidence which relates these algae to the ancestors of the higher land plants is

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based on the mechanism of nuclear and cell division, the structure of the flagellate motile cells, the nature and subcellular location of the glycolate oxidizing enzyme and (probably) the enzyme involved in urea breakdown (Leftley and Syrett, 1973; Jones et aE., 1973; Thompson and Muenster, 1974; M$estrup, 1974; Rckett-Heaps, 1975; Stewart and Mattox, 1975; Taylor, 1976; Stabenau, 1975; Tolbert, 1976). Stewart and Mattox (1975) have indeed proposed that the Chlorophyta be divided into the Chlorophyceae and the

Fig. 1 . (A) A young plant of the charophyte alga Cham sp. The diploid zygospore (z) gives rise t o the haploid plant. This has a colourless rhizoidal system (rh) which penetrates into the substratum; a chlorophyllous erect system (es) which can be a metre in height; and a short-lived chlorophyllous basal system (bs). The thallus consists of elongate internodal cells (100 m m x 1 mm) separated by a parenchymatous nodal complex of much smaller cells. A whorl of branches, again consisting of internodes and nodes, is borne at the nodes of the main axis. Growth is apical. [Figure 15-24A of Scagel et al. (1965).] (B) Longitudinal section of the apex of the thallus of Cham sp. Division of the apical cell (ac) gives rise t o alternating nodal (no) and internodal (in) regions. [Figure 15-24B of Scagel et ul. 19651.1

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157

Charophyceae s.1. This expanded Charophyceae includes not only the Charales (Fig. 1) but also Chaetosphaeridium (MGestrup, 1974), Klebsormidium, Coleochaeta and the Zygnematales. This recalls the view expressed by Fritsch (1916) that Coleochaeta resembles the ancestors of the vascular land plants. The more recently discovered Fritschiella (Fig. 2: Iyengar, 1932; Singh, 1941; Fritsch, 1945b; McBride, 1970) has been widely regarded as resembling the ancestors of the land plants. However, it is definitely a Chlorophyte sensu

Fig. 2. A young plant of the chlorophyte alga Frifschiella tuberosa. The rhizoid (r) and the cluster of desiccation-resistant cells(c1) are subterranean parts of the prostrate system. The chlorophyllous, aerial system (pr, sec) is analogous to the erect system of submerged algae (e.g Cham, Fig. I). [Fig. 3 of Singh (1941): Nem Phytol. 40, p. 173.1

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(Stewart and Mattox, 1975), so it cannot be on the direct evolutionary sequence to the higher plants. How far have the charophyte (and chlorophyte) algae, as represented by extant forms, achieved the complexity of structure characteristic of the simplest vascular land plants (Section III)? Much of the complexity shown by these land plants can be interpreted as a selective response to a spatially non-uniform supply of water, minerals, light and C 0 2 (the first two from the soil, the last two from the aerial environment). However, despite the absence of this extreme polarity of material and energy supply, many green algae have attained considerable structural complexity. Aquatic algae are completely surrounded by an aqueous medium. Even in the most massive green algae each cell has fairly immediate access to the bathing medium. This is important in terms of the supply of water, mineral salts and COz to the cells. Also important, but less commonly emphasized, is the use of the bathing medium as a sink for toxic by-products of metabolism. Quantitatively important toxic products of essential (primary) metabolism are the H' produced in excess of the cells requirements when using ammonium ion as N-source and excess OH- generated during nitrate assimilation (Raven and Smith, 1973, 1974, 1976a, b). Similar considerations apply to secondary metabolism (Freudenberg and Neish, 1968; Doyle, 1970). Further, all cells are illuminated (with the exception of rhizoids which penetrate unconsolidated substrates, e.g. in the Charales), and so, if they have the necessary metabolic machinery, can photosynthesize. Thus the cells of multicellular submerged algae might be expected to show considerable metabolic independence. Nai've expectation is that each cell can make its own reduced carbon, take up its own minerals and water and excrete its own toxic products. However many multicellular algae show much more cellular interdependence than this: indeed, such interdependence may be considered a sine qua non of a multicellular organism rather than a colony (Raven, 1976a). Both the charophyte and the chlorophyte lines of green algae have multicellular representatives with localized growing points, non-green cells, parenchymatous construction, and with plasmodesmata linking the cytoplasm of individual cells into a symplasm (Fraser and Gunning, 1969, 1973; Stewart and Mattox, 1975; Pickett-Heaps, 1975; Robards, 1975; Gunning and Robards, 1976a, b; Raven, 1976a; Table I, Figs 1, 2 and 3). The plasmodesmata (Fig. 3) probably function in these organisms in the transfer of photosynthate from mature photosynthetic cells to non-green cells (Raven, 1976a). As has been pointed out by Raven (1976a), demand for photosynthate in the dividing and expanding cells in algae with localized growing points exceeds the supply from local photosynthesis, again demanding intercellular transport. Similar consideration may apply to inorganic solutes (Tyree et al., 1974; Hope and Walker, 1975). The symplast may also function in the transmission of information (electrical or chemical) required for differentiation.

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The chlorophyte and charophyte algae have apparently not developed a “super-symplast” transport system such as is found in the phloem of vascular land plants and in large phaeophyte algae and, in a less structurally complicated form, in bryophytes and in rhodophyte algae (Eschrich, 1970; Richardson, 1975). The cytoplasmic streaming found in those chlorophyte and charophyte algae which possess giant cells may be an alternative method of increasing nutrient fluxes around the thalli of these algae (Tyree et al., 1974; Hope and Walker, 1975; Bostrom and Walker, 1976).

Structural

and

Functional ~

TABLE 1 Characteristics of Charophyte Algae ~~

~

Extant ~

Chlorophyte

~~

and ~

Examples from Characteristic

Chlorophyt e

Charophyte

Fritschiella Stigeoclonium Schizomeris Bulbochaete Dra par na ld ia

Coleochaete Chara Nitella

Non-green vegetative cellsa

Bulbo chaete Fritsch iella

Chara Nitella

Localized growth

Stigeo clonium b Drapartzaldia Cladophora

Parenchymatous aowth

Sch izo meris Fritschiella (?)

Plasmodesmata

Colaeochaete Chara Nitella Chars Nitella

] nodes

a There is also a tendency toward loss of photosythetic competence in reproductive cells of algae; this is found in many male reproductive cells, a number of female reproductive cells, and in relatively few asexual reproductive cells. b Localized growth (apical) is found in the basal, not the erect portions of this heterotrichous plant.

The alternative to symplastic transport of solutes around algal thalli is apoplastic transport in the free space (Tyree, 1969); since all the cells of these plants have ready access to the aqueous medium as source and sink for solutes, apoplastic transport would be very leaky and inefficient (Raven, 1976a). The division of labour found in the more complicated chlorophyte and charophyte algae, and the associated symplastic transport systems, are essential aspects of the functioning of an efficient homoiohydric land plant (Walter and Stadelmann, 1968). Also the production of an efficient intercellular gas-phase

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Fig. 3. Plasmodesmata in the chlorophyte alga Bufbochaete hiloensis. (A) Electron micrograph of a longitudinal section of a filament of B. hitloensis. Plasmodesmata (arrowed) are found in the walls separating the chlorophyllous cells. Growth is localized (intercalary) in this alga, so t h e plasmodesmata may be involved in the nutrition of growing cells and in regulation of growth. [Fig. 2 of Fraser and Gunning (1969).] (B) Electron micrograph of

EVOLUTlON OF VASCULAR LAND PLANTS

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the cell wall between a chlorophyllous axial cell and a chaeta-bearing, lateral “bulb” cell. Plasmodesmata ( m o w e d ) are also present in this wall. The “bulb” cell lacks plastids, and hence chlorophyll and (since it is a chlorophyte) starch; the plasmodesmata are probably involved in t h e nutrition of this heterotrophic cell (Frascr and Gunning, 1973). [Fig. 3 of Fraser and Gunning (1969).]

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transport system, which is another essential feature of the homoiohydric land plant, requires a parenchymatous plant body at least as bulky as that found in the freshwater chlorophyte alga Schizomeris (Mattox et al., 1974; see Section V). Before proceeding to examine what other structural and functional attributes characterized the earliest land plants (Section III), it is perhaps worth pointing out that the fossil record is not very helpful in demonstrating what chlorophyte and charophyte algae preceded, or were contemporaries of, the earliest land plants in the uppermost Silurian deposits (Banks, 1970, 1972, 1975). Certainly the Charales, which probably shared a Coleochute (or Trentopohlia?-like) ancestor with the vascular plants (see above; Stewart and Mattox, 1975), are found in earlier Silurian rocks than the vascular plants (Grambast, 1974). Siphonaceous marine chlorophyte algae, mainly calcified, have been found in Ordovician deposits (Banks, 1970); however, these cannot have been on the main line of land plant evolution. A further search of freshwater Silurian deposits for remains of chlorophyte and charophyte algae is required (Banks, 1970; Wickarder and Schopf, 1974; h a l l and Barghoorn, 1975; Kazmierczak and Golubic, 1976).

111. THE STRUCTURE OF EARLY VASCULAR PLANTS

The geological stages of the Upper Silurian and Lower and Middle Devonian periods in which the earliest vascular plants were found are shown in Fig. 4, together with the classes and orders of plants found in them, following the classification of Sporne (1975). The earliest vascular plant is the Downtonian Cooksonia (Psilopsida, Rhyniales, Fig. 5 ) . This plant is known from aerial axes which were smooth, dichotomizing and with terminal sporangia. It had tracheids (Fig. 5 ) , a cuticle but no stomata (Lang, 1937; Banks, 1970, 1972, 1975; Chaloner, 1970, 1975; Sporne, 1975). Cooksoniu is also present in the next stage (Dittonian or Gedinian); another genus of vascular plants in these deposits is ZosterophyZlum (Lele and Walton, 1961; Fig. 6) which is a member of the Zosterophyllales (Psilopsida) which have lateral sporangia in contrast to the terminal sporangia of the Rhyniales. Zosterophyllum had axes with a complex branching pattern and a central vascular strand, a cuticle and stomata of the single-celled, moss type: preservation is inadequate to establish if intercellular air spaces occurred (Lele and Walton, 1961; Walton, 1964; Edwards, 1969, 1975; Chaloner, 1970). In the two later stages (Siegenian and Emsian) of the Lower Devonian many more genera of vascular plants are found, belonging to the Rhyniales ( e g Rhynia), Zosterophyllales (e.g. Asteroxylon), and Trimerophytales (e.g. Psilophyton). Many of these plants are known from petrified materials, which allows their anatomy as well as their external morphology to be investigated (Fig. 7; Kidston and Lang, 1917, 1920a, b, 1921a, b; Lyon, 1964; Banks etal.,

Period

Vascular plants represented

Stage

Terrestrial plant features represented

Sphenopsids Pteropsids

/

/

Lycopsids I

I

I

I

370

I

Emsian 374

Devonian Siegenian

390 Gedinian (Dittonian)

/

39

/

-

Downtonian ?400

Silurian

Ludlovian Wenlockian 41 5 -

Fig. 4. The occurrence of different orders of vascular plants and of terrestrial plant-structures in the Upper Silurian and Lower Devonian (see

Chaloner, Chaloner, 1970; 1970;Banks, Banks, 1974; 1974; Sporne, Sporne, 1975). 1975).

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1975). These investigations show that these plants have all of the features essential to a homoiohydric vascular plant (Sections IV, V and VII; Walter and Stadelmann, 1968). These plants had a branched parenchymatous axis with a protostele of tracheids. The underground portion may, in some cases, have been mycorrhizal (Kidston and Lang, 1921b; Baylis, 1974; Pinozynski and Malloch, 1975; Nicolson, 1975). The aerial portions had a cuticle, with stomata of the type with two guard cells characteristic of extant vascular plants. These allow gaseous diffusion between the atmosphere and an extensive intercellular space system in the cortex of the aerial axes, where photosynthesis is presumed to occur, and in the underground portion (see Appendix B). Anatomical evidence for a true phloem (i.e. with sieve pores) in these plants is inconclusive; the

(A) Fig. 5. Cooksonia, the earliest known vascular plant (Rhyniales). [Micrographs from D. Edwards.] (A) Micrograph of a compression fossil of the whole plant (pin as a scale object); note the dichotomizing axes with terminal sporangia. (B) Micrograph of a spore; note the triradiate marking. The walls of these spores probably contained sporopollenin. (C) Micrograph of a tracheid; note the annuiar thickening (lignin?).

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occurrence of sieve pores in later (Carboniferous) petrifications shows that the preservation of sieve plates is possible (see Section VIB). There also appears to be no direct evidence for plasmodesmata in any plant fossil, although petrifaction of such small (Gunning and Steer, 1975; Robards, 1975) structures might not be expected.

Fig. 6. A single stoma, showing the apparently undivided annular guard cell surrounding the pore. [Fig. 22 of Lele and Walton (1961).]

(B) Fig. 7.Rhynia spp. and Gosslingia breconensis (Rhyniales), and Psilophyton princeps (Trimerophytales). (A) Reconstruction of R. Gwynne-vaughanii. Note the erect, dichotomizing aerial axes bearing terminal sporangia, and the horizontal subterranean axis. [From D. Edwards.] (B) Micrograph of a transverse section of the central region of an axis of R. major. In the centre is the endarch xylem. Surrounding this is the thin-walled “phloem” then comes the inner cortex with large intercellular air spaces. [From D. Edwards.] -Continued.

168 J. A. RAVEN

Fig. 7-Continued. (C) Micrograph of a surface view of epidermis showing a stone with two guard cells (cf. Fig. 6B). [I:rom D. Edwards.] (D) Micrograph of a transverse section of G. breconensis, showing the central xylem strand, the cortex and the (supporting?) hypodermis. [From D. Edwards.]

(E) Micrograph of a longitudinal section of the xylem of P.princeps, showing annularly thickened tracheids. [From D. Edwards.] (F) Micrograph of a longitudinal section of the “phloem” of R. m j o r , showing “pits” which may correspond to sieve areas. [Fig. 2 of Satterthwaite and Schopf (1972).]

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IL

I

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IV. THE XYLEM AND LIQUID-PHASE WATER TRANSPORT A. THE TRANSPIRATIONAL FLUX OF WATER

The aerial portions of the vascular land plant have a large demand for water, in addition to that used directly in growth. Net C 0 2 fixation in the chloroplasts of the aerial portions of the plant requires a diffusion pathway for C 0 2 between the atmosphere and the carboxylation enzyme in the chloroplast. The COz concentration gradient which acts as the driving force for this flux (see Section VA) is produced by the fixation of C 0 2 in the chloroplasts during photosynthesis. However, there is generally a driving force for water vapour diffusion out of the leaf, since the water vapour concentration in the bulk air is less than that in equilibrium y i t h the wet cell walls of the photosynthetic cells, and the existence of a low-resistance pathway for gas diffusion allows this driving force to produce a net efflux of water vapour from the shoot (Heath, 1969; Raven, 1970; Nobel, 1974; Raschke, 1976). This situation is aggravated by the requirement for light absorption during photosynthesis. Some of the absorbed light, even at low irradiances when light is limiting the photosynthetic rate, is unavoidably degraded to heat. This raises the temperature of the photosynthetic cells, and hence the driving force for water loss. Neither man nor nature has been able to produce a material which has a high permeability to C 0 2 but a low permeability to HzO (Martin and Juniper, 1970; Poljakoff-Mayber and Gale, 1972). Thus normal photosynthesis with simultaneous aquisition of light energy and atmospheric C 0 2 involves considerable water loss (Black, 1973). The magnitude of the water lost in transpiration is, for a C3 plant, some 500 g per g net dry weight increase (Black, 1973; Raschke, 1976), and thus far exceeds the water retained within the plant during growth (about 5 g per g dry weight). In the steady state, the water lost in transpiration is replaced by the transport of liquid water from soil w h c h flows into the subterranean parts of the plant, and up the axis to the sites of evaporation. This mass flow of liquid water is driven by a gradient of water potential generated by the transpirational loss of water from the photosynthetic cells. Applying the Gradmann-Van den Honert catenary formulation (Gradmann, 1928; Van den Honert, 1948), the flux through each of the three components of the soil-plant-atmosphere continuum is the same, and equal to the flux through the whole system thus:

'

where Jw = water flux in cm3 liquid water (cm' leaf surface)-' s- , and the subscripts refer to the components of the system which is under consideration. The flux through each portion of the system can be expressed as the quotient

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of the driving force for water movement for that component and the resistance to water flow in that component, thus:

-A$ Jw =rW

where -A$ = the driving force (MPa difference in water potential between the ends of the portion of the pathway) and r, = the resistance to water flow through that portion of the pathway (MPa s-' em-'). Combining eqns (1) and (2): - -A $ (gas) - -A $(plant) J, = -A$ (soil) rw(soi1)

rw(p1ant)

(3)

rw(gas)

Application of this equation is complicated by the fact that the most appropriate units for the liquid-phase movement of water are not the same as those for the gas-phase movement. Thus a linear relationship between flux and driving force in the liquid phase is found if the driving force is expressed in terms of a difference in water potential, while in the gas phase such a linear relationship results from expressing the driving force in terms of differences in water-vapour concentration. This difficulty has been ably discussed by Phillip (1 966). By working in conductances (permeabilities) rather than resistances, it is possible to express all of the driving forces in the same units; when this is done for well watered plants it is found that the driving force in the gas phase is much higher (10-100 times) than the driving force in the liquid phase, i.e. the main resistance to the transpirational flux occurs in the gas phase (Meidner and Sherriff, 1976; Weatherley, 1976). This has important implications for the location of variable resistances to transpirational water flux which regulate water loss from the plant without large changes in plant water content (see Section V). For the subsequent discussion in this Section, however, the important point is that the low resistance to water transport within the plant requires an efficient (low-resistance) water-conducting system within the plant: the xylem. The occurrence of this low-resistance pathway is what makes possible the high transpirational flux of water through the plant without developing impossibly large (negative) water potentials in the photosynthetic cells in equilibrium with the upper portions of the pathway, or impossibly high tensions in the conducting pathway. The low-resistance pathway for longitudinal conduction is the xylem. It consists of dead, lignified, longitudinally elongate cells arranged end t o end, with varying degrees of loss of the end walls (Fig. 8). Water moves through the lumina of these dead cells by mass flow in response to the gradient of negative pressure generated by transpirational water loss. The movement of water by diffusion in the gas phase (in intercellular air spaces, or gas-filled xylem elements) is restricted to the pathway from the sites of evaporation to the bulk

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atmosphere, at least as far as making a significant contribution to the transpirational flux is concerned. The resistance to mass flow of liquid water is much less than is that to gaseous diffusion of water (Section VB (3); eqn. (3)), which is in turn much less than that to aqueous phase diffusion (self-diffusion) of water (Levitt, 1956). B. THE XYLEM AS A LOW-RESISTANCE PATHWAY FOR MASS FLOW OF WATER

The pathway of liquid water transport through the vascular land plant may be considered in three parts. Water moves radially inwards from the soil to the xylem across the living cells of the subterranean axis; it then moves longitudinally in the xylem; and finally it moves radially outwards from the xylem to the sites of transpiration through the living tissue of the

Fig. 8. Scanning electron micrographs of a transverse section (A) and a longitudinal section [From J. 1. Sprent] (B) of the xylem- in the stem of Ricinus conmunis. - n e large cells are vessels; note the reticulate pitting and (in B) the end of vessel elements with only the centrifugal portions of the end walls remaining. [From J. I. Sprent].

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photosynthetic portion of the aerial axis. Following the argument used in Section IVA, during steady-state transpiration the water flux through each portion of the plant pathway is the same, and the plant part of eqn. (3) can be expanded as follows:

J,

=

-A$(plant) r,(plant)

- -A$(root) -

r,(root)

-- -A$(xylem) -- -AiL(leaf) r,(xylem)

(4)

r,(leaf)

Boyer (1 971 ) has investigated these resistances in Helienthus and Glycine. The total resistance to liquid water flux within the plant was about 6 lo4 MPa s cm-' in Helianthus. The xylem of the root and stem contributed about 3 lo4 MPa s cm- , while the living tissue of the root and leaf each contributed about 1.5 * lo4 MPa s cm-' . In Glycine total plant resistance was higher (up to 16 lo4 MPa s cm-'), largely because the resistance of the pathway through living roots tissue is higher (6 lo4 MPascm-I). The magnitude of these

-

-

'

-

-

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resistances has also been discussed by Rawlins (1 963), Boyer (1 969), Weatherley (1970, 1975) and by Neumann et ul (1974). In herbaceous angiosperms the xylem generally contributes less than half of the total resistance; the radial pathway in the root is usually the highest resistance part of the plant resistance, and varies more with water flux and metabolic factors than the other resistances. While these absolute resistance values are of great importance in considering the regulation of water flux through the plant as a whole, the specific resistivity (or its reciprocal, the specific conductivity) of the xylem when compared with living tissue is more relevant to a discussion of the evolution of the xylem. The specific conductivity may be defined as the volume flux of liquid water (cm3(cm2 area of tissue normal to the water flux)-' s-') divided by the water potential gradient driving the water flux (MPa cm-'); it thus has the units cm2 s-l MPa-l. Hydraulic conductivity, L,, which is appropriate to measurements of systems in which the driving force gradient is not known, is the ratio of the water flux (cm s-') to the water potential (not the water potential gradient) driving the flux, and thus has the units cm s- Mpa- . The specific conductivity refers to unit cross-sectional area of a conducting pathway and to unit gradient of driving force, and thus may be used to compare the efficiency of various water transport pathways. A number of authors have discussed, or attempted to measure, the specific conductivity of xylem compared with that of the apo- and sym-plastic pathway of living parenchyma (e.g. Briggs, 1967; Slatyer, 1967; Kozinka and Luxova, 1971; Nobel, 1974; Newman, 1976; Weatherley, 1976). The specific conductance of the xylem of woody plants varies from 10 cm2 s-l MPa-' for conifers which contain only tracheids to 500 cm2 s-l MPa-' for ring-porous dicotyledonous trees and vines with large vessels (Briggs, 1967; Zimmermann, 1971 ). Explanation of these differences in terms of Poiseuille's equation, which predicts that the specific conductance of wood is proportional t o the fourth power of the radius of the conduits, has been attempted (Zimmermann, 1971, Table IV-3), but is complicated by the influence of the frequency of cross-walls in the conduits (Briggs, 1967; Nobel, 1974). Measurements of the specific conductance of the xylem of herbaceous plants is much more difficult, since isolation of xylem from other tissues is not easy in most cases (e.g. the Zea roots investigated by Kozinka and Luxova, 1971). In material such as the petiole of Heracleum mantegazzianum it is, however, possible to obtain relatively undamaged xylem segments, and these exhibit specific conductances of 50 to 255 cm2 s-' MPa-', i.e. within the range of values found in forest trees (Tyree and Fensom, 1970). In parenchymatous tissue the situation is much more complex. There exists the possibility of movement not only in the apoplast (i.e. the water-filled extracellular spaces), but also via the cells. This latter could involve movement through individual cells and the intervening apoplast (cell wall), or through a continuum of symplast, i.e. cells and the plasmodesmata which connect them.

'

'

EVOLUTION OF VASCULAR LAND PLANTS

175

This is discussed by Briggs (19671, Slatyer (19671, Tyree (1969, 19701, Weatherley (1970, 1975, 1976), Nobel (1974), Molz and Ikenberry (1974), Dainty (1976), Newman (1976) and by Gunning and Robards (1976a, b). Measurements of the specific conductivity of pieces of parenchymatous plant tissue are often complicated by injection of intercellular air spaces with solution (Briggs, 1967; Meiri and Anderson, 1970), leading t o overestimation of the specific conductivity by the production of spurious apoplastic pathways for water movement (e.g. Kozinka and Luxova, 1971). Experiments on roots avoid this problem and give values of L, for radial water transport of 0.2-2.10-’ cm s-l MPa-’ (Brouwer, 1954; House and Findlay, 1966; Ginzburg and Ginzburg, 1970; Meiri and Anderson, 1970; Hay and Anderson, 1972; Newman, 1973; Milthorpe and Moorby, 1974). These values of L , correspond to specific conductivities of 10-7-10-6 cm2 s-’ MPa-’ (Newman, 1976). It is unwise to take these values as typical for the specific conductivity of parenchymatous tissue in longitudinal water transport in comparison with the specific conductivity of xylem due to two peculiarities of the radial transport in roots. One is that the presence of suberization in the endodermis may greatly impede apoplastic transport, both in the purely apoplastic pathway in radial cell walls and as part of a cell-to-cell transport involving the tangential cell walls (Slatyer, 1967; Clarkson and Robards, 1975; Newman, 1976). The other peculiarity is that the short path-length involved in radial water transport in the root will magnify the “end effects” of transmembrane apoplast-symplast exchange involved in water transport through the symplastic pathway which the blocking of the apoplastic route may involve (see Spanswick, 1976). Thus the specific conductivity of up t o cm2 s-’ MPa-’ may be an underestimate for the “mid-stream’’ specific conductivity of parenchymatous tissue in longitudinal transport, although it is unlikely t o be more than an order of magnitude t o o low (see below). Considering first the purely apoplastic pathway through longitudinal cell walls, the specific conductivity of plant cell walls is probably up to cm2 s-’ MPa-’ (Briggs, 1967; Tyree, 1968; Lauchli, 1976; Newman, 5* 1976). If the cell walls occupy 5% of the cross-sectional area of the tissue, then the specific conductivity of the tissue due to this pathway alone is 0.05) or 2.5 * l o p 7 cm2 s-l MPa-’. (5 A “cell-to-cell” pathway in parallel with this involves water movement across two plasma membranes per cell; if there are 200 cells cm-’ (Tyree, 1969) and L , for the plant membrane is 2 * lo-’ cm s-’ MPa-’ in higher plant parenchyma (Clarkson, 1974; Newman, 1976) this pathway will contribute lo-’ cm2,s-’ MPa-’ t o the specific conductivity of parenchymatous tissue (ignoring the fact that this pathway only occupies about 90% of the cross-sectional area of the tissue, and that there are intracellular and cell-wall resistances to water transport through it). Thus a parallel apoplastic and cell-to-cell pathway could give a specific

-

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conductivity for the tissue as a whole of (2.5 + 1.0) or 3.5 * lo-’ cm2 s-’ MPa-’. While this is in the range reported for the specific conductivity for radial transport in root parenchyma, it must be remembered that both of these pathways, involving as they do water movement through the cell wall, may be seriously impeded in the root (see above). cm2 s-’ MPa-’ as the upper Tyree (1970) has computed a value of limit for the specific conductivity of the symplastic pathway of water transport in parenchymatous tissue. However, there is little evidence that the capacity to permit mass flow of solution through plasmodesmata is a widespread attribute of the symplast (Spanswick, 1976; Robards and Gunning, 1976a, b). Furthermore, Tyree’s analysis does not include intracellular resistances to water flow. Water movement from one array of plasmodesmata to those in the opposite cell wall is (depending on the conductivity of the two possible pathways) through the small amount of viscous peripheral cytoplasm, which only occupies a few per cent t o the cross-sectional area of the tissue, and through the vacuole, which involves traversing the tonoplast ( L p of about 2 * lo-’ cm s-l MPa-’) twice in crossing a single cell. Thus it seems more realistic to take cm2 s-’ MPa-’ as an absolute upper limit on the specific conductivity of the symplastic pathway for water movement for water already present in the symplast, i.e. ignoring the “end effects” of entry and exit (see above). Such a value is consistent with the abserved specific conductivity of the radial pathway in roots when the “end effects” of water entry into the symplast from the bathing medium, and water exit into the xylem exudate are taken into account. Taking l o p 5 cm2 s - l MPa-’ as the upper limit for the specific conductivity of parenchymatous tissue in longitudinal conduction, we see that the specific conductivity for longitudinal water flux of xylem consisting of tracheids alone (10 cm2 s-l MPa-’) is l o 6 higher. Since in Downtonian and Lower Devonian vascular plants the dimensions of both the tracheids and the parenchyma cells are similar to those of the extant plants for which these specific conductivity figures were derived (Figs 5 and 7; Kidston and h g , 1917, 1920a, b ; Lang, 1937; Edwards, 1969; Zimmermann, 1971; Banks et al., 1975), it will be assumed that this ratio of lo6 also applied to these early vascular plants. Plants such as Zosterophyllum and Rhynia had a xylem which occupied about 1% of the total axis cross-sectional area (Fig. 7) (Edwards, 1969; Kidston and Lang, 1917, 1920a), while in Psilophyton the value is up to 10% (Banks et al., 1975). Taking the lower ratio for xylem : parenchyma, and the ratio of specific conductivities for the two tissues, the ratio of water movement through the xylem t o that through the parenchyma when the driving force is the same for both tissues is lo6 or l o 4 . Thus even a small xylem strand composed entirely of tracheids is lo4 times more efficient in water transport than is the remaining, parenchymatous, tissue of the axis. Since an individual xylem strand of Zosterophyllum has about 100 tracheids in transverse section, we may conclude that even a single file of tracheids in such an axis would carry much

-

EVOLUTION OF VASCULAR LAND PLANTS

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more water than the remaining, parenchymatous, tissue. This would present an unstable situation, in that the tension in such a single file of tracheids would be high during transpiration, and if cavitation (Milburn and McLaughlin, 1974) occurred there is no “back-up” system available. However, this does suggest that the very small xylem strand found in the earliest vascular plant, Cooksoniu (Lang, 1937), may have been of considerable selective advantage (cf. Passioura, 1972). A similar ratio obtains in the early vascular plant fossils mentioned above, provided plants with similar axis diameters are compared (Fig. 9; Metcalfe and Chalk, 1950; House and Findlay, 1966). The angiosperms have vessels in their xylem, which probably increases the specific conductivity of the xylem and hence (in view of the similar fraction of the axis occupied by the xylem) the specific conductivity of the stem as a whole compared to the early fossil vascular plants. However, it is likely that, prior to the evolution of plant leaves, the transpiring surface associated with an aerial axis of a certain radius was smaller than in extant plants; further, it is found that in extant trees the increased specific conductivity of the xylem due to the presence of large vessels is frequently offset by a smaller fraction of the xylem being functional in conduction (Zimmermann, 197 1). Thus the xylem is the predominant tissue involved in the long-distance transport of liquid water in vascular plant axes. However, water transport from the soil to the xylem, and from the xylem to the transpiring surface, involves transport through living parenchymatous tissue over a distance of several cell diameters (see above). The overall resistance of the parenchymatous pathway in herbaceous angiosperms is probably greater than the overall resistance of the xylem (Rawlins, 1963; Boyer, 1969, 1971; Neumann et ul., 1974). However, the specific resistance of the parenchymatous pathway is about lo6 greater than that of the xylem pathway. This difference between absolute and specific resistance is explained if the lengths and areas of the two diffusion paths are examined. Thus the parenchymatous pathway at either end of the pathway is only a few millimetres in total length, while the xylem pathway of an herbaceous angiosperm such as Zeu or Heliunthus can be 1 m or more long. Further, the area over which the radial flux occurs through parenchyma at each end of the pathway is lo3 or so greater than the area over which longitudinal movement occurs in the xylem (Brigs, 1967; Sherriff and Meidner, 1975; Tyree et ul., 1975). These two factors of up to lo3 each can explain the similar absolute resistances of the parenchyma and the xylem parts of the pathway in herbaceous angiosperFs despite the difference of about lo6 in their specific conductivities. C. THE SIGNIFICANCE OF LIGNIFICATION

The cell walls of certain xylem elements are lignified, i.e. contain lignin as well as polysaccharide and protein. The significance of lignification is generally sought in mechanical support of aerial shoots. While unlignified cell walls can

I

500pm

i Y

4 00

Fig. 9. Micrograph of a transverse section of a young stem (not secondarily thickened) of the dicotyledon Ricinus communis. Note the peripheral arrangement of the vascular bundles, the large vessels (see Fig. 8) in the xylem, and the phloem region (see Fig. 12).

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withstand tensile forces, they are relatively weak in compression. Compressive forces are imposed by gravity on aerial shoots by virtue of their high density relative to the surrounding air. The large surface area required for photosynthesis means that wind exerts large lateral forces on aerial shoots, which results in compression of the leeward side. Lignification has enabled the plant to withstand compressive forces and thus to overcome the size restrictions imposed by the mechanical properties of a hydrostatic skeleton of turgid parenchymatous tissue (Haberlandt, 1914; Wainwright, 1970; Carlquist, 1975; Wainwright et al., 1976). The nature of the stresses in aerial shoots suggests that the greatest economy of structural materials is achieved by peripheral distribution of this lignified tissue: such is indeed the case in many extant vascular plants (Haberlandt, 1914; Carlquist, 1975; Wainwright et d., 1976). However, it appears that lignified tissue was not a major mechanical support in the earliest vascular plants (Upper Silurian and Lower Devonian). In these plants the only lignified tissue appeared to be confined to the xylem. This was protostelic and consisted entirely of tracheids. As was pointed out above (Section IVA; Figs 7 and 9) the fraction of the stem occupied by lignified tissue in the early vascular plants was not much less than in extant herbaceous angiosperms with an axis of similar cross-sectional area. The mechanical efficiency of the lignified tissue in the angiosperms is greater since it contains fibres and is in a peripheral position; it has to withstand greater compressive forces from the action of wind and gravity, since an axis of a given size generally supports a larger area of photosynthetic tissue of greater lateral extent in angiosperms than in the early vascular plants. The main mechanical support in early vascular plants (and in such possible ancestors as the non-vascular Echostirnella) was a hypodermal tissue of isodiametric, thck-walled cells (Schopf et al., 1966; Carlquist, 1975). This thick-walled tissue did not form a complete hypodermal layer in plants (e.g. Rhynia, Psilophyton) with stomata and intercellular gas spaces (Section V). Between the stomata and the inner cortex, which has large intercellular gas spaces and was probably the site of photosynthesis, were thin-walled hypodermal cells with large intercellular spaces (Fig. 7); this maintained a pathway for gaseous diffusion between the atmosphere and the sites of C 0 2 fixation (Kidston and Lang, 1917, 1920a; Banks et d., 1975). Thus the peripheral strengthening tissue probably did not greatly inhibit C 0 2 supply for photosynthesis, although it may have attenuated the light supply to a small extent by scattering. . A more important structural role for lignified tissue is found in the Middle Devonian progymnosperms, which are the probable ancestors of extant seed plants (Banks, 1970). These plants had cambial activity of the “normal” type found in conifers and dicotyledons (Eames and MacDaniels, 1947; Corner, 1964; Barghoorn, 1965). The extensive secondary xylem of these plants contained lignified xylem and unlignified parenchyma (Scheckler and Banks, 1971a, b).

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They were also the first plants to have lignified cells specialized for the mechanical supporting rather than the conducting role (fibres) in the secondary phloem and the cortex. In Upper Devonian and later strata the mechanical role of lignin was further emphasized by the tendency for a peripheral location of the primary vascular tissue during stelar evolution, and ‘the occurrence of peripheral schlerenchyma in axes lacking secondary thickening. Finally, with the evolution of angiosperms in the Jurassic and Cretaceous periods, fibres occur in the xylem. This provided additional mechanical support in the xylem when the specialization of tracheids for efficient conduction gave rise to the mechanically less strong vessels (Foster and Gifford, 1974; Carlquist, 1975; Eames and MacDaniels, 1947). Thus support of the aerial axis did not appear to be the original function of lignified tissue in vascular plants. The occurrence of lignin in the water-conducting tissue suggests that lignification may have a role in water transport per se. Such a role could be in resisting the tensile forces generated during transpiration. The tension which develops within the lumen of the dead conducting cells (Zimmermann, 197 1) is translated into compression in the cell walls of these cells (Crafts, 1961; Wainwright, 1970; Carlquist, 1975). Lignification would help to resist this force and prevent collapse of the conducting cell (Molz and Klepper, 1973), and it is suggested that the earliest mechanical role of lignin is to be found here. The tracheids found in Upper Silurian and Lower Devonian sediments generally have annular or spiral thickenings (Kidston and Lang, 1917, 1920a, b ; Lang, 1937; Edwards, 1969; Banks et al., 1975). The absence of such thickenings, e.g. in Rhynia major, has been attributed to poor preservation (Kidston and Lang, 1920a). This pattern of thickening is consistent with a role in preventing collapse of the conducting elements under tension, although direct proof of lignification has not so far been forthcoming. The “rubbery wood” disease of apple trees is of interest in relation to the mechanical role of lignin (see literature cited by Nelmes et al., 1973). This disease is probably caused by a mycoplasma, and involves an altered pattern of lignification (decreased lignin content, changes in the substituents in the aromatic nucleus, and dissociation of lignin from wall polysaccharides) in the vessels and fibres of the wood. It results not only in the structural defect from which the name of the disease is derived, but also in the collapse of vessels (Beakbane and Thompson, 1945). This is consistent with a role for lignification in resisting collapse of water-conducting xylem elements. The protoxylem of many plants is disrupted during axis elongation. The lacuna which is left (e.g. the carinal canal of Equisetum) is an intercellular space of lysigenous and schizogenous origin, in contrast t o the intracellular, lysigenous xylem elements. It has been suggested that these canals function in longitudinal water transport; however, the occurrence of a transpiration stream in them has only been demonstrated under small water tensions (Bierhorst, 1958). Since it is

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to be expected that such channels have very limited protection against cavitation due t o the entry of air or through collapse under tension, it would be of interest t o compare water transport in them with transport in normal xylem elements under various water potential gradients. Thus the lignified tissue of extant plants at once provides mechanical support for photosynthesizing and transpiring surfaces up t o 100 m above the soil surface, and prevents implosion of the xylem elements which carry water to these surfaces under the tension which is implicit in the cohesion mechanism of water transport. Differentiation of dead cells has no precedent in the algae (Fritsch, 1935, 1945a; Foreman, 1976); some bryophytes have an analogous conducting tissue to xylem in the form of hydrome (Doyle, 1970; Hebant, 1970). This differentiation of dead tissue may have further evolutionary significance in that the hydrolysis of proteins and nucleic acids which it involves may produce growth substances such as indoleacetic acid and cytokinins (Sheldrake, 1973). Biochemical aspects of the evolution of lignification are considered in Appendix A.

V. TRANSPORT IN THE G.4S PHASE A. THE PROB1,EhI OF H 2 0 LOSS AS A CONCOMITANT OF PHOTOSYNTHETIC C 0 2 FIXATION: POIKILOHYDRY

Photosynthetic assimilation of atmospheric C 0 2 requires a low-resistance diffusion path for C 0 2 between undepleted air and the photosynthetic cells: in the absence of a substance with a much greater permeability t o C 0 2 than HzO, this involves a loss of water (Section IV). When soil water is plentiful this water loss is acceptable, provided the water conduction pathway is efficient enough to allow exploitation of this water without requiring too low a shoot water potential. However, when soil water is depleted, an efficient xylem is of no avail in supplying the transpiring shoot with water. Poikilohydric plants cannot regulate water loss ftom their aerial parts, and desiccate under water stress. Such plants, which can tolerate desiccation of the vegetative plant body, include terrestrial algae and bryophytes, and “resurrecrion plants” among the vascular plants (Bertsch, 1966; Walter and Stadelmann, 1968; Levitt, 1972; Gaff and Hallam, 1974). Growth can occur at varying degrees of water loss in these plants; only survival is possible at the lowest water contents. Poikilohydry has a number of metabolic and structural implications for the plant (Hsaio, 1973; Gaff and Hallam, 1974). One is that poikilohydric land plants tend t o have less extensive vacuoles than their piesumed green algal ancestors (Walter and Stadelmann, 1968). This may be related to the greater mechanical stresses in the cytoplasm as water is lost from a large vacuole (Walter and Stadelrnann, 1968; Giles et al., 1976; Greenway and West, 1973). Possible selective advantages of the occurrence of large vacuoles in land plants are discussed by Raven and Smith (1976a, b) and in Section IV.

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B. HOMOIOHYDRY IN VASCULAR LAND PLANTS AND THE INTERCELLULAR SPACE-CUTICLE-STOMATACOMPLEX

1. General Considerations Many vascular land plants are homoiohydric. This means that they can regulate their rate of water loss to such an extent as to remain hydrated when water supply is considerably restricted (Maximov, 1929, 1931; Walter and Stadelmann, 1968). This regulatory system involves a variable resistance to gas exchange between the external atmosphere and intercellular gas spaces; the variable resistance takes the form of stomata1 pores of variable aperture which perforate a cuticularized, relatively gas-impermeable epidermis (Troughton and Donaldson, 1972; Fig. 10). When water supply to the shoot is adequate to

Fig. 10. (A) Scanning electron micrograph of a transected mature leaf of Viczh faba. The upper and lower epidermes (E) enclose the photosynthetic tissue consisting of spongy mesophyll (M) on the lower side and palisade cells (PI, elongated at right angles to the epidermis. The leaf has an extensive system of intercellular air spaces such that the area of cells exposed to the internal atmosphere is about ten times the external surface area of the

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replace a large transpirational loss, the stomata open, allowing net water loss and also, in the light, net entry and fixation of atmospheric CO,. When water supply to the shoot is restricted by depletion of soil water, the stomata close. This greatly restricts water loss in transpiration, but also restricts net COz entry for photosynthesis. The position of the stomata as the major variable resistance in the soil-plant-atmosphere water flux pathway (Section IV) is crucial; when their resistance increases, water loss is reduced and the whole plant remains hydrated. If the major variable resistance were somewhere in the liquid-phase water pathway t o the plant, then increase in this resistance under water stress would only allow those tissues on the soil side of the resistance to remain hydrated. Further, the stomata are in the gas-diffusion part of the water-transport

leaf. [Plate 38, p. 48 of Troughton and Donaldson (1972).] (B) Scanning electron micrograph of a portion of the lower surface of a leaf of Cucumis sativus, showing the smooth cuticle, and an open stomate. The photosynthetic cells of the spongy mesophyll czn be seen through the stomata1 pore. [Plate 29, p. 39 of Troughton and Donaldson (1972).]

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pathway; this has the largest resistance to water flow of any part of the plant water-flux pathway (Section IVA) so that a given fractional change in resistance has a much greater effect on the overall water flux in the gas phase than in the liquid phase (see Slatyer, 1967, pp. 233-4). It must be emphasized that the homoiohydry achieved in this way is never absolute, in that even plants with the most water-resistant cuticles still have some net water loss when the stomata are closed and no water is being supplied via the roots (Maximov, 1929, 1931; Larcher, 1975, Tables 27 and 32). Many homoiohydric plants can avoid the effects of prolonged water deficit on their vegetative body by producing desiccation-resistant seeds or spores (Maximov, 1929; Walter and Stadelmann, 1968).

2.

Resistance to Gas Exchange Imposed by Stomata and Cuticle Gas exchange between the aerial shoot and the bulk atmosphere in vascular plants can be analysed by the Gradmann-Van den Honert method (Section IVA; cf. Jones, 1973). Thls approach has been widely applied to water vapour loss in transpiration, and to COz uptake in photosynthesis; it has been less widely used in considerations of O2 exchange (Heath, 1969; Raven, 1970; Samish, 1971, 1975; Nobel, 1964; Raven, 1 9 7 7 ~ ) . Water vapour loss during transpiration when the stomata are open can be expressed as:

where the transpirational flux is measured in nMole (cm2 external surface)-' s-' ; DF= driving force (nMole cm-3 difference in water vapour concentration between the ends of the portion of the pathway considered); r = resistance (s cm-') of the portion of the pathway considered, and the subscripts, g, bl and s refer to the total gas phase transfer of water vapour, and to its two components, the external boundary layer and the intercellular gas spaces plus stomata, respectively. The value of rg is determined from the measured transpirational flux and the measured total driving force for water vapour diffusion between the intercellular air spaces and the external bulk atmosphere. Determination of rbz involves measuring "transpirational" water flux from a paper model of the transpiring organ, saturated with water, and the driving force for water vapour loss from the model. The stornatal resistance can then be computed by difference as r, = rg - rb[. The minimum stornatal resistance for vascular plants is about 0.3 s cm-' for herbaceous angiosperms, and about 2 s cm-' for annually deciduous dicotyledonous trees (Holmgren et al., 1965; Slatyer, 1967; Larcher, 1975). The value o f rbl is a function of wind speed, dropping from 1-3 s cm-' at a wind speed of 0.1 m s - I to 0.1-0.3 s cm-' at a wind speed o f 10 in s - l .

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A similar analysis can be carried out when the stomata are closed, resulting in an estimate of the cuticular resistance, rc. This is generally at least an order of magnitude higher than the stomata1 resistance, i.e. it is rarely less than 10 s cm-’ (Holmgren et al., 1965). For crop plants it is commonly 20-80 s cm-’, while it can be greater than 200 s cm-’ in xerophytes (Slatyer, 1967; Larcher, 1975). a s range of values for r, helps to explain the variation between plants in their ability to curtail water loss when the stomata are closed (Section VB(1)). The path of C 0 2 in photosynthesis involves the transport of C 0 2 from the bulk air through the boundary layer and the stomata and intercellular gas spaces to the cell walls bounding the intercellular spaces. There it dissolves in the cell water and diffuses in the liquid phase to the site of carboxylation, where it is fixed in the biochemical reactions of photosynthesis. If true (gross) photosynthesis is considered, i.e. net photosynthesis corrected for all CO, production in “dark” respiration and photorespiration, then the C 0 2 concentration at the end of this reaction sequence is zero (Raven, 1970). All of these portions of the reaction sequence-the biochemical as well as the transport processes-can be incorporated into a formulation analagous to eqn. (5). OF; DFAI - DF; - OF; - OF; p=---=--------(6) r; rbl rk r; rb Units and symbols are the same as in eqn. (4); additional symbols are the prime, indicating that C 0 2 rather than water is being considered; and the subscripts t, 1 and b which refer to the total reaction sequence, and its liquid phase diffusion and biochemical reaction components, respectively. A value of the photosynthetic C 0 2 flux, P, of 1.5 nMole C 0 2 (cm2 external surface)-’ s-’, and a ratio of internal to external area of the photosynthetic organ of 10, will be used in illustrating this approach; these values are typical ones for C3 pathway “sun” plants (see Raven, 1970; and Table 4 of Raven and Glidewell, 1975). The flux of C 0 2 in the gas phase follows essentially the same pathway, but in reverse, to that taken by water vapour during transpiration (Slatyer, 1967; Jones and Slatyer, 1972). The resistance to gas diffusion is, for water vapour,

k

1;

I

CO,,rg=, Dg

where 1 = effective len’gth of the gaseous diffusion path (cm), and D = diffusion coefficient (cm’ s-’); the subscript g indicates, as before, that it is the gas phase = r g D;/D,. Thus, with which is being considered. Since lg = I;, Dg = 0.16 cm2 s-l and D i = 0.24 cm2 s-l we can compute the minimal values for rbl and ri from the corresponding values of rbl and r, as being 0.2 and 0.5 s cm-’ respectively. For the value o f flux of 1.5 nMole (cm2 external

ri

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surface)-' s-' , this means that the total C 0 2 gradient between the bulk air and the cell walls bounding the intercellular spaces inside the leaf is 1.5/0.7 = 1 nMole cm- j . Thus the CO? concentration at the start of the aqueous phase diffusion pathway is only 1 nMole cm-3 less than the 10 nMole cm-3 in the atmosphere and the resistance imposed by gaseous diffusion of C02 in a C3 vascular land plant can account for as little as 10% of the total resistance to C 0 2 fixation. With respect to the remaining components of the pathway of C 0 2 fixation (eqn. (6)) which can comprise up to 90% of the total resistance to C 0 2 fixation, the value of r; can be calculated from the relationship of r; = 1;D;.For a typical C3 plant with a single layer of chloroplast just inside the plasmalemma of the mesophyll cells, the average value of 1; is 1.5 * l o p 4 cm if the chloroplast thickness is up to 3 pm and the cell wall is of a normal thickness for a mesophyte (see Rackham, 1966; Raven, 1970; Nobel, 1974; Jacobsen et al., 1975). The value of D; will be taken as 1.5 lo-' cm2 s-l (Raven, 1970; Nobel, 1974; Jacobsen et al., 1975). Thus r; is 1.5 104/1.5 lo5 or 10 s cm-' on the basis of internal exposed area; on the basis of external exposed area it is 1 s cm-' (since the external area is 1/10 of the internal; Raven, 1970). With the flux of 1.5 nMole (cm2 external area)-' s - l , the driving force is 1.511 or 1.5 nMole crnp3. Since the C 0 2 concentration in the intercellular spaces (which is equal t o that in the liquid phase of the cell wall) has already been computed as being 9 nMole cniP3, this means that the C 0 2 concentration at the site of carboxylase (ribulose diphosphate carboxylase) activity is 7.5 nMole cm- 3. Thus in the case considered here, 75% of the resistance t o overall photosynthesis is in the biochemical reactions, provided that the stomata are wide open and the boundary layer is dispersed by wind. Under these conditions, there is a large biochemical resistance in series with the gas-diffusion resistance to C 0 2 uptake, while the resistance to liquid-phase water-flux from a moist soil up t o the transpiring surface, involving mass flow of water, is very small relative to the series resistance to water movement by gas-phase diffusion (Section IV). This has important implications for the relative effects on photosynthesis and transpiration of changes in gas-phase resistance (wind speed or stomata1 aperture); transpiration will be affected more than COz fixation (see Black, 1973). Estimates of cuticular resistance to C 0 2 movement (r:) show that it is greater than the cuticular resistance to water movement, r, (Holmgren et al., 1965). Thus lL/DL exceeds lc/D,; it is not clear whether this reflects 1; > 1 2 , or 0:< D, (Holmgren et al., 1965). At all events, that data are consistent with the inability of nature to devise a material with D' 3 D (see Section VB(1)).

-

3.

-

The Czucial Role of the Intercellular Air Spaces in the Homoiohydric VascularPlant The intercellular gas spaces constitute an essential (Whitehouse, 1952; Corner, 1964, p. 161; Hays, 1975) but frequently neglected part of the homoiohydric gas exchange system. Stomata opening onto a tissue without such spaces would

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not constitute an effective system for the supply of atmospheric C 0 2 to more than the ch!oroplasts within about 10 p m of the stoma. Such a tissue would have a photosynthetic rate in air, at light saturation, of not more than 10% of that found in a dimensionally similar tissue with a typical intercellular air space system, such as that considered in Section VB(2). This conclusion is based on a consideration of the diffusive and biochemical resistances to COz fixation in such a tissue. We shall consider a photosynthetic organ lacking air spaces with chloroplasts of the same dimenstions and biochemical activity as those in the plant considered in Section VB(2) arranged in a single layer as close as possible to the plant-air interface. Then, if the plant has no cuticle, the resistances to C 0 2 fixation (s cm-') are r i l (0.2), r; (10) and r; (50), all on the basis of the external surface area of the plant. With an external CO2 concentration of 10 nMole, we can compute a photosynthetic rate of 10/(0.2 + 10 + 50) or 0.17 nMole, (cm2 external surface)-' s-' (eq. ( 5 ) ) . This rate can be increased if a further layer (or layers) of chloroplasts are added as near t o the air-plant interface as possible, thereby minimizing the value of 1; and hence r;. With the size of photosynthetic organ necessary to permit the ratio of internal to external surface (10) quoted in Section VB(2), it may be assumed that the area of each layer of chloroplasts is equal to the external area of the organ (regardless of its planar or cylindrical shape). Addition of successive layers of chloroplasts yields successively smaller increments in photosynthesis. This is because (a) the aqueous phase diffusion path from the outside air to the chloroplast (1;) is greater for each successive layer; ( b ) the available C 0 2 is depleted by photosynthesis in chloroplast layers closer to the external air; and (c) the rate of C 0 2 fixation may be taken as linearly related to the C 0 2 concentration at the carboxylation site in the range 0-10 nMole cmW3(Raven, 1970; Lilley and Walker, 1975; Laing e l ul., 1974); this is implicit in the use of rb, eqn. (6). These considerations may be quantified by application of eqn. (6) to each successive layer of chlorop!asts. When this is done for five closely packed layers of chloroplasts, it is found that the total photosynthesis by the five chloroplast layers does not exceed 0.45 nMole (cm2 external surface)-' s-'. Since the COz concentration in the centre of the fifth layer of chloroplasts (13.5 pm from the plant-air interface) is less than 0.3 nMole cni-3, the carboxylase in this layer is operating at less than 5% of the rate in the first layer. It will be assumed for the purposes of further discussion that there would be no selective pressure for the addition of further chloroplast layers, since the very small return in increased photosynthesis must be considered in relation to the large investment of material and energy in constructing and maintaining chloroplasts. One possibility for increasing the rate of photosynthesis in such a tissue would be t o increase the concentration of photosynthetic enzymes in the chloroplast. This would increase the activity of the chloroplast on a volume basis, and hence decrease rb and would allow the carboxylase to be nearer to the

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source of C 0 2 , thereby decreasing 1; and hence r;. However, there appears to be very little scope for such an increase in concentration. A potentially rate-determining enzyme, ribulose diphosphate carboxylase, has a very. large molecule with a very low turnover number (Raven, 1970; Lilley and Walker, 1975; Takabe and Akazawa, 1975; Kung, 1976). To achieve the rates of photosynthesis per unit chloroplast volume that are observed, the enzyme is present at high concentration (about 5 mMole) and sometimes forms paracrystalline arrays rather than a true solution (Gunning et al., 1968; Holdsworth, 1971). The protein is less enzymically active in these arrays than it is in solution (see discussion in Raven, 1977a, c). This analysis of a vascular plant tissue without intercellular air spaces relates to a poikilohydric plant with very little diffusion resistance for C 0 2 between the outside air and the chloroplasts. If the plant is to be homoiohydric with stomata as a variable resistance in parallel with a fixed cuticular resistance, then some fraction of the external plant surface must have a very low permeability for C 0 2 . It is difficult to conceive of an epidermis with operational stomata in which the fully open pore occupies more than one-third of the epidermal area. So our hypothetical homoiohydric plant lacking intercellular gas spaces has a further restriction on photosynthesis, even with the stomata fully open, compared with the poikilohydric plant discussed above. Thus with negligible gas-phase resistance to C 0 2 transport, our hypothetical homoiohydric plant without intercellular gas spaces can only photosyfithesize at 0.45 nMole (cm2 external surface)-' s - l times 1/3 (the upper limit for the f r a c t i y of the epidermis occupied by stomata), or about 0.15 riMole (cm2 nal surface)-' swhile the homoiohydric plant with intercellular air ces can reach 1.5 nMole (cm2 external surface)- sOur hypothetical homoiohydric plant tissue without air spaces, but with the same size and shape as a ventilated tissue with a ratio of internal to external surface area of 10, will have the same dry weight as the ventilated tissue, and half of the photosynthetic machinery (five layers of chloroplasts per unit external area as compared with ten). The rate photosynthesis achieved in air by the ventilated tissue is ten times greater than that of the unventilated tissue on a surface area or dry weight basis, and about five times greater on the basis of the quantity of photosynthetic machinery present. This is significant in view of the high cost in energy and materials of producing and maintaining chloroplasts (Raven and Glidewell, 1975). These conclusions are based on a consideration of extant terrestrial angiosperms with the C 3 pathway of C 0 2 fixation. However, what evidence is available suggests that they are equally applicable t o the earliest vascular land plants. Thus the atmospheric C 0 2 concentration has probably been very close to its present value for at least the past 500 million years (Miller and Orgel, 1974). Evidence from the ratio of stable carbon isotopes in fossil organic matter suggests that the C3 pathway was the means by which early vascular plants and

',

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their algal ancestors fixed C 0 2 photosynthesis (Troughton, 1971; Pardue et al., 1976). The close similarity between the properties of all of the eukaryote ribulose diphosphate carboxylase enzymes so far investigated suggests that this is an evolutionsrily conservative enzyme (Takabe and Akazawa, 1975; Kung, 1976) whose properties relevent to the argument presented above have not changed appreciably over the last 500 million years. Finally, the anatomy of such plants as Rhynia, Psilophyton and Asteroxylon (see Section 111) seems generally in accord with the assumptions made in the quantitative analysis concerning internal : external area, and stomatal geometry and hence stornatal resistance. In discussion the possible origins of this elaborate system which regulates gas exchange in the aerial shoots of vascular land plants, its three major components (cuticle, stomata and intercellular air spaces) will be considered separately.

4. Structure, Chemistry and Evolution of the Cuticle In the terminology of Crisp (1963) this waterproof component of vascular plant cell walls is both water-resistant (has a low permeability to water) and is water-repellent (is not readily wetted). The cuticle (see Fig. 10) is structurally and chemically coinplex (Martin and Juniper, 1970; Van den Erde and Linskens, 1974). The basic chemical component of the cuticle of aerial shoots is cutin, a polymer of a-hydroxy C16 and C18 fatty acids, with some in-chain hydroxyand epoxy-groups; it contains co-valently bound phenolic acids, and a small amount of long-chain alcohols and of C20 or longer-chain o-hydroxy- or dicarboxylic fatty acids (Kolattakudy and Walton, 1973; Riley and Kolattakudy, 1975). The suberin of underground organs differs in having a predominance of the longer-chain fatty acid derivative alcohols (Kolattakudy et al., 1975). The cuticle of the outer surface of aerial shoots has a superficial wax layer (Martin and Juniper, 1970). There seems to be no precedent in the green algae for the synthesis of the monomers of cutin or suberin (Wood, 1974); the structure referred to as the “cuticle” on the outer surface of the cell walls of some green algae is probably largely composed of protein (Hanic and Craigie, 1969). However, the outer surface of the aerial portions of terrestrial green algae is water-repellent (Bertsch, 1966). This is probably related to decreasing the aqueous diffusion path for CO, influx from the air when liquid water is present in the environment. Further, some submerged chlorophyte and charophyte algae have a hydrophobic cell wall component; this appears to be similar to the terpenoid derivative sporopollenin found in the walls of bryophyte and tracheophyte spores (Brooks, 1971; Atkinson et al., 1973; Syrett and Thomas, 1973; Gunnison and Alexander, 1975). Thus while the proposed ancestors of vascular land plants can produce a waterproofing cell wall component, it is not clear how closely this resembles chemically the waterproofing component of the vegetative portions of vascular land plants.

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5. Unique Features and Evolution of Stomata There are no direct structural precedents for stomata (see Fig. 10) in algae (cf. Corner, 1964, p. 162). The gametophytes of some liverworts (Marchantiales) have intercellular gas spaces communicating with the external atmosphere via air pores in the upper epidermis of the thallus (Fig. 11). These pores do not show the subtle responses to light, C 0 2 and changes in water content of the intercellular gas which are characteristic of the true stomata of the sporophytes of land plants, but they share with stomata a role in preventing the entry of liquid water from the plant surface into the intercellular air spaces, and a tendency to close when the plant is suffering from a severe water deficit (Walker and Pennington, 1939; Sifton, 1945, 1957; Campbell, 1965; Schonherr and Ziegler, 1975). Functional stomata occur in all aerial photosynthetic sporophyte tissue of land plants with intercellular air spaces (Sifton, 1945, 1957; Garen and Paolillo , 197 3).

Fig. 11. Scanning electron micrograph of a transverse section of the thallus of the liverwort Marchantia polymorpha. The ventral tissue above the lower epidermis has few chloroplasts and is relatively close-packed. Above this is an air chamber, epidermis, perforated by an air pore. [From J. I. Sprent.]

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Stomata1 opening is “powered” by an increase in guard-cell osmotic pressure: this is generated by a similar mechanism to the one involved in turgor regulation in other walled plant cells (Willmer and Pallas, 1973; Dayanadan and Kaufmann, 1975; Raschke, 1975; Meidner and Willmer, 1975). The change in pore area which results from changes in guard-cell osmotic pressure (and hence turgor pressure) results from specialized wall anatomy in the guard cells (Raschke, 1975; Meidner and Willmer, 1975). A further requirement of functional stomata is the regulatory mechanism which relates resistance to gas transfer t o C 0 2 consumption within the tissue and to water stress. Stomata tend to open when conditions are favourable for photosynthesis: the signals involved are a decreased intercellular space concentration of C 0 2 brought about by net C 0 2 fixation, and more direct effect(s) of light not mediated by C 0 2 assimilation. Stomata tend to close when a potential water stress, signalled by a high atmospheric water vapour saturation deficit, or an actual water stress, signalled by a highly negative leaf water potential, occur. The combined operation of these regulatory mechanisms allows the plant to regulate its gas exchange with the atmosphere such that C 0 2 fixation can occur when water is plentiful, but water loss (and hence desication) is reduced when water loss begins to exceed water uptake by the leaf (Meidner and Mansfield, 1968; Heath, 1975; Neilson and Jarvis, 1975; Raschke, 1975, 1976). Thus the special features of stomata relate to the sensors which regulate the processes which alter guard-cell turgor, and the structures which produce a gas-filled space between the guard cells and an aperture which varies with cell turgor.

6. Origin and Maintenance of Intercellular Air Spaces Intercellular gas spaces are present in vascular plant sporophytes (Fig. 10) including completely submerged aquatics, with the exception of much reduced aquatics like the thalloid Podostemaceae (Sifton, 1945, 1957; Sculthorpe, 1967). There are no analogous spaces in algae; the gas bladders of Phaeophyceae do not function in the distribution of metabolic gases, and they arise lysigenously (i.e. by tissue dissolution) in pseudoparenchymatous tissue (Fritsch, 1945a; Sifton, 1945, 1957; Walsby, 1972; Foreman, 1976). By contrast, the gas spaces in photosynthetic land plants are functional in the distribution of metabolic gases, and they arise in parenchymatous tissue by separation of cell walls as turgid cells expand, i.e. schizogenously (Sifton, 1945, 1957; Dengler et al., 1975). The formation an2 maintenance of these gas spaces has received relatively little attention (Sifton, 1945, 1957; Crisp, 1963; Hays, 1975). They are lined by a water-repellent internal cuticle (Crisp, 1963; Scott, 1966; Martin and Juniper, 1970; Van den Erde and Linskens, 1974), and pressures of the order of 0.1 MPa are required to infiltrate them with liquid water (Ursprung, 1924; Lewis, 1948; Crisp, 1963; Sifton, 1945, 1957; Pitman et al., 1974; Hays, 1975). Thus their

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formation and maintenance in transpiring tissues, where the water in the cell walls bounding the air spaces is under tension, is fairly readily explained. In photosynthetic tissues at night, however, root pressure can produce a positive pressure in the xylem and the communicating leaf cell walls. In some cases the vascular tissue and hydathodes are surrounded by a suberized barrier which may help to prevent injection of intercellular spaces by guttation fluid (Schnepf, 1974). Even if such injection does occur at night, subsequent photosynthetic production of O2 can displace the water (Ursprung, 1924; Lewis, 1948; Sifton, 1945, 1957; Crisp, 1963; Pitman et al., 1974). The problems of producing and maintaining intercellular gas spaces are compounded in submerged non-photosynthetic organs. The water surrounding such organs is under pressure, and metabolism replaces 0, with the more water-soluble C 0 2 . The converse problem of the maintenance of liquid water in the xylem despite the occurrence of tensions which can cavitate it has received much more attention. The xylem differs from the intercellular gas space system in being lysigenous, i.e. made up of dead cells; thus the conduits are water-filled when they are formed. The occurrence of gas embolism is prevented by the absence of both non-wettable substances and large pores in the cell walls (Zirnmermann? 1971) although lignin is somewhat hydrophobic (Siegel, 1962: cf. Scott, 1966). The occurrence and functioning of this intercellular gas space system in photosynthetic and other tissues (Appendix B) of a homoiohydric vascular plant requires that the plant has a certain minimal bulk. A plant which is less than three cells thick in its smallest dimensions can neither be truly parenchymato6 nor have a functional intercellular gas space system. This imposes certain restrictions on the structure of an alga from which such land plants could have evolved. The necessary bulk of parenchymatous tissue is found in Schizomeris (Mattox et al., 1974) among the cklorophyceae, and in the nodal tissue of the Charales (Pickett-Heaps, 1975; Fig. 1) in the Charophyceae.

VI. TRANSPORT OF DISSOLVED SOLUTES A. XYLEM

In addition to water (Section IV), the aerial shoot of the vascular land plant obtains minerals from the soil solution. It is generally accepted that the major pathway for transfer of solutes from the soil solution to the shoot involves the xylem and the transpiration stream (Peel, 1974; Clarkson, 1974; Pitman, 1975). The solutes are actively or passively transported to the xylem, whence they are carried up the xylem in the transpiration stream or by operation of the root pressure mechanism. A fraction of the solutes are removed by root, stem and petiole tissue, but most of these solutes end up at the transpirational termini in

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photosynthetic tissue. Mature non-growing photosynthetic tissue does not have a net requirement for these solutes; the metabolic sinks in the shoot which have a net requirement (vegetative nieristems, fruits) have relatively low rates of transpiration. Transport from the mature photosynthetic organs t o these growing regions is largely by phloem transport (Pate, 1971; Liuchli, 1972; Clarkson, 1974). Metabolic transforinations occur en route in both the root and the mature leaf for some elements. This is particularly so for nitrogen (Pate, 1971, 1973); the significance of this for pH regulation is considered elsewhere (Raven and Smith, 1976a, b). This discussion of redistribution of mineral nutrients within the shoot implies that supply of minerals to the shoot from underground parts of the plant is regulated. This is indeed the case; aspects of this regulation are discussed by Davidson (1969), Thornley (1972, 1975, 1976), Hunt (1975), Hunt etal. (1975) and Pitrnan (1975). Solution of problems of excess of certain solutes in the aerial shoots, arising inter ulia from the absence of adequate regulation of the solutes transported to the shoot in the xylem are discussed in Section VID. 13. PHLOEM

This is the other major pathway for long-distance transport of solutes in vascular plants. This appears to be an elaboration of the symplast (Gunning and Steer, 1975; Fig. 12); in contrast to the apoplastic xylem, the functional conducting cells of the phloem are alive. There is still a great deal of controversy over the mechanism of phloem transport (e.g. Eschrich, 1970; Crafts and Crisp, 1971; MacRobbie, 1971; Anderson, 1974; Canny, 1973; Richardson, 1975; Wardlaw, 1974; Zirnniermann and Milburn, 1975). The most frequently considered aspect of phloem function is the transport of photosynthetically produced carbohydrate t o heterotrophic sinks in the subterranean parts of the plant, and to such mainly heterotrophic sinks as developing leaves and fruits in the aerial part of the plant. The carbohydrate translocated is a non-reducing oligosaccharide (usually sucrose or a sucrose derivative) or a polyol (Ziegler, 1974). Translocation of carbohydrate provides evidence for the very large capacity of the phloem “super-symplast” compared with the ordinary symplast from which it probably evolved. Thus Kuo et al. (1974) quote values for-sucrose flux in the symplast during phloem loading (see below) of 230 pmol c m P 2 sP1 in Triticum, and 640 pmol cmP2 s-l in Viciu. The sucrose flux along the sieve tubes of Triticum is about 16.5 pmol cm-’ s-l, where the area refers to the cross-sectional area of the sieve tubes (data of Evans et. al., 1970; method of calculation, and assumptions involved, from MacRobbie, 1971). Even higher fluxes of sucrose in Tnticum sieve tubes, up t o 175 pmol sP1, can be computed from the data of Passioura and Ashford (1974), who grew their plants with only one seminal root and measured dry weight transfer along this root. It is thus clear that the fluxes in the “super-symplast” of the phloem-are at

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least lo4 higher than those in the “ordinary” symplast, although in the absence of a clear knowledge of the mechanisms involved in these transport processes it is not possible to compare the efficiencies of the two systems (MacRobbie, 1971; Anderson, 1974). As MacRobbie (1971) points out, the flux along the phoem is much greater than ordinary transmembrane fluxes in plants (or, indeed, than “ordinary” symplastic fluxes), and loading and unloading of the phloem system requires a very large lateral surface area compared with the cross-sectional area of the conducting cells in the phloem. Assuming

Fig. 12. (A) Light micrograph of sieve plates in a transverse section of the phloem of the petiole of Ricinus communis. [From J. I. Sprent] (B) Scanning electron micrographs of the same sieve plates as are shown in (A) after trypsin digestion. Note the sieve pores. [From J. I. Sprent] (C) Transmission electron micrograph of a longitudinal section of the petiolar phloem of Lupinus albus. ST = sieve tubes, SP = sieve plates. Note the much greater size of the sieve pores in the sieve plates than of the plasmodesmata (arrows) connecting the sieve tubes to the adjacent companion cells (see also B, C and I)). Scanning electron micrograph of a sieve plate of stem phloem of Ricinus communis. [Plate 8a of Gunning and Steer (1975).] -Continued.

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Fig. 12-Continued. (D) Transmission electron micrograph of a sieve plate of Lupinus albus. ER = endoplasmic reticulum; C = callose in an electron-lucent ring around each pore; P = P-protein fibrils. [Plate 8b of Gunning and Steer (1975).j (E) Transmission electron micrograph of three plasmodesmata in the wall between two meristematic cells'in Brassica oleracea root. Small arrowheads indicate the continuity of the plasmalemma through the pore; large arrowhead indicates the axial strand present in all higher plant, but not in all algal, plasmodesmata (contrast Fig. 3). [Plate 14a of Gunning and Steer (1975).]

(MacRobbie, 1971) that 20% of the phloem of herbaceous dicotyledons is occupied by sieve cells, the conducting elements in the phloem occupy not more than 1% of the cross-sectional area of the organs of these plants (Metcalfe and Chalk, 1950). In respect to both the small fraction of the total plant volume occupied by the conducting cells, and the large area needed for loading and unloading by transport systems of lower capacity, the phloem is analogous to the other specialized long-distance transport system, the xylem (Section IV). It is not clear when in the history of the vascular land plants a fully functional phloem evolved. The earliest vascular land plants with well preserved anatomy ( e g . Rhyniu and Psiloplzyton) from the Lower Devonian: see Section 111) had longitudinally elongate cells in the region of the axis where phloem

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might be expected; the cells resemble sieve cells except that they appear to lack well defined. sieve pores (e.g. Kidston and Lang, 1917, 1920a, b, 1921a; Satterthwaite and Schopf, 1972; Banks et al., 1975, Fig. 7). Later (Carboniferous) vascular plants have phloem with sieve pores (e.g. Eggert and Gaunt, 1973; Eggert and Kanemoto, 1974; Wilson and Eggert, 1974), so sieve pores can be preserved. Interpretation of this anatomical data in terms of phloem function remains in doubt in the absence of a convincing mechanism for phloem transport (see above): the minimal structural requirements for “phloem-type” translocation, i.e. a large solute flux with high apparent velocities of transport -(see Canny, 1973), are not well established. Thus a high-velocity solute transport (approaching 100 cm h - * ) is found in some Rhodophyceae and Bryophyta, although there is no true phloem structure in these plants (Eschrich, 1970; Hebant, 1970; Richardson, 1975). However, it has not yet been established that this high-velocity transport is associated with high solute fluxes: this latter criterion is perhaps of greater significance to the plant than the velocity. Thus the significance of the “phloem without sieve-pores” of the early vascular plants, which resembles in some respects the leptome of some mosses (Hebant, 1970), remains in doubt. C. SYMPLAST AND APOPLAST

The “ordinary” symplast (Fig. 12) plays a role in phloem loading in many plants (Kuo et al., 1974); the symplast is also involved in many other “short-distance” (i.e. to the order of mm) transport processes in higher plants as well as in green algae (Fig. 3; Tyree, 1970; Clarkson, 1974; Dick and Ap Rees, 1975; Gunning and Robards, 1976a, b; Section 11). Unlike the algae in their extensive aqueous milieu, apoplastic transport can occur in some situations in vascular land plants without undue risk of loss of solute. This is particularly the case with shoot tissues, where loss to the soil solution by diffusion through the apoplast is very slow (Tyree, 1969), and the cuticle is a barrier to solute loss to the shoot surface (see Raven and Smith, 1976a,b). However, some solute leakage from aerial shoots does occur (Martin and Juniper, 1970; Tukey, 1970). Apoplastic transport is involved in the loading of solutes into the phloem in photosynthetic organs of many plants (e.g. Pate and Gunning, 1972; Anderson, 1974; Geiger et al., 1974; Gunning et al., 1974; Kuo et af., 1974). Apoplastic transport may also be involved in solute unloading from phloem in bulky underground organs, e.g. the storage “roots” of Beta (Kursanov, 1974), but not in the more typical absorptive and anchoring root (not morphologically specialized for storage) of Pisum, where phloem unloading appears t o be symplastic (Dick and Ap Rees, 1975). Tissues in vascular plants (and bryophytes) which mediate large symplast-apoplast solute exchanges have anatomical specializations termed “transfer cells” (Pate and Gunning, 1972; Gunning et al., 1974). In these cells

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wall ingrowths increase the area of membrane (and hance the flux of any solute which is limited by transport capacity on a unit membrane area basis) associated with unit cell surface area. D. EXCRETION

This discussion of long-distance transport in vascular land plants has concentrated on problems of solute supply. However, the shoot of the vascular land plant also has problems of disposal of a toxic excess of certain solutes. While the vacuole and, to a smaller extent, the shoot apoplast can act as sinks for these excess solutes, in many cases other transport processes are involved. A major disposal problem is that of the excess Hf or OH- generated in primary metabolism notably in relation to assimilation of the N-sources ammonium and nitrate. This has been discussed by Raven and Smith (1976a, b). pH can be regulated during N assimilation by carrying out the Ht- or OH--generating portions of N assimilation in the subterranean parts of the plant, where there is ready access to the solute sink of the soil solution. In this case the nitrogenous compounds transported to the shoot in the xylem are such as to not lead to pH stress when they are used in shoot growth. Alternatively, in the case of nitrate the nitrate reduction and assimilation process, generating excess OH-, can occur in the shoot. The excess OH- is neutralized by organic acid synthesis. One way in which this organic acid anion can be disposed of is by translocation to the roots in the phloem, with decarboxylation in the root which regenerates OH-; the OH- can then be excreted to the soil solution. Another disposal problem may be associated with the synthesis of toxic secondary metabolites; these may be excreted from the aerial parts of the plant via special glands (Liittge, 1971 ; Schnepf, 1974; Luttge and Schnepf, 1976), or, to a limited extent, transported to the roots via the phloem prior t o excretion to the soil solution (Appendix A). The concept of “excess” production of secondary metabolites is not widely accepted (Appendix A). Finally, in halophytes transpiration may supply more of certain solutes from the rooting medium (particularly Nat and Cl-) than the shoot requires. The mechanisms by which this excess is dealt with are discussed by Sutcliffe (1962), Von Willert (1968), Luttge (1971, 1974), Waisel(1972), Levitt (1972), Clarkson (1974), Schnepf (1974), Pitman (1975) and Hill and Hill (1976). Two of these mechanisms are analogous to those described for disposal of excess secondary metabolites, i.e. excretion from the aerial shoot (salt glands) and excretion to the soil solution after translocation from the shoot to the roots in the phloem. Thus excretory glands, phloem and xylem are all involved in the alleviation of stress due to excess solutes in the shoot of land plants. Excretory glands can directly remove Nacl (and secondary metabolites) from the shoot. The extent t o which the phloem can remove solutes which are toxic to the cytoplasm (excess H‘ or OH-, NaC1, CaZt and secondary metabolites) is limited by the cytoplasmic nature of the transport pathway (Raven, 1977b). The xylem is

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important in that selective loading of the xylem with solutes can avoid solute stress in the shoot (salt exclusion in halophytes; selection of suitable nitrogenous compounds for supply to the shoot in the case of pH; and the absence of transfer of root-produced compounds to the xylem in the case of secondary metabolites). VII. THE EVOLUTION OF VASCULAR LAND PLANTS: AN HYPOTHESIS We start with a multicellular green alga living in shallow, transient bodies of fresh water. This would have resembled Stigeoclonium, but with charophyte rather than chlorophyte cellular characteristics. It would thus have symplastic transport, localized growth, a differentiation into a photosynthetic portion in the water and a rhizoidal, anchoring portion in the unconsolidated substratum. When such a pool dries out, such an alga could survive for a time as does the present-day Fritschiella. While growing on damp soil, a Fritschiella-like alga would photosynthesize using atmospheric COz , with symplastic transport of photosynthate from the photosynthetic cells to growing points and non-green cells as occurs in the submerged state. An additional transport requirement is the supply of water and minerals from the soil solution to the aerial portion. Over the short distance involved this could be symplastic or apoplastic (including ectohydric movement of solution as a surface film). It is likely that difficulties of excretion from the aerial parts of the plant to the soil solution of H+ or OH(which are not transported in the symplast to a quantitatively significant extent) in the face of a net upward mass flow in the apoplast lead to restrictions on the location of the reactions of N assimilation which lead to pH stress (Section VI; Raven and Smith, 1976a). Ultimately, as the “soil” dried out, insufficient water would be available to allow the poikilohydric plant to remain hydrated. Its survival requires either resistance t o desiccation in the vegetative state, or the production of desiccation-resistant spores (Walter and Stadelmann, 1968). Such spores permit not only survival, but also dispersal from one transient habitat t o another (Jeffrey, 1962, 1968). Such a resistant spore could be a zygospore of the type found in charophyte algae such as Coleochaete and the Charales (Fig. 1; Fritsch, 1935). However, the requirement for external water for the fertilization required to produce a zygospore means that there would be selective pressure for such spores to be produced near ground level, while efficient aerial dispersal demands that the spore be released into turbulent air above the boundary layer (Jeffrey, 1962, 1968; Ingold, 1974). In view of the advantages of sexual reproduction (Williams and Mutton, 1973), it is likely that this provided a selective pressure for an alternation of a sexual generation (genetic recombination) and an asexual generation (dispersal of spores). It is significant that aerial spores, apparently impregnated with sporopollenin and produced by meiosis, are found in the Wenlockian (Silurian), some 15 million years before the first fossil evidence for

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vascular plants (Chaloner, 1970; cf. Fig. 5). The hypothesis presented here (derived from that of Jeffrey, 1962, 1968) is that these spores were produced by a pre-vascular sporophyte of some stature living on a damp substratum. Such a plant would have to be more robust than a single filament in order to release spores outside the boundary layer. Pseudoparenchymatous cortication or, more significantly, parenchymatous construction may have evolved at this stage. A possible example of this stage of evolution is Eohostimellu from the Lower Silurian (Schopf et ul., 1966). This plant had axes 1-2 mm in diameter with no evidence of vascular tissue, but with the outer cortex apparently specialized for mechanical support. A larger plant has, as a concomitant of releasing its spores into turbulent, dry air, an increasing tendency to lose water from this erect portion (Sections IV and

Fig. 13. (A) Scanning electron micrograph of upper leaf surface of the moss DQwsOniQ superba. The ridges are the tops of plates (one cell thick and up t o six cells high) of longitudinally-running photosynthetic lamellae. [Plate 40 of Troughton and Sampson (1973).] (B) Higher magnification of (A), showing flakes of wax on the surface of the cells. [Plate 41 of Troughton and Sampson (1973).]

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V). This leads to a selection pressure for a more efficient water supply from the soil to the upper portions of the plant. This takes the form of a “super-apoplast” of dead cells within the living plant axis (i.e. xylem, or the hydrome of an endohydric bryophyte; see Figs 5, 7 and 8). Lignification prerumably evolved at the same time; initially the structural role of this polymer would have been to prevent the collapse of the tracheids under the influence of the tension generated during transpiration (Section IV, Appendix A). The greater distance involved in the transport of nutrients in this larger plant suggests that a “super-symplast” (leptome/phloem, Fig. 12) evolved at this stage. It must be emphasized that such a plant would resemble extant bryophytes in being poikilohydric. Its endohydric “super-apoplast” can increase water supply from moist soil to the transpiring surface, but the plant cannot impose a variable resistance to gas exchange with the air such as can a homoiohydric plant (Section V). Some extant bryophytes have a cuticle on their astomatous gametophyte (Doyle, 1970; Grubb, 1970; Fig. 13); However, since this does not

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show a greater permeability to COz than to H 2 0 , it cannot be sufficiently water resistant (Seccion IV) to allow a photosynthetic plant to be homoiohydric. Such a plant, when subjected to water stress, ultimately can only prevent further water loss by desiccating to air-dryness. In terms of the fossil record the earliest vascular plant (Cooksonia from the Downtonian strata of the uppermost Silurian) may well represent such a poikilohydric stage in land plant evolution (see Section 111; Fig. 5); it has xylem and a cuticle, but lacks stomata. The potential for synthesis of waterproofing materials in green algae is considered in Section V. The possible occurrence of plant cuticle in older strata is discussed by Banks (1 975). Homoiohydry requires the intercellular space-cuticle-stomata system (Section V). The cuticle was already present in Cooksonia, although in such an astomatous organism it could not have had the low gas permeability typical of the cuticle of homoiohydric plants, otherwise photosynthesis would have been severely restricted (e.g. Ogawa, 1975). Furthermore, although the gas permeability of the cuticle decreases with dehydration (Martin and Juniper, 1970), the magnitude of this effect is inadequate to give a plant like Cooksonia the variable resistance to gas exchange typical of the homoiohydric plant. Possible selective pressures involved in the evolution of the cuticle in poikilohydric plants could be related to defence against pathogens (Martin and Juniper, 1970), or to the water-repellent rather than the water-resistant aspect of waterproofing (Crisp, 1963). This latter property may have been advantageous in hastening run-off of rain and dew which would impose a barrier t o C 0 2 diffusion from the atmosphere to the chloroplasts (see Section IVA (2)). A possible evolutionary parallel is found in the astomatous gametophytes of mosses. Here the presence of a water-repellent cuticle on the photosynthetic shoot is correlated with internal water conduction and the presence of a hydrome, i.e. the endohydric condition; a cuticle is lacking in the ectohydric mosses in which water supply to the transpiring shoot is by means of an external surface film (Doyle, 1970; Grubb, 1970; Watson, 1971; Troughton and Sampson, 1973; Fig. 13). Here the cuticle in the ectohydric plants may function in preventing the persistence of an external film of water which would impede C 0 2 entry and is not required for water supply to the shoot; such a cuticle in an ectohydric moss would inhibit water supply t o the shoot by disrupting the external water film. Since stomata can confer no obvious selective advantage in the absence of an intercellular air space system (Section V), it is likely that functional stomata did not evolve before intercellular air spaces. The selective advantage of such an intercellular gas-space system connected to the atmosphere via pores which do not function as stomata might be in increasing the surface : volume ratio for gas exchange in a parenchymatous axis. This would be analogous to the system found in the Marchantiales (extant poikilohydric bryophytes: Doyle, 1970, Fig. 11). While other ways of increasing the surface : volume ratio are found in

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bryophyte gametophytes (e.g. the lamellate leaves of Polytrichum and Dawsonia: Doyle, 1970; Bazzaz et at., 1970; Bayfields, 1973; Troughton and Sampson, 1973; Fig. 13); the occurrence of intercellular air spaces with pores providing continuity with the external atmosphere shows greater potential in terms of the evolution of homoiohydry (Section V) and in supplying O2 to subterranean organs (Appendix B). As was discussed above (Section V), a certain minimal bulk of tissue is required for an intercellular space system to occur, and to be functional in exchange of metabolic gases. It is suggested that the necessary bulk of tissue was the end-product of selective pressure for release of spores well above ground level (see above, and Jeffrey, 1968) rather than following from the derivation of the vascular plants from massive marine algae (Church, 1919; Corner, 1964), or as a result of fungal symbiosis (Jeffrey, 1962, 1968). Such an air-space system increases both the potential rate of photosynthesis and the potential rate of water loss (on a tissue volume basis), but at the same time provides the material for the evolution of homoiohydry whereby these two gas-exchange rates can be reversibly decreased (Section V). This requires that the pores connecting the air-space system with the outside air should open and close in response to water stress and net COz fluxes (i.e. behave as true stomata); and an impermeable cuticle which severely restricts gas exchange when the stomata are closed. A plant with xylem, intercellular gas spaces, stcmata and a cuticle is potentially homoiohydric (Walter and Stadelmann, 1968). These attributes were possessed by such plants as Rhynia and AsteroxyZon from the Siegenian (Lower Devonian: see Section 111, Fig. 7). Plants from the preceding stage (Gedinian, e.g. Zosterophyllum, Fig. 6) had xylem, cuticle and (primitive) stomata, although the preservation is not such as to allow a decision to be reached as to the occurrence of intercellular gas spaces (Section 111). Thus the fossil record (Section 111; Bassett and Edwards, 1973) is consistent with the evolutionary sequence for vascular plants suggested in this paper. Corner lists “four great inventions of land plants: intercellular air spaces between cells, cuticle, lignin and seeds” (Corner, 1964, p. 162). The Siegenian plants had the first three of these attributes, to which I would add stomata as being ofequal importance in conquering the land. The seed evolved later in the Devonian (Banks, 1970), and was important in making sexual reproduction in land plants as independent of water supply as the homoiohydric characteristics of the Siegenian sporophyte had made vegetative growth. No gametophyte is homoiohydric (Walter and Stadelmann, 1968), and archegoniate sexual reproduction requires external water for fertilization to occur. The seed habit eliminates both the independent (poikilohydric) gametophyte phase, and eventually the motile male gamete swimming in a surface water film, from the life cycle of the vascular land plant. Important vegetative advances also occurred later in the Devonian. One

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involves improved mechanical support, related to the occurrence of primary vascular tissue in the outer portion of cylindrical aerial axes, and of secondary vascular tissue (Section IV). This permitted the evolution of plants of larger stature, using lignified tissue for support. Another was the differentiation of aerial axes into stems and planate megaphylls, and subterranean axes into roots and rhizomatous stems (Banks, 1970; Foster and Gifford, 1974). Without involving any increase in metabolic capacity at the cell level in terms of photosynthetic rates in shoots (on a chlorophyll or cell surface area basis) or of water and solute uptake in subterranean organs, these structural advances allowed significant increases in community light interception an exploitation of soil water and nutrients, and hence in productivity and water use efficiency (Slatyer, 1967; Horn, 1971; Loomis et al., 1971; Parkhurst and Louks, 1972; Paltridge, 1973; Clarkson, 1974). The final perfection of water relations of land plants was not however, reached until the rise of the angiosperms (Walter and Stadelmann, 1968; Carlquist, 1975). The argument developed here is summarized in Fig. 14. Stage of evolution

Selective pressure

Submerged, filamentous poikilohydric charophyte alga, with plasmodesmata, non-green cells, localized growth. Rhizoids in mud, shoot in water. Biochemical potential for sporopollenin, cinnamate synthesis. Drying out of shallow bodies of fresh water. Fuitschiella-like alga growing on damp mud; symplastic supply of photosynthate to rhizoids, sym- or apo-plastic (ectohydric) supply of water and minerals to aerial portion. Location of N metabolism determined by considerations of pH regulation. Need for dispersal between temporary bodies of fresh water, hence release into turbulent air rather than from oogonia near ground where fertilization can occur, hence robust plant to support sporangia. Alternation of generations produces meiospores as resting, resistant spore instead of zygospore. Meiospores with sporopollenin, produced at apex of parenchymatous axes. (Ech ostim ella)

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Larger plant requires more water supplied to transpiring surface, and more mechanical support, than can be coped with by living, parenchymatous turgid cells; also needs more efficient symplast . Evolution of endohydric water and mineral transport (super-apoplast or xylem, with lignin), and super-symplast (phloem). (Coofsonia) Larger plant needs better surface : volume ratio for COz exchange. Intercellular gas spaces plus pores connecting them to the outside air (but not functional as stomata for regulrtion of gas exchange). (Zosrerophyllurn?) Kee,p plant alive and hydrated during water shortage, even if net photosynthesis cannot occur (alternatives are survival only as resting spores, or desiccation-resistance in vegetative stage, which implies lack of large vacuoles with repercussions for SjV for CO, and light absorption, and storage of organic anions generated in pH regulation during nitrate assimilat ion) ~

Pores functional in regulation of gas exchange (i.e. stomata): epidermis of aerial portions cutinized. (Xh.vnia) Maximizing photosynthesis : transpiration ratio. Secondary thickening, evolution of megaphyllous leaves (planate, determinate) from branches; improved light interception and photosynthesis/transpiration ratio. Freedom from requirement for external water for sexual reproduction. Seed habit. ~

~~

~~

Fig. 14. Summary of the hypothesis as to the evolution of vascular land plants presented in Section VII.

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VIII. APPENDIX A: SECONDARY PLANT PRODUCTS IN RELATION TO VASCULAR PLANT EVOLUTION, WITH PARTICULAR REFERENCE TO LIGNIFICATION Secondary metabolites are compounds not involved in the “central core” of metabolism common to the majority of organisms, and which are specific to certain taxa (Bu’lock, 1965). Chemically these compounds are polyketides, isoprenoids, phenolics and alkaloids; they are best known from higher plants, although all except the alkaloids are also known from the algae (Ogino, 1962; Bu’lock, 1965; Goodwin, 1974a, b ; Hellebust, 1974). The waterproofing agents, sporopollenin and cuticle components (which are polyketide and terpenoid compounds), and the compression-resistant structural component lignin (a polyphenolic compound) are examples of secondary products which are important in the evolution of the homoiohydric land plant (Sections IV and V). Green algae may be regarded as biochemically “pre-adapted” for the synthesis of certain of these compounds. Thus sporopollenin is found in some chlorophyte and charophyte algae (see Section V). Lignin, defined as a polymer of hydroxylated and methoxylated phenylpropane) does not occur in algae (Freudenberg and Neish, 1968). However, the cell walls of the chlorophyte Staurastrum contain a polymer of protocatechuic acid derivatives which shares some of the physicochemical characteristics, and associated resistance to biodegradation of lignin (Gunnison and Alexander, 1975). Green algae do have certain of the enzymes needed for the synthesis of lignin. The starting point for lignin biosynthesis involves the conversion of the primary metabolite phenylalanine into transcinnamic acid, using the enzyme phenylalanine ammonia-lyase (Stafford, 1974). Freudenberg and Neish (1968) suggested that the key evolutionary step needed for the production of lignin in the ancestors of vascular plants was the synthesis of this enzyme. While this was reasonable on the basis of what was then known of the distribution of the enzyme in photolithotrophs, it is now clear that the enzyme is found in all organisms capable of producing ubiquinone (Stafford, 1974). It has been found in the chlorophyte DunaZiella (Loffelhardt et ul., 1975), and flavonoid products of its activity accumulate in the charophyte Nitella (Markham and Porter, 1969). The monomers produced from cinnamic acid are polymerized to produce lignin in a reaction involving H 2 0 2 and peroxidase. Siegel and Siegel (1970) have shown that, alone among the algae which they tested, the green algae contained a peroxidase with a similar specificity to that from higher plants, i.e. capable of bringing about lignin synthesis. It has been suggested that lignin originated as a detoxication product for excess soluble phenolics (Freudenberg and Neish, 1968). This implies that these toxic compounds were produced in excess of any requirement which the plant may have for them, and in excess of the capacity of other detoxication mechanisms. Neither of these conditions is met by phenolics in extant land

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plants. Thus there is considerable evidence that phenoiic biosynthesis is regulated, and that many of the low molecular weight phenolics accumulated or excreted by plants have selective advantage rather than being waste products (e.g. Rice and Pancholy, 1969; Sondheimer and Simeone, 1970; Zeevart, 1975; Halligan, 1975; Reynolds, 1975; Walker, 1975). Even if phenol biosynthesis exceeded the capacity of the vacuole to store them away from the cytoplasm, it is clear that “inefficient plumbing” (Freudenberg and Neish, 1968) is not a complete barrier to removal of phenolics from the shoot, in which extracellular space constitutes a very small sink (Section VI). At least some phenolic secondary products can move in the phloem of land plants (Schultz, 1969; Cleland, 1974; Cleland and Ajami, 1974; Zeevart, 1974). However, the cytoplasmic nature of the transport pathway, together with the toxicity of many phenolics, means that the capacity of the phloem to transport phenolics is limited when compared with the capacity of the shoot to synthesise them. Thus there is a limited but finite capacity for transport of phenolics from shoot to root, whence they could be excreted to the large sink of the soil solution. In aquatic algae, excretion of phenolics to the large extracellular sink as well as their storage in the vacuole occurs (Ogino, 1962; Evans and Holligan, 1972; Hellebust, 1974; Provasoli and Carlucci, 1974). Thus whle it is not possible to disprove the hypothesis of Freudenberg and Neish (1 968) on the basis of the evidence discussed here, it is clear that none of its premises is invariably true. As an alternative, it might be suggested that the apoplastic accumulation of phenolics was initially related to defence against pathogenic micro-organisms (Gunnison and Alexander, 1975; Ride, 1975). Polymerization of these soluble phenolics might have constituted a more effective mode of defence; eventually, the polymerized phenolics might accumulate in sufficient quantities to have a structural role. Thus it might be possible t o account for the evolution of lignin without having to postulate the unregulated synthesis of functionless phenols. Initially, selective pressures would be related t o defence against micro-organisms; subsequently, additional selection would occur in relation to mechanical properties.

IX. APPENDIX B: ROLE OF INTERCELLULAR GAS SPACES IN RESPIRATORY GAS EXCHANGE OF VASCULAR LAND PLANTS In addition t o their role in photosynthetic gas exchange in aerial tissues, intercellular gas spaces have further roles in extant vascular plants (Raven, 1970, 1972a, b). One appears to be the distribution of such gaseous growth regulators as ethylene (Jackson and Campbell, 1975); another, which will be discussed in more detail, is in the supply of O2 for respiration. Photosynthetic eukaryotes are obligate aerobes (Nuhrenberg ef ~ l . , 1968; Morris, 1976). While such angiosperms as Oryza can carry out limited germination processes in the absence of 0 2 , they cannot grow to a state of photosynthetic competence which would, in the light, be able to generate O2 (Kordan, 1976a, b, c).

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Photosynthetic cells of land plants are unlikely to suffer from lack of O2 for respiration in the dark in air, even with the stomata almost completely shut. Thus O2 diffuses through air faster than does C 0 2 , and an aqueous solution in equilibrium with air contains 250 pM O2 at 25"C, but only about 10 pM C 0 2 (see Samish, 1971, 1975). Further, the diffusion coefficients of O2 and CO, within living cells are probably almost identical, and little less than the respective values in free solution (Raven, 1 9 7 7 ~ ) Finally, . the affinity of plant cytochrome oxidase for O2 (K% of about 0.1 p ~ Bonner, , 1965) is much higher than that of ribulose diphosphate carboxylase for C 0 2 in uivo K% of about 10-20 p M : Lilley and Walker, 1975), while the rate of C 0 2 consumption in net photosynthesis is often more than ten times the rate of O2 uptake in dark respiration when both are measured in air. Taken together, these factors suggest that a tissue which can photosynthesize in the normal air concentration of C 0 2 should have no problem with O2 supply from air for dark respiration. Even much more bulky and less well ventilated aerial organs (e.g. fruits, woody stems) are probably rarely anaerobic in air (James, 1953; Forward, 1965). The situation is different in subterranean plant parts, especially if the soil is waterlogged and there is no appreciable net water flux through the soil. Armstrong (1974) gives a well-reasoned account of the role of intercellular gas spaces in the subterranean parts of plants. He shows that the intercellular gas spaces of roots of normal length in mesophytic herbs are inadequate to carry sufficient O2 from the atmosphere, via the aerial shoot, for the metabolic requirements of the apical part of the root, and concludes that such plants normally obtain a portion of the O2 for root metabolism from the soil atmosphere. When the soil is waterlogged and stagnant, however, it is rapidly depleted of 0 2 by the respiration of roots and aerobic micro-organisms. The aerobic micro-organisms are succeeded by anaerobes, whose activities lead to an accumulation of reduced inorganic compounds such that the waterlogged soil acts as an O2 sink, rather than as an O2 source for plant roots and rhizomes. Under such conditions the extensively developed intercellular gas spaces play an important role in supplying O2 to these underground parts from the atmosphere via the aerial shoot. This O2 not only provides for the respiratory requirements of the roots but also, by radial leakage, provides beneficial oxidising conditions in the rhizosphere. These arguments (Armstrong, 1974) suggest that intercellular gas spaces are important for oxygenation of subterranean plant parts in O 2-deficient soils. This may have been relevant to the early vascular land plants, many of which are thought t o have lived in soils subject to more or less frequent waterlogging (Kidston and Lang, 1917, 1921b). Any O2 supply problems due to waterlogging would have been exacerbated if the atmospheric O2 concentration was appreciably lower than it is at present. Estimates of the atmospheric 0 2 concentration in the early palaeozoic, based on a number of lines of evidence, vary from 10 to 100% of the present value. While an O2 concentration of

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one-tenth the present value would not have presented great problems for O2 supply to aerial, photosynthetic tissues in the dark, it would have imposed a major restriction on O2 supply to subterranean organs of a plant the size of Rhynia major living in a waterlogged soil. The quantitative argument on which this conclusion is based is as follows. Fenn's equation (see James, 1953, p. 144) described the 0, concentration at a specified locus in a cylinder of tissue with a uniform distribution of respiratory capacity. An implicit assumption in its use is that the reaction which consumed 0 2 within the tissue is zero order with respect to the O2 concentration. This is not a serious source of error with respiration, granted the very low Kt/, of cytochrome oxidase for 0 2 . Its use is not permissible for photosynthetic C 0 2 fixation (Section IV), since C 0 2 consumption by ribulose diphosphate carboxylase is first order with respect to C 0 2 concentrations at and below the atmospheric level. In a form which uses the diffusion coefficient of 0, in the units used for C 0 2 in Section V, rather than as Krogh's invasion coefficient, Fenn's equation is:

where A = radius of the tissue (cm), [O,] = external 0, concentration, expressed in terms of the concentration in tissue equilibrated with the external atmosphere (nMole ~ m - ~ ) , [02].i = 0, concentration in the respiring tissue at a distance a cm from the centre (nMole cmP3), D = diffusion coefficient for 0, in the tissue, cm2 s- and tissue) sm = respiration rate, nMole O2

'.

',

Taking A = 0.25 cm for the subterranean axis of Rkyizia major (Kidston and Lang, 1917, 1921a), a = 0.05 cm (i.e. somewhere in the inner cortex/phloem region of the axis), D = 1.5 cm2 s-' (probably an overestimate for a tissue without air spaces, see above) and m = 0.1 nMole cm- s-' (probably an underestimate for a non-dormant, vacuolate plant tissue at 20°C: James, 1953) then if [0210is 2 5 0 n M o l e ~ m - ~ ,[O,]i is 1 5 0 n M o l e ~ r n - ~Thus . the innermost living tissue of the axis would be adequately oxygenated at present atmospheric levels of [O,] O , even if the axis had no intercellular gas spaces. However, if [ 0 2 ] 0 is less than 100 nMole cm-3 (i.e. an atmospheric 0, level less than 40% of the present value, and/or soil depleted of 0, relative to the bulk atmosphere) the innermost living tissue would be completely anaerobic. James (1953, p. 145) discusses ebldence that even a very small fraction of air space in such a tissue can dramatically improve its oxygenation. Thus it is likely that an important selective pressure involved in the evolution of intercellular gas spaces in vascular land plants, in addition to that related to photosynthetic gas exchange (Section V), was O2 supply to subterranean parts of the plant. Intercellular gas spaces have evolved independently in large,

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subterranean fungal structures such as the rhizomomorphs of Armillaria mellea. Here they are in pseudoparenchymatous tissue, as in the phaeophyceae; but, in contrast t o the algae, they are functional in the transport of respiratory gases (Sifton, 1945, 1957; Griffin, 1972).

X. SUMMARY AND CONCLUSIONS The homoiohydric vascular land plant has three major transport systems at the supracellular level: the apoplast (including the xylem), the symplast (including the phloem) and the intercellular gas space system. The aquatic green algal ancestors of these plants use only the symplast for transport at the supracellular level. The other two transport systems, together with the elaboration of the symplast into the much more efficient phloem, are essential components of the homoiohydric land plants. A terrestrial plant, carrying out photosynthetic C 0 2 fixation in the light, loses large quantities of water from its aerial parts. The initial function of the xylem in vascular land plants was to conduct this water from the soil to the sites of transpiration; the specific conductivity of the xylem is about lo6 times greater than that of parenchyma. Lignin in the xylem probably evolved in relation to the prevention of xylem element collapse under tension, with mechanical support of aerial axes as a later function. When the soil dries out, the vascular plant can prevent water loss from its aerial parts b y increasing the value of a variable resistance (the stomata-cuticle complex) to gas exchange between the shoot and the atmosphere. At the expense of preventing net C 0 2 fixation, this allows the plant to remain hydrated under conditions of restricted water supply-the homoiohydric condition. The essential (but often neglected) role of the intercellular gas spaces in the homoiohydric vascular plant must be emphasized, together with the unsolved problems of the origin and maintenance of these spaces. Long-distance solute transport in vascular land plants involves both the xylem and the phloem. As well as the well known roles of the xylem in supplying soilderived nutrients to the shoot, and the phloem in redistributing these nutrients from transpirational termini and the movement of assimilates, these two transport systems are involved in maintaing the solute balance of the shoot. Selectivity of xylem loading can avoid the accumulation to toxic levels in the shoot of NaCl (in halophytes), of root-synthesized secondary products, and of H+ or OH- (produced in excess by the assimilation of ammonium and nitrate respectively). The shoot has no large extracellular sink for such excess solutes: a method of disposing of excess NaCl shoot-produced secondary products, and excess OH- produced during nitrate assimilation in the shoot, is transport to the root followed by excretion to the soil solution. Short-distance solute transport in the shoot of vascular land plants can involve the apoplast as well as the symplast without great risk of solute loss from

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the plant, as would occur in aquatic algae and many subterranean parts of land plants. These characteristics of extant homoiohydric land plants were all present in such Lower Devonian plants as Rhynia and Psilophyton. A sequence of acquisition of these characters, starting from a terrestrial charophyte green alga at the level of organization of the chlorophyte Fritschiella, involving plausible selective pressures, and generally consistent with the fossil record of the Silurian and Lower Devonian, is as follows: aerially dispersed meiospores; parenchymatous axes; cuticle; xylem; phloem; intercellular gas spaces communicating with the atmosphere via pores not functional as stomata; functioning of these pores as stomata. The extant green algae show some biochemical “pre-adaptations” t o the synthesis of lignin and waterproofing materials. ACKNOWLEDGEMENTS

I am extremely grateful t o Dr D. Edwards of the Botany Department, University College, Cardiff, for invaluable advice on palaeobotany and for criticizing the manuscript; and to Drs S . M. Glidewell, G. 0. Kirst, T. H. Nicolson, J. I . Sprent, F. A. Smith and S. E. Smith for reading the manuscript and for much helpful discussion.

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Author Index Page numbers in roman figures are text references; page numbers in italic are bibliographical references.

A

B

Aaronson, S., 11 7, 1 4 8 Abdul-Baki, A. A,, 136, 137, 1 4 3 Abrams, M., 65, 8 7 Adelman, M. R., 116,743 Ajami, A., 207, 212 Akazawa, T., 188, 1 8 9 , 2 1 8 Albersheim, P., 58, 87, 91, 92, 93, 94, 95, 140, 143, 144, 146, 147, 150, 151 Alexander, M., 189, 206, 207,214 Allen, D. M., 99, 1 0 1 , 1 4 3 Allen, J. C., 60, 88 Allen, R. D., 110,144 Amar-Costesec, A., 1 16, 144 Anderson, J. M., 48, 49 Anderson, N. G., 1 13, 118, 144 Anderson, W. P., 175, 193, 194, 197, 211,214,215 Andreae, W. A., 6 1 , 8 5 Andrews, H. N., 179, 2 0 0 , 2 1 7 Anfinsen, C. B., 66, 86 Ap Rees, T., 197, 212 Armstrong, W., 208, 21 1 Arnold, R., 140, 146 Arnold, W., 5, 6 , 4 9 , 50 Ash, J. F., 134,150 Ashford, A. E., 193,216 Ashworth, D., 1 10, 148, 180,216 Ashwood-Smith, M. J., 2 0 , 4 9 Aspinall, G. O., 91, 95, 1 4 4 Atkins, E. D. T., 9 7 , 1 4 8 Atkinson, A. W., 106, 144, 189, 211 Azou, Y.,6 3 , 8 8 Azzi, J . R., 6 , 4 9

Bailey, R. W., 5 9 , 8 7 Ball, E., 191, 192, 216 Ballschmiter, K., 23, 50 Bamberger, E. S., 62, 85 Bandori, R. J., 155, 1 5 6 , 2 1 7 Banerjee, D., 128,149 Banks, H. P., 162, 163, 176, 179, 180, 197, 202, 2 0 3 , 2 0 4 , 2 1I , 21 7 Barber, G., 136, 140, 1 4 4 Barghoorn, E. S., 162, 179, 211, 214 Barnett, J. R., 102, 144 Bartnicki-Garcia, S., 105, 136, 138, 144,147,149 Bassett, M. G., 203, 211 Batt, S., 72, 73, 74, 75, 76, 77, 78, 19, 80, 81, 82, 85 Bauer, S., 130,144, 145, 151 Bauer, W. D., 58, 87, 91, 92, 93, 94, 95,143,144,146,150 Bayfield’s, N. G., 203, 211 Bayley, S. T., 134, 1 4 4 Baylis, G. T. S., 164,211 Bazzaz, F. A ., 203,211 Beakbane, A. B., 1 8 0 , 2 1I Beams, H. W., 101,144 Beardsall, M. F., 1 8 1 , 2 1 3 Beasley, C. A., 137, 141, 1 4 5 Beaufay, H., 116,144 Becker, J. F., 3 2 , 4 9 Beevers, H., 114, 116, 1 1 8 , 1 4 7 Behnke, O., 1 1 1 , 1 4 4 Bendana, F. E., 6 1 , 8 5 Ben-Hayyim, G., 138,144 Bergstrand, A., 128,145

22 1

222

AUTHOR INDEX

Berridge, M. V., 65,86 Berthet, J., 116, 144 Bertsch, A., 181, 189,21I Biely, P., 130, 144,145, 151 Bierhorst, D. W., 180,211 Biggs, W. D., 179,218 Billmire, E., 117,148 Bischoff, E., 130, 146 Biswas, B. B., 63,64, 70, 85,86, 8 7 Bittiger, H., 98, 125, 146 Black, C. C., 170, 186,211 Blackwell, J., 98, 145 Blobel, G., 1 16,143 Bluemink, T. F., 100,144 Boardman, N. K., 25, 2 7 , 4 2 , 4 9 Bonner, J., 62, 86 Bonner, W. D., 8,49, 208,211 Borisy, G. G., 109,144 Bostrom, T. E., 159,211 Bouck,G. B., 105, 110,144, 145 Boucot, A. J., 179, 200,217 Bowles, D. J., 112, 119, 120, 121, 122, 126, 127, 128,144,151 Boyer, J. S., 173, 174, 177,212 Bracker, C. E., 117, 132, 145, 146, 147,150 Bradley, M. O., 134,150 Branton, D., 104,144 Breidenbach, W., 65,88 Bresciani, F., 78, 8 7 Bressan, R. A., 6 1, 86 Breton, J., 32, 49 Brett, C. T., 112, 141,144 Brian, R. C., 59,86 Briggs, G. E., 154, 174, 175, 177, 179, 212 Bril, C., 25,49 Brock, B. L. W., 6 0 , 8 6 Brody, M., 13, 2 7 , 5 0 , 5 2 Brody, S. S., 2 , 4 , 26, 27,50 Brooks, J., 189,212 Brouwer, R., 175,212 Brown, D. H., 136,145 Brown, D. L., 110,144 Brown, J. S., 22,50 Brown, O., 121,146 Brown, R. M., 96,98, 99,144, 1 4 6 Broyde, S. B., 27, 50 Brummond, D. O., 136,144 Brysk, M. M., 119,144 Buchala, A. J., 97, 144

Bu’lock, J. D., 206,212 Burke, D., 95,144 Butcher, F. R., 134,144 Butler, W. L., 10, 26, 29, 31, 42, 43, 44,45,47,49,50,51,52 C Cabib, E., 136, 139,144, 1 4 7 Caldwell, C., 177,218 Callow, M. E., 101,144 Calvin, M., 2, 6 , 5 2 Campbell, D. J., 207,214 Campbell, E. D., 190,212 Canny, M. J., 193,197,212,215 Cardell, R. R., 101, 145 Carlquist, S., 179, 180, 204,212 Carlucci, A. F., 207,216 Castle, J. D., 135, 144 Cardini, C. E., 136,147 Chalk, L., 177, 196,216 Chaloner, W. G., 162, 163, 200, 212 Chambers, J. E., 112, 121, I 4 8 Chandra, G . R., 54,88 Chapman, D., 32,50 Cheadle, V. J., 105, 145 Cheetham, R. D., 132, 1 4 5 Cherry, J. H., 62, 63, 69, 70, 84, 85, 86, 87, 88, 117,146 Cherry, R. J., 32,50 Cho, F., 8, 10, 17, 18, 19, 21, 27, 29, 50 Chrispeels, M. J., 117, 118, 119, 134, 144,145,149 Chu, E. H. Y., 17,51 Church, A. H., 203,212 Clarke, A. E., 96,144 Clarkson, D. T., 154, 175, 192, 193, 197,198,204,212 Clayton, R. K., 2 , 5 0 Cleland, C. F., 207, 21 2 Cleland, R., 58,86, 88, 134,144, 155, 213 Cleland, R. E., 58, 61, 86 Clendenning, K. A., 2 0 , 5 0 Clermont, Y., 101,150 Clowes, F. A. L., 155,212 Cochrane, M. P., 188,213 Cocking, E. C., 135, 144 Cohen, D., 181,213

223

AUTHOR INDEX

Cohen, W., 1 3 , 5 0 Colvin, J. R., 138, 140, 141, 144, 145, 146,147 Cooper, D., 138,144 Copeland, M., 134,144 Corner, E. J. H., 179, 186, 190, 203, 212 Cotton, T. M., 23,50 Crafts, A. S., 154, 180, 193,212 Craigie, J. S., 189, 214 Crisp, C. E., 154,193,212 Crisp, D. J., 154, 189, 191, 192, 212 Cronquist, A., 155,214 Cronshaw, J., 105,145 Cuatrecasas, P., 66, 86 Cummins, W. R., 134,149 Currey, J. D., 179,218 D Dainty, J., 158, 159, 175, 177, 212, 218 Dankert, M., 141, 145 Das, A., 63, 64, 70, 85,86 Dashek, W. V., 118, 134,145,149 Datko, A. H., 5 5 , 86 Dauwalder, M., 100, 126, 145 Davidson, R. L., 193,212 Davies, E., 55,86 Davies, P. J., 54, 58, 61, 86 Dayanandan, P., 191,212 Deamer, D. W., 104,144 Decker, K. F. A., 130, 146 De Lange, R. J., 6 2 , 8 6 Delmer, D. P., 137, 141,145, 1 4 6 Dengler, N. G., 191,212 Dennis, D. T., 98, 138, 145 Deuel, H., 91,148 Deumling, B., 132,145 Dewar, J., 2, 50 Dick, P. S., 197,212 Donaldson, L. A., 182, 183,218 Donze, M., 33,50 Doyle, W. T., 158, 181, 201, 202, 203, 212 Duckett, J. G., 155, 212 Dunstone, R. L., 193,213 Dutton, P. L., 7 , 5 0 Duve, C., de, 112,145 Duysens, L. N. M., 33,50

E Eames, A. J., 179, 180,212 Early, R. W., 62, 8 7 Eckhoff, A., 33,51 Edwards, D., 162, 176, 180, 203,211, 212,213 Eggert, D. A., 197, 213,219 Eisinger, W. R., 113, 114, 116, 117, 119, 120, 121, 122, 123, 124, 126, 127,128, 133,148,149 Elbein, A., 136, 140, 141,144, 145 Elnagy, M. A., 137,145 Emerson, R., 5 , 3 7 , 4 8 , 5 0 Engels, F. M., 125,145 Ericsson, J. L., 128, 145 Erion, J. L., 65, 86 Ermin, B., 99,145 Esau, K., 105, 145 Eschrich, W., 159, 193, 197, 21 3 Evans, L. T., 193,213 Evans, L. V., 101,144, 207,213 Evans, M. L., 58, 59, 60, 86, 88 F Fambrough, D. M., 62, 86 Fan, D. F., 5 5 , 86 Farkas, V., 130, 139, 144,145, 151 Feingold, D. S., 140,145 Fellenberg, G., 62, 86 Fellows, R. J., 197,213 Fensom, D. S., 174,218 Figier, T., 101, 145 Filner, P., 54, 86 Findlay, N., 175, 177, 214 Fischer, R. A., 158, 159,218 Fischer, W.,130, 1 4 5 Fletcher, R. A., 60, 86 Flores-Carreon, A., 136, 138, 1 4 7 Floyd, G. L., 162, 192,215 Fong, F., 186,214 Foreman, R. E., 181, 191,213 Forsee, W. T., 141,145 Forster, T., 6, 50 Forward, D. F., 208,213 Foster, A. S., 180, 204,213 Fosket, D. E., 105, 146, 150 Fox, J. E., 65, 86 Franck, J., 41, 50

224

AUTHOR INDEX

Franke, W. W., 96, 98, 99, 110, 125, 132,134,144,145,146 Frantz, C., 117, 145 Franz, G., 137, 1 4 5 Fraser, T. W., 126, 145, 158, 160, 161, 2 1 3 Frei, E., 102, 1 10, 1 4 5 French, C. S., 8, 11, 21, 22, 2 3 , 5 0 Freudenberg, K., 158, 206, 207, 213 Friedman, H. T., 101,145 Friend, D. S., 1 0 1 , 1 4 5 Fritsch, F. E., 155, 157, 181, 191, 199,213 G Gaff, D. F., 1 8 1 , 2 1 3 Gaffron, H., 5, 1 3 , 5 0 , 5 1 , 5 2 Gale, J., 170, 216 Galston, A. W., 54, 61, 85, 86, 87, 88 Ganguly, A., 63, 64, 70, 8 5 , 8 6 Garcia, R. C., 141, 1 4 5 Gardiner, M., 1 17, 1 1 8 , 1 4 5 Gardner, K. H., 98, 1 4 5 Garen, D. L. B., 1 9 0 , 2 1 3 Gaunt, D, D., 1 9 7 , 2 1 3 Gawlik, S. R., 134,149 Geacintov, N. E., 3 2 , 4 9 Gibbons, A. P., 136, 1 4 4 Geiger, D. R., 197, 213 Gifford, E. M., 180, 204,213 Giles, K. L., 1 8 1 , 2 1 3 Gill, R. H., 105, 1 4 5 Ginsburg, V., 136,145 Ginzburg, B. Z., 1 7 5 , 2 1 3 Ginzburg, H., 1 7 5 , 2 1 3 Glaser, L., 136, 138,145 Glaumann, H., 1 2 8 , 1 4 5 Glidewell, S. M., 185, 188, 21 7 Goedheer, J. C., 9, 25, 27, 33, 5 0 Goldman, R. H., 134, 1 4 4 Golubic, S., 162, 214 Good, N. E., 42, 51 Goodman, R. N., 1 0 2 , 1 4 8 Goodwin, T. W., 206,213 Gorham, P. R., 20, 26, 50 Gosline, J. M., 179, 218 Gotelli, I. B., 155, 213 Govindjee, 8, 10, 11, 17, 18, 19, 21, 25, 27, 3,29, 30, 33, 36, 40, 50, 51, 52

Govindjee, R., 3 0 , 5 1 Gradmann, H., 1 7 0 , 2 1 3 Green, P. B., 110, 11 1, 1 4 5 Greenfield, J. C., 80, 8 7 Greenway, H., 1 8 1 , 2 1 3 Gregoire, J., 137,145 Gregory, L. M., 191,212 Gregory, R. P. F., 32, 51 Griffin, D. M., 210, 213 Grimm, I., 100, 105,145, 1 4 9 Grove, S. N., 132, 1 4 5 Grubb, P. J., 201, 202, 213 Gunning, B. E., 126, 1 4 5 Gunning, B. E. S., 106,144, 158, 160, 161, 166, 175, 176, 188, 189, 193, 194,196,197,211,213,216 Gunnison, D., 189, 206, 207,214 H Haberlandt, G., 179, 214 Hagemann, R. H., 1 8 7 , 2 1 5 Hager, A., 58, 8 6 Hall, A. E., 204, 215 Hall, M. A., 96, 1 3 6 , 1 4 5 , 1 4 8 Hallam, N. D., 1 8 1 , 2 1 3 Halligan, J. P., 207, 214 Hanic, L. A., 189, 214 Hanke, D. E., 1 3 5 , 1 4 6 Hanoune, J., 78, 8 7 Hanson, J. B., 62, 70, 8 7 Hardin, J. W., 62, 63, 69, 84, 85, 86, 117,146 Harnischfeger, G., 13, 33, 34, 35, 36, 39,51 Harris, P. J., 119, 1 4 6 Hartree, E. F., 2, 51 Hartridge, H., 2 , 51 Harvey, J . M., 59,87 Hassid, W. Z., 136, 140, 141, 144, 145,146,150 Havinga, E., 59, 8 6 Hay, R. K. M., 175,214 Hays, R. L., 186, 191,214 Heath, I. B., 110 , 1 4 6 Heath, M. F., 93, 9 8 , 1 4 6 Heath, 0. V. S., 170, 184, 1 9 1 , 2 1 4 Heath, R. L., 40,51, 186,214 Hebant, C., 181,197,214 Heber, U., 20, 51 Heiniger, U., 137, 146

225

AUTIIOR INDEX

Hellebust, J. A., 206, 207, 214 Heller, J. S., 140, 1 4 6 Hepler, P. K., 105, 134,146, 1 5 0 Hermville, E., 71, 8 6 Herscovics, A., 101, 151 Hertel, R., 54, 58, 71, 72, 75, 86, 87, 88, 1 1 7 , 1 4 7 Herth, W., 96, 98, 99, 105, 109, 110, 125, 1 3 4 , 1 4 4 , 1 4 5 , 1 4 6 , 1 4 9 Hess, W. M., 1 17, 150 Hestrin, S., 138, 146 Heyes, J. K., 58, 59, 8 7 Heyn, A. N. J., 9 8 , 1 4 6 Higashi, T., 141,150 Higgins, T. J. V., 55, 8 6 Highkin, H. R., 29, 52 Hill, A. E., 198, 214 Hill, B. S., 198, 214 Hind, G., 40, 51 Hinman, M., 9 6 , 1 5 0 Ho, D. T-H., 55, 86 Hoch, G. E., 39, 52 Hodges, T. K., 117 , 1 4 6 Holdsworth, R. H., 188, 214 Holligan, P. S., 207, 21 3 Holmgren, P., 184, 185, 1 8 6 , 2 1 4 Hope, A. B., 158, 159,214 Horn, H. S., 204,214 Horst, D. J., van der, 25, 49 House, C. H., 175, 177, 214 Houwink, A. L., 11 I , 1 4 9 Howell, K., 128, 149 Hsaio, T. C., 181,214 Hsu, Kwan, 32, 50 Huang, C. M., 1 0 1 , 1 4 6 Hueber, F. M., 162, 176, 179, 180, 197, 21 1 Hunt, R., 193, 214 I

Ikenberry, E., 175, 216 Ingold, C. T., 199,214 Itoh, S., 42, 52 Iyengar, M. 0. P., 1 5 7 , 2 1 4 Izawa, S . , 42, 51 J JablonoviE, M., 78, 86 Jackson, M. B., 207,214 Jackson, P., 6 1 , 8 6

Jacobi, G., 23, 51 Jacobs, M., 9 5 , 1 4 6 Jacobsen, B. S., 186,214 Jacobsen, J. V., 54, 55, 86, 8 7 Jagels, R. H., 203, 214 James, W. O., 208, 209, 214 Jamieson, J. D., 90, 101, 128, 129, 135,144,146 Jarasch, E-D., 132, I 4 5 Jarvis, B. C., 62, 70, 8 7 Jarvis, M. S., 184, 185, 1 8 6 , 2 1 4 Jarvis, P. G., 184, 185, 186, 191,214, 216 Jeffrey, C., 199, 200, 203, 214 Jilka, R., 121, 146 John, P. C. L., 106,144, 1 8 9 , 2 1 1 Johnson, B. F., 130, 1 4 6 Johri, M. M., 65, 87 Joliot, A., 39, 51 Joliot, P., 39, 51 Jones, D. D., 99, 101, 118, 119, 1 4 7 Jones, H. G., 184, 1 8 5 , 2 1 4 Jones, R. A . , 156, 214 Jones, R. L., 118, 119, 127, 1 4 8 Junge, W., 3 3 , 5 1 Juniper, B. E., 155, 170, 189, 191, 197, 202,212, 21 5

K Kagawa, T., 114, 116, 11 8, 1 4 7 Kanemoto, N. Y., 1 9 7 , 2 1 3 Kartenbeck, J., 132, 1 4 5 Kasamo, K., 60, 78, 8 7 Katz, J. J., 23, 50 Kaufman, P. B., 95, 144 Kaufmann, P. B., 191,212 Kaur-Sawhney, R., 61, 85, 86, 8 7 Kauss, H., 140, 141, 146, 1 4 8 Kazmierczak, J., 162, 21 4 Ke, B., 27, 51 Keegstra, K., 58, 87, 91, 92, 93, 94, 95,143,144,146,150 Keenan, T. W., 101, 117,146 Kefford, N. P., 61, 86, 87 Keilich, G., 98, 125, 1 4 6 Keilin, D., 2 , 5 1 Kennedy, C. D., 5 9 , 8 7 Kephart, J. E., 100, 130, 132, 145, 150 Keppler, D. 0. R., 1 3 0 , 1 4 6

22 6

AUTHOR INDEX

Kessel, R. G., 101, 144 Key, J. L., 54, 8 7 Kidby, D. K., 117,149 Kidston, R., 162, 164, 176, 179, 180, 197,208, 209,214 Kiermayer, O., 135, 146 Kindinger, J. I., 117, 125, 136,150 Kindl, H., 206,215 King, G. G. S., 138,146 Kirshner, A. G., 101,146 Kirshner, N., 101,146 Kitajima, M., 29, 31, 42, 43, 44, 45, 47,49,50, 51 Kjosbakken, J., 138, 141, 1 4 7 Klambt, D., 7 1, 86 Klein, R. M., 155,214 Klepper, B., 180,216 Knall, A. H., 162,214 Knofel, H. D., 7 0 , 8 7 Knox, R. S., 6, 32,51 Koch, W., 101,149 Koerper, M. A., 8, 1 1, 21,50 Kok, B., 27, 29, 39, 51 Kolattakudy, P. E., 189,214, 21 7 Kooiman, P., 95,147 Kordan, H. A., 207,215 Kortum, G., 1 0 , 5 1 Kovar'ik, J., 130,144 Kozinka, V., 174, 175,215 Kramell, R., 70, 8 7 Kramer, D., 191,192,216 Kratky, Z., 130, 144 Krauss, A., 58,86 Kreger, D. R., 125,145 Kreutz, W., 3 1 , 5 1 Kronmann, K., 189,214 Kulow, C., 137, 146 Kuo, J., 193, 197,215 Kung, S-D., 188, 189,215 Kupper, A., 96, 125,146 Kursanov, A. L., 197,215

L Labavitch, J. M., 95, 147 Lacombe, M. L., 7 8 , 8 7 Laetsch, W. M., 117,150 La Fountain, J., 134,147 Laing, W. A., 187,215

Lamport, D. A., 155,215 Lamport, D. T. A., 91, 93,94, 1 4 7 Lang, W. H., 162, 164, 176, 177, 179, 180, 197,208, 209,214,215 Larcher, W., 184, 185, 215 Euchli, A., 154,175, 193,215 Lavin, G. I., 2, 51 Lawrence, M. C., 22,50 Leblond, C. P., 101, 128, 148, 150, 151

Leclercq, S., 162, 176, 179, 180, 197, 211 Ledbetter, M., 1 10, I 4 7 Ledbetter, M. C., 100, 105, 110, 11 1, 147 Leftley, J. W., 156, 215 Leibo, S. P., 1 7 , 5 1 Le John, H. B., 71, 8 7 Lele, K. M., 162, 166,215 Leloir, F., 136,147 Leloir, L. F., 136,147 Lembi, C. A., 54, 61, 71, 84, 85, 86, 87, 88, 117, 125, 136, 146, 147, 149,150 Lennarz, W. J., 141,147 Leonard, N. J., 80,87 Leonard, R. T., 117, 146, 1 4 7 Leopold, A. C., 1 1 , 8 8 Lesemann, D., 207,216 Letham, D. S., 65,86 Levine, R. P., 33, 36, 52 Levitt, J., 172, 181, 198, 215 Lewis, D. R., 104, 148 Lewis, F. J., 191, 192,215 Li, J. S., 40, 51 Libbenga, K. R., 64, 8 7 Lierop, J. H., van, 3 1, 52 Likholat, T. V., 64, 85, 8 7 Lilley, R. McC., 187, 188, 208, 215 Limozin, N., 137,145 Lin, P. P-C., 61, 8 7 Lin, T-Y., 140, I 5 0 Lindenmayer, G. E., 60, 88 Linskens, H. F., 189, 191,218 Lippman, E., 138,144 Litvan, G. G., 16, 20,51 Litvin, F. A., 22, 23, 24, 5 2 Litvin, F. F., 22, 23, 26, 27,51 Loffelhardt, W., 206,215 Loomis, R. S., 204,215 Lord, J. M., 114, 116, 118,147

AUTHOR INDEX

227

Meiri, A., 175,215 Mencher, E., 179, 200,217 Menzel, H., 58, 86 Merrit, E., 2, 52 Merryman, H. T., 16, 52 Mertz, J., 112, 147 Metcalfe, C. R., 177, 196,216 Metter, R., von, 32, 49 Meudt, W. J., 61,86 M Meyer, B., 2,51 Meyer, K., 101, 147 Meyer, K. H., 97,147 McBride, G. E., 157,215 Michel-Villaz, M., 32, 49 McComb, R. B., 130,147 Milburn, J. A., 177, 193,216,219 MacDaniels, L. H., 179, 180,212 Miller, D. H., 93,147, 155,215 McElroy, W. D., 2 , 5 2 Miller, S. L., 188, 216 Mackay, L. B., 191,212 Milthorpe, F. L., 175,216 Mackie, W., 155,215 Maclachlan, G. A., 55, 86, 1 17, 123, Minaker, E., 128, 150 Minchin, F. R., 197,213 132, 136, 137,150 Misch, L., 97, 147 McLaughlin, M. E., 177,216 McMurrough, L., 136, 138,147 Mitranik, M. M., 128, 150 McNab, J. M., 140,147 Mfiestrup, P. F., 156, 157,216 McNeil, M., 95, 144 Mohanty, P., 26, 2 7 , 4 0 , 5 1 MacRobbie, E. A. C., 193, 194, 196, Mollenhauer, H. H., 99, 101, 112, 118, 215 119, 121, 130, 132, 133, 142, 147, Malkin, S., 39, 51 148,150 Malloch, D. A., 164,216 Molz, F. J., 175, 180,216 Mandel, R. K., 63, 85, 8 7 Mondal, H., 63, 8 5 , 8 7 Mandels, M., 123, 149 Monro, J., 5 9 , 8 7 Montague, M. J., 132, 147, 156, 214 Manley, R. St.J., 138, 144 Mansfield, T. A., 191,215 Moor, H., 14, 15, 16,51, 102, 1 4 7 Moorby, J., 175,216 Manton, I., 99, 147, 155,215 Moore,T. S., 114, 116, 118,147 Mapson, L. W., 139,150 Morozova, T. M., 64, 8 5 , 8 7 Mar, T., 27, 33, 36,51 MorrC, D. J., 54, 61, 71, 84, 85, 86, Marchant, H. J., 109, 110, 1 4 7 87, 88, 99, 101, 112, 117, 118, 119, Maretzki, A., 11 7, 150 121, 125, 130, 132, 133, 134, 136, Markham, K. R., 206,215 142, 145, 146, 147, 148, 149, 150 Marks, I., 197,213 Morris, J. G., 207,216 Marshall, D., 58, 59, 8 7 Martin, J.T., 170, 189, 191, 197, 202, Moscarello, M. A., 128, 150 Moyed, H. S., 60, 8 7 , 8 8 215 Muenster, A-M. E., 156,218 Mascarenhas, J. P., 134, 1 4 7 Muhlethaler, K., 102, 1 4 7 Matthysse, A. G., 62, 63, 64, 65, 8 7 Mattox, K. R., 156, 158, 162, 192, Muller, P., 70, 8 7 Miiller, S., 71, 75, 88 215,218 Miiller, W., 32,51 Maximov, N. A,, 182, 184,215 Murakami, S., 42,51 Mayer, F., 105, 149 Murata, N., 27, 38, 3 9 , 4 2 , 5 1 , 52 Mazur, P., 17, 19,51 Murmanis, L., 104, I48 Megnet, R., 130, 147 Meidner, H., 171, 177, 191, 215, 218 Mutton, J. B., 199, 219 Myers, J., 37, 52 Meier, H., 97, 137, 144, 145

Louks, 0. K., 204,216 Lowe, M., 69, 88 Luduena, M. A., 134,150 Luttge, U., 191, 192, 198,215,216 Ludwig, B., 206,215 Luxova, M., 174, 175,215 Lyon, A, G., 162,215

228

AUTHOR INDEX

N

Nadeau, R., 65, 88 Nathanson, B., 13, 27, S O , 5 2 Neilson, R. E., 191, 216 Neish, A. C., 158, 206, 207,213 Nelmes,B. J., 110, 148, 1 8 0 , 2 1 6 Neufeld, E. F., 140, 145 Neukom, H., 9 1 , 1 4 8 Neumann, H. H., 174, 1 7 7 , 2 1 6 Neutra, M., 101, 128, 1 4 8 Newcomb, E. H., 105, 1 4 6 Newman, E. I., 174, 175,216 Nichols, E. L., 2 , 5 2 Nicolai, E., 98, 102, 148 Nicolson, T. H . , 164,216 Nieduszynski, I. A . , 97, I 4 8 Nishimura, M., 27,52 Nissl, D., 60, 88 Nobel, P. S., 170, 174, 175, 184, 186 216 Nola, E., 78, 8 7 Nooden, L. D., 7 8 , 8 6 Nordin, P., 112, 121, 137, 145, 1 4 6 147 Norris, K. H., 10, 50 Northcote, D. H., 90, 93, 98, 99, 100 101, 104, 112, 119, 120, 121, 122 126, 127, 135, 141, 143, 144, 1 4 6 148,150 Northcote, D. H. N., 155,216 Northrop, J. N., 2 , 5 1 Nostrand, F. van, 3 2 , 4 9 Nuhrenberg, B., 207,216 Nyquist, S., 132, 148 0

O’Brien, T. J., 62, 63, 69, 70, 84, 8 6 87 O’Brien, T. P., 98, 99, 105, 111, 142 148, 193, 1 9 7 , 2 1 5 Odzuck, W., 1 4 0 , 1 4 6 , 1 4 8 Ogawa, T., 202, 21 6 Ogino, C., 206, 207,216 Ogren, W. L., 187,215 Ohad, I., 138, 144 Okada, O., 4 2 , 5 2 Okayama, S., 4 2 , 5 2 Oliver, J. M., 110, 148 Oostrom, H., 6 4 , 8 7

Oquist, G., 41, 52 Ordin, L., 96, 117, 125, 134, 136, 137, 1 4 1 , 1 4 5 , 1 4 8 , 1 5 0 Orgel, L. E., 188,216 P Packer, L., 4 2 , 5 1 Paillotin, G., 32, 49 Palade, G. E., 90, 101, 114, 128, 129, 135,144,146,149 Paleg, L. G., 59, 88 Palevitz, B. A., 134, 146 Pallas, C. E. Jr., 191, 219 Paltridge, G. W., 204,216 Panayotatos, N., 140, 1 4 8 Paolillo, D. J., 1 9 0 , 2 1 3 Paolillo, D. R., Jr., 203, 21 1 Papageorgiou, G., 2 7 , 5 2 Pardue, J. W., 189,216 Parker, P. L., 1 8 9 , 2 1 6 Parkhurst, D. F., 204,216 Parrish, F. W., 123, 149 Passioura, J. B., 177, 193,216 Pasternak, G. W., 7 8 , 8 8 Pate, J. S., 193, 197,213, 216 Patni, N. J., 117 , 1 4 8 Paull,R.E., 118, 119, 127, 148 Paulouse, A. J., 189,214 Pedersen, K., 70, 8 5 , 8 8 Peel, A. J., 154, 192, 216 Penner, D., 62, 8 7 Pennington, W., 190, 219 Penny, D., 58, 59, 8 7 Penny, P., 58, 59, 61, 8 5 , 8 7 Penon, P., 6 3 , 8 8 Perlin, A. S., 123, 149 Phillip, J. R., 171, 216 Phillips, C . , 62, 63, 64, 8 7 Pickett-Heaps, J. D., 90, 100, 104, 105, 109, 110, 147, 148, 150, 156, 158, 1 9 2 , 2 1 6 Pincholy, S. K . , 207,217 Pinozynski, J. K. A., 1 6 4 , 2 1 6 Pirrson, A., 207,216 Pitman, M. G., 191, 192, 193, 198, 21 6 Poljakoff-Mayber, A., 170,216 Pollard, J. K., 118, I 4 8 Poort, S. R., 25,49 Porter, K. R., 100, 105, 110, 11 1, 1 4 7

229

AUTHOR INDEX

Porter, L. J., 206,215 Porter, W. L., 83, 8 7 Pospelov, V. A., 64, 8 5 , 8 7 Preston, R. D., 91, 97, 98, 99, 102, 105, 110, 143, 144, 145, 148, 149, 155, 1 8 0 , 2 1 5 , 2 1 6 Provasoli, L., 207,216 Puca, G. A., 7 8 , 8 7 R Rabinowitch, E., 4 , 5 0 Racey, P. A., 155,212 Rackham, O., 1 8 6 , 2 1 7 Ralph, R. K., 65, 8 6 Rappaport, W., 6 5 , 8 8 Raschke, K., 154, 170, 1 9 1 , 2 1 7 Rattray, J. B. M., 117, 1 4 9 Rause, G. E., 155, 156, 2 1 7 Raven, J. A., 154, 155, 158, 159, 170, 181, 184, 185, 186, 187, 188, 193, 197, 198, 1 9 9 , 2 0 7 , 2 1 7 Rawlins, S. L., 174, 1 7 7 , 2 1 7 Rawson, H. H., 1 9 3 , 2 1 3 Ray, P. M., 58, 60, 6 1 , 8 6 , 87, 95, 96, 97, 112, 113, 114, 116, 117, 119, 120, 121, 122, 124, 126, 127, 128, 130, 132, 133, 134, 135, 136, 137, 143,146,147,148,149 Rayle, D. L., 58, 59, 86, 88 Raymond, Y., 137,150 Read, D. J., 193,214 Recondo, E., 14 1, 145 Redman, C. M., 128,149 Rees, D. A., 95, 149 Reese, E. T., 123, 149 Reynolds, T., 2 0 7 , 2 1 7 Ricard, J., 63, 88 Rice, E. L., 2 0 7 , 2 1 7 Richards, E. L., 91, 9 5 , 1 5 0 Richardson, J. P., 68, 88 Richardson, M., 159, 193, 1 9 7 , 2 1 7 Ride, J. P., 2 0 7 , 2 1 7 Rideal, E. K., 59, 86 Riehle, U., 15, 16, 1 7 , 5 2 Riley, R. G., 189,217 Rivera, E., 132, 148 Rizzo, P. J., 70, 85, 88 Robards, A. W., 104, 149, 154, 158, 166, 175, 176, 197, 212, 213, 217 Robbi, M., 116, I 4 4

Robertson, R. N., 154,212 Robinson, D. G., 97, 100, 102, 105, 112, 113, 114, 116, 117, 119, 120, 121, 122, 123, 124, 126, 127, 128, 130, 133, 1 3 4 , 1 4 5 , 1 4 8 , 1 4 9 Roderer, G., 105, 109, 110, 149 Roelofson, P. A., 105, 11 1 , 1 4 9 Roland, J-C., 104, 117, 145, 149 Romanovicz, D., 96, 98, 99, 1 4 4 Rose, D., 17, 52 Rosen, W. G., 134,149 Ross, C., 1 5 4 , 2 1 7 Ross, C. W., 61, 8 6 Roux, E., 3 2 , 4 9 Roy, P., 63, 64, 70, 85, 8 6 Rudigier, J. F. M., 130, 1 4 6 Ruiz-Herrera, J., 105, 138, 144, 149 Rurainski, H. J., 3 9 , 5 2 Russo, V. E. A., 54, 72, 75, 86

S Sabatini, D. D., 114, 116, 143, I 4 9 Sachs, H., 100, 105, 145, 1 4 9 Sadava, D., 118, 119, 149 Salganik, R. I., 64, 8 5 , 8 7 Salisbury, F. B., 1 5 4 , 2 1 7 Salomon, Y., 69, 88 Samish, Y. B., 184, 2 0 8 , 2 1 7 Sampson, F. B., 200, 202, 2 0 3 , 2 1 8 Sandermann, H., 141, I50 Sarkissian, I. V., 60, 88 Satterthwaite, D. F., 169, 197, 21 7 Sauer, K., 42, 52 Savorick, S. A., 197,213 Scagel, R. F., 155, 156, 21 7 Scala, J., 101, 149 Scalan, R. S., 189,216 Scheckler, S. E., 1 7 9 , 2 1 7 Scher, M. G., 141, 1 4 7 Schmalstieg, F. C., 60, 88 Schmid, G. H., 5 , 5 2 Schmitz, R. Y., 8 0 , 8 7 Schnepf, E., 101, 105, 109, 110, 111, 130, 132,149, 198,215 Schnepf, G., 192, 1 9 8 , 2 1 7 Scholfield, W. B., 155, 1 5 6 , 2 1 7 Schomer, U., 6 2 , 8 6 Schonherr, J., 1 9 0 , 2 1 7 Schopf, J. M., 179, 2 0 0 , 2 1 7 Schopf, J. W . , 162, 169, 197, 21 7,219

230

AUTHOR INDEX

Schramm, M., 138,146 Schroter, K., 135, 149 Schultz, G., 207,217 Schwab, D. W., 101, I 4 9 Schwartz, A., 60,88 Schwerz, F. A., 11,52 Scott, F. M., 191, 192,217 Sculthorpe, C. D., 191,217 Seely, G. R., 4 7 , 4 9 , 5 2 Seliger, H. H., 2,52 Selinger, Z., 69,88 Sembdner, G., 70,87 Setterfield, G., 134, 144 Shack, T. L., 197,213 Shapiro, J., 17,52 Shavit, N., 3 9 , 5 1 Sheldrake, A. R., 181,217 Sherriff, D. W., 171, 177,215,218 Shiberi, A., 117, 149 Shields, R., 110,150 Shininger, T. L., 96, 112, 113, 117, 136,148 Shore, G., 117, 123, 132, 136, 137,

150 Shybin, V. V., 22, 23, 24,52 Sica, V., 78, 8 7 Siderer, Y., 39,51 Siegel, B. Z., 206,218 Siegel, S. M., 61, 88, 192, 206,218 Siegesmund, K. A., 134, 149 Sievers, A., 135, 149 Sifton, H. B., 190, 191, 192, 210,218 Simeone, J. B., 207, 21 8 Simonis, E., 101, I 4 9 Sineshchekov, V. A., 22, 23, 24, 26, 27,51,52 Sing, V. O., 105, 138,144, 149 Singh, R. N., 157,218 Skoog, F., 80, 8 7 Slatyer, R. O., 154, 174, 175, 184, 185,204,214,218 Smakula, E., 123, 149 Smith, A. D., 101,150 Smith, E. L., 6 2 , 8 6 Smith, F. A., 158, 181, 193, 197, 198, 199,217 Smith, M. M., 137, 150 Snyder, S. H., 78, 88 Soifer, D., 11 1, 150 Sondheimer, E., 207,218 Spanswick, R. M., 154, 175, 176,218

Spencer, J., 8, 29,50 Spooner, B. S., 134, 150 Sporne, K. R., 162, 163,218 Stabenau, H., 156,218 Stadelmann, E. J., 159, 164, 181, 182, 184, 199, 203, 204,219. Stadler, J., 98, 146 Staehelin, L. A., 104, 150 Stafford, H. A., 206,218 Stahl, C. A., 59,88 Steer, M. W., 166, 188, 193, 194, 196, 213 Stein, J. R., 155, 156,217 Stevenson, K. R., 174, 177,216 Steward, F. C., 118, 148 Stewart, K. D., 156, 158, 162, 192, 215,218 Stewart, W. D. P., 155,218 Stockman, V. E., 143,150 Stoddart, J., 65, 88 Stoddart, R. W., 93,150 Stoessl, A., 55, 88 Stone, B. A., 96, 137,144, 150 Stone, K. J., 141, 150 Storm, D. L., 141, 150 Stribley, D. P., 193,214 Strobel, G., 117, I50 Strominger, J. L., 139, 141, 150 Sturgess, J. M., 128, 150 Siidi, J., 55, 88 Sun, A. S. K., 42,52 Susi, R. F., 101, 150 Sutcliffe, J. F., 198, 218 Svoboda, A., 130,145 Swanson, A. L., 140, 146 Syrett, P. J., 156, 189,215,218 Szalay, L., 11, 52 Szollosi, D., 100, 150

T Takabe, T., 188, 189,218 Takamiya, A., 27,52 Takegami, R., 70,88 Takegami, T., 7 1, 88 Talmadge, K . W . , 58, 87, 91, 92, 93, 94,95,143,144, 146,150 Tashiro, Y., 114,149 Tavares, J. E., 71, 75, 88 Taylor, E. L., 134, 150

23 I

AUTHOR INDEX

Taylor. E. W., 109,144 Taylor, F. J. R., 156,218 Taylor, J., 156,214 Taylor, T. M. C., 155, 1 5 6 , 2 1 7 Teissere, M., 63, 88 Ten Ham, M., 3 1 , 5 2 Thimann, K. V., 8 3 , 8 7 Thines-Simpoux, D., 116, 1 4 4 Thom, M., 117,150 Thomas, E. A., 189,218 Thomas, J. B., 25, 31, 4 9 , 5 2 Thompson, E. C., 180,211 Thompson, J. F., 156,218 Thomson, K. S., 54, 71, 72, 75, 86, 87,88,117,147 Thornber, J. P., 26, 2 9 , 5 1 , 5 2 Thorne, S. W., 27,42,49 Thornley, J. H. M., 193,218 Thurtell, G. W., 174, 177,216 Timell, T. E., 95, 150 Tolbert, N. E., 156,218 Tollin, G., 2,52 Torok, M., 11,52 Torres-Pereira, J., 42, 51 Torrey, J . G., 105, I S 0 Trebst, A., 3 1, 52 Tributsch, H., 6 , 5 2 Trifunac, A. D., 23, SO Troughton, J. H., 182, 183, 189, 200, 202, 203,218 Tsai, C. M., 136,150 Tukey, H. B., Jr., 197,218 Tuli, V., 60,88 Tyree, M. T., 158, 159, 174, 176, 177, 197,218 U

Uhr, J. W., 101,150 Ursprung, A., 191, 192, 218

v Valent, B. S., 91, 150 Van den Berg, L., 17,52 Van, L. V., 137, 145 Van Baalen, C., 189,216 Van den Erde, G., 189, 1 9 1 , 2 1 8 Van den Honert, T. H., 1 7 0 , 2 1 8 Vandepeute, J., 61,86 Vanderhoef, L. N., 59, 88

Van Der Woude, W. J., 61, 88, 105, 117, 125, 132, 134, 136, 138, 144, 145,147,149,150 Van Huystee, R. B., 6 3 , 8 8 Van Loopik-Detmers, M. A , , 64, 8 7 Van Ysselstein, M. W., 61, 85 Varner, J. E., 54, 55, 65,86, 87, 88 Veldstra, H., 59, 86 Venis, M. A., 55, 56, 57, 59, 61, 66, 67, 68, 70, 73, 74, 75, 76, 77, 78, 79, 80, 81, 8 2 , 8 5 , 8 8 Vernon, L., 27,51 Villemez, C . L., 96, 137, 140, 146, 147,148,150 Von Willert, D. T., 198,218

W Wainwright, S. A,, 179, 180,218 Waisel, Y . , 198,219 Walker, D. A., 187, 188, 208, 215 Walker, J. R. L., 207,219 Walker, N. A., 158, 159,211,214 Walker, R., 190,219 Walsby, A. E., 19 1,219 Walter, H., 159, 164, 181, 182, 184, 199,203,204,219 Walton, J., 162, 166,215,219 Walton, T. J., 189,214 Wanka, F., 109,150 Wardlaw, I. F., 193,219 Wardrop, A. B., 98,148 Warby, C., 20,49 Wartenberg, A., 32,51 Watson, E. V., 202,219 Weatherley, P. E., 171, 174, 175, 219 Webb, J. L., 130, 150 Wehry, E. L., 5 , 5 2 Weidemann, G., 130, 145 Weinstock, M., 101, 150 Wessells, N. K., 134, 1.50 West, K. R., 181,213 Whaley, W. G., 100, 126, 130, 132, 145,150 Whistler, R. L., 91, 95, 150 White, R. K., 105,149 Whitehouse, H. L. K., 186, 219 Whitmarsh, J., 33, 36, 52 Whur, P., 101, IS1 Wibo, M., 116, 144

232

AUTHOR INDEX

Wickarder, E. R., 162,219 Wiessner, W., 22, 5 0 Wight, N. I., 95, 149 Wilchek, M., 66, 6 9 , 8 6 , 8 8 Wilder, B. M., 9 5 , 1 5 1 Wilkins, M. B., 72, 73, 74, 75, 80, 82, 85 Williams, G. C., 139, 219 Williams, R. F., 193, 2 1 3 Williams, R. J., 16, 5 2 Williams, W. A., 204, 215 Williamson, F. A., 117, 145 Williamson, V., 6 0 , 8 7 Willmer, C. M., 1 9 1 , 2 I 5 , 2 1 9 Willoughby, E., 141, 250 Wilson, D. F., 7, 50 Wilson, H. A., 78,88 Wilson, L., 105, 151 Wilson, M. L., 197,219 Wohl, K., 5,50 Wolken, J. J., 11, 52 Wood, A., 5 9 , 8 8 Wood, B. J. B., 189,219 Wooding, F. B. P., 100, 151

Wrenn, J. T., 134, 1 5 0 Wright, K., 112, 151 Wydrzynski, T., 40,51 Y Yamada, K . M., 1 3 4 , 1 5 0 Yamaki, T., 60, 78, 8 7 Yamashita, T., 4 2 , 5 2 Yang, L., 10, 27, 28, 51 Yoshida, K., 70, 71, 88 Yushok, W. D., 130, 1 4 7 Z Zeevart, J. A. D., 207,219 Zemek, J., 130, 151 Zenk, M. H., 6 0 , 8 8 Zentgraf, H-W., 132, 1 4 5 Ziegler, H., 190, 1 9 3 , 2 1 7 , 2 1 9 Zilinskas Braun, B., 2 6 , 5 1 Zimmermann, M. H., 174, 176, 177, 180, 192, 193,219 Zwar, J. A . , 55, 86, 8 7

Suhjeet Index

A Abscisic acid (ABA), and membrane binding, 7 1, 84 Absorption spectra, 23 Acetobacter .xyZinurn, in vitro cellulose synthesis, 138, 140, 141 Achyla, isolation of cytokinin binding glycoprotein, 71 N-acylaspartate synthetase induction by auxins in pea, 55-56 Affinity chromatography adsorbents benzyl adenine coupled to cyanogen bromide-activated zgarose, 70 2,4-D-lysine coupled t o cyanogen bromide-activated agarose, 66 IAA-lysine coupled to cyanogen bromide-activated agarose, 70 isopentenyl adenosine coupled to epoxy-activated agarose, 7 1 applications in auxin-binding effect of column eluates o n RNA synthesis, 66-69 effects of rifampicin on isolated factor stimulation, 67-68 isolation of protein factors from Maize, 6 6 Pea, 6 6 Soybean, 7 0 isolation of receptors from coconut, 70, 85 mode of action of the isolated protein factor, 68-70 applications in gibberellin and cytokinin-binding, 70-7 1

Affinity labelling techniques for probing hormone active binding sites amino auxin analogues 2-chloro-4-aminophenoxyacetic acid (CAPA)-diazo salt, 80-84 diazo-chloramben (2,s-dichloro3 aminobenzoic acid), 81-84 photoaffinity labelling with arylazides, 8 0 Algae, see Chlorophyceae and Charaphyceae Alternation of generations, in evolution of vascular land plants, 199 Ammonium acetate, use as a cryoprotective agent, 20 Anabaena, emission spectrum, 23 Aizacystis nidulans, 41 Apoplastic water movement and endodermal suberization, 175 in algal thalli, 159 Avmillavia rnellea, intercellular gas spaces of rhizomorphs, 2 1 0 Asteroxyion fossil evidence for existence of homiohydry, 203 ratio of internal and external areas, 189 Auxins affinity chromatography, 66-70 benzoylmalate synthesis in pea, 55-57 binding to purified histones, 61-62 cycloheximide, effect on auxin induced growth, 59 membrane binding specificity of sites 1 a n d 2 , 72-75 rapid cell elongation, 5 8 specificity of N-acylaspartate synthetase of peas, 55-56 233

234

SUBJECT INDEX

Auxins-cont. stimulation of glucan synthetase, 61 see also IAA, NAA, 2,4-D Avena, structur,e of cellulose, 96

B Benzoic acid aspartate conjugate formation in peas pre-treated with auxin, 55 specificity of sites 1 and 2 of auxin binding, 75, 78 Benzoylmalate synthetase induction in pea, 55-56 Benzyladenine binding to ribosomes, 65 Beta, apoplastic phloem unloading, 197 Bovine serum albumin, use as a cryoprotective agent, 20 Brassica oleracea, plasmodesmata, 196 BSA see bovine serum albumin Bulbochaete hiloensis, plasmodesmata, 1 60-1 61

C Carbon dioxide fixation resistances in biochemical reactions, 186 flux and its relationship to water flux in plants, 170-181 flux in C3 pathway ‘sun’ plants, 185-1 86 Cell fractionation studies analysis of cell wall protein fraction, 1 18-1 19 analysis of polysaccharide-bearing fraction, 11 9-1 25 identification of cell fractions, 1 1 2-1 18 preparation of cell fractions, 11 1-1 18 use of glutaraldehyde, 113, 114, 116 useof Mg2+, 114, 116, 117

Cellulose composition of cellulose, 96-97 demonstration of prescence, 96 orientation of cellulose, 105-1 11 structure of cellulose in Avenu, 96 Cladophora, 97 Pisum, 96 Pleurochrysis scherffelii, 9 8 Valonia, 97 Cell wall microfibril intermediates, pathways of synthesis, 136 Chaetomorpha melagonium orientation of cellulose, 105 site of synthesis of cell wall components, 102 Charophyceae Chaetosphaeridium, evolution of land plants, 157 Chara, structure, 159 Charales evolution of intercellular gas spaces, 192 evolution of land plants, 155-1 56 zygospores, 199 Coleochaet e structure, 157, 159 zygospores, 199 Klebsormidium, evolution of land plants, 157 Nitella, phenylalanine ammonia-lyase, 206 structure, 156, 159 Zygnemetales, evolution of land plants, 157 Chitin synthetase, three forms, 139 Chlamydornonas emission spectrum, 23 polarization, 34 Chlorella emission spectrum, 21, 23, 24, 27, 34, 35, 40 excitation spectrum, 23, 24, 27 polarization, 34, 36 spectral distortions, 1 1, 18, 19, 21 Chlorophy ceae Bulbochaete, structure, 159 Cludophora , localized growth, 159

235

SUBJECT INDEX

Chlorophyceae-cont. orientation of cellulose, 105 structure of cellulose, 97 Draparnaldia, structure, 159 Dunaliella, phenylalanine ammonia-lyase, 206 Fritschiella tuberosa, structure, 157,159 Schizo m eris evolution of intercellular gas spaces, 192 structure, 159, 162 Staurastrum, cell wall composition, 206 Stigeoclonium, structure, 159 Trentepholia, structure, 159 Chrysochromulina chiton, site of synthesis of cell wall components, 99 Citrate synthase activation and change in enzyme conformation by IAA, 60 Cooksonia fossil structure, 162, 164, 165 poikilohydry, 202 xylem, 177 Cryoprotective agents ammonium acetate, 20 bovine serum albumin (BSA), 20 dextran, 20 dimethylsulfoxide (DMSO), 20, 21 glycerol, 20, 21 polyvinylpyrrolidone (PVP), 20 sucrose, 20 use in prevention of freezing damage, 19-21 Cucumis sativus, leaf surface structure, 183 Cuticle, structure chemistry and evolution, 189 Cuticular resistance to C 0 2 movement, 186 to water movement, 185 Cycloheximide, effect on auxininduced growth, 59 Cytochrome oxidase, affinity for 0 2 , 208 Cytokinins binding by ribosomes, 65 binding glycoprotein from Achyla, 71 mediator protein in pea chromatin, 65

Cytoplasmic streaming, in giant cells of Algae, 159

D Dawsonia superba, leaf surface, 200 Devonian land plants, 162, 163 Dextran-charcoal techniques in IAA binding, 64 Dextran, use as a cryoprotective agent, 20 2,4-dichlorophenoxyacetic acid (2,4-D) induction of aspartate conjugates of NAA, 55-56 inhibition of 3-MO reduction, 60 protein mediators in stimulation rf chromatinbound RNA polymerase in soybean, 62-63 in stimulation of RNA synthesis by tobacco and soybean nuclei, 62 stimulation of glucan synthetase activity of onion plasma membrane, 61 2,6-dichlorophenoxyacetic acid (2,6-D), induction of aspartate in conjugates of NAA, 55-56 Dimethylsulfoxide, use as a cryoprotective agent, 20, 21 DMSO, see dimethylsulfoxide DNA melting temperature (T,) alteration by hormones, 62

E Electron spin-conservation rule, 3 Emission spectrum, 18, 19, 21-27, 34-40 Endoplasmic reticulum (ER) and auxin binding, 78 Eohostimella, sporophyte structure, 200 Ethylene in intercellular gas spaces, 207 Evolution of vascular land plants, an hypothesis, 199-205 Excitation spectrum, 23-24, 27-28

236

SUBJECT INDEX

F Fluorescence basic features, 34 definition, 3 determination of structure and composition of photosynthetic apparatus, 22-3 1 Freezing process and spectral distortion artefacts caused by freezing, 17-19 freezing of water, 14 ice-crystal formation, 14-1 7 use of cryoprotective agents, 19-21 Fritschiella, drought survival, 199

G

Golgi apparatus-cont. ation studies, 11 2, 113, 114 polysaccharide content, 1 19 synthesis of cell wall components, 99,100, 111, 119 various secretory functions, 101 Golgi membranes and auxin binding, 78 Closslingia breconensis, fossil xylem structure, 168 Gradmann-Van den Honert catenary formation applied t o CO, flux, 185-186 applied to water flux, 170, 171, 184-1 85

H

Halophytes, NaCl excretion, 198-199 Gas phase transport in vascular land Helzanthus, total resistance to water flux, 173 plants homiohydry and the intercellular Heracleum mantegazzianum, measurespace-cuticle-stomata complex ment of specific conductance, 174 cuticle, 189 Homiohydry, 182-184, 201-203 general considerations, 182-1 84 Hormonal alteration of DNA melting mtercellular air spaces, 186-189, temperature (Tm), 6 2 190-191 Hormonal control of enzyme synresistance imposed by stomata thesis, 54-59 and cuticle, 184-1 86 Hormonal regulation of cyclic AMP, stomata, 190-1 91 61 poikilohydry, 181 Hormone action sites Gel filtration studies of solubilized effects on macromolecular synorganelle fractions, 121 thesis, 54-56 General concepts of interaction beevidence for two sites of auxin tween light and matter, 3-5 action, 58-59 General influence of low temperature rapid effects of hormones, 56-58 on fluorescence, 5 Hormone receptors Glucan synthetase stimulation by auxdirect interaction with enzymes, 60-6 1 ins, 61 in animals, 54 Glycerol, use as a cryoprotective membrane-bound receptors agent, 20, 21 binding of naphthylphthalamic Glycine, total resistance t o water flux, acid (NPA), 71-72 173 model systems, 59-60 Glycolipids as intermediates in cell recognition process, 54 wall polysaccharide synthesis, ‘soluble’ nuclear/cytoplasmic re140-142 ceptors Glycosyl transfers in vitro, substrate applications of affinity chromaconcentration and glucan-formation, tography, 66-71 136-1 37 auxin-binding proteins, 63-64 Golgi apparatus auxin mediator proteins, 62-63 identification from cell fraction-

SUBJECT INDEX

Hormone receptors - cont. binding of gibberellins and cytokinins, 65-66 early studies, 61 histones and DNA, 61-62

I 3-Indolylacetic acid (IAA) citrate synthase activation, 60 complexes with protein in pea, 61 glucan synthetase stimulation in pea, 61 plasma membrane ATPase activation, 6 0 protein mediator of stimulated RNA polymerase, 6 3 reactive intermediate, 3-methyleneoxindole (3-MO), 6 0 receptor protein (IRP) and enhanced RNA synthesis, 63-64 regulation of cellulase activity, 55 Instrumentation for measurement of fluorescence emission, 7-9 Interactions between photosystems, 37-47 Intercellular gas spaces in evolution of homiohydry, 202-203 in respiratory gas exchange, 207-21 0 in subterranean organs, 208-209 In vitro synthesis bacterial cellulase, 136-1 37 chitin, 138-139 higher plant cellulose, 136-1 3 7 lipid intermediates, 140-142 noncellulose polymers, 139-140

237

Lignin biosynthesis, 206 synthesis in chlorophytes and land plants, 155 Lilium longiflorum, alkali-insoluble p-1,3 glucan in pollen tubes, 125 Lupinus albus phloem structure, 195 sieve plate structure, 196 M Marchantia polymorpha, structure of thallus, 1 9 0 Membrane binding at sites 1 and 2 function of site 1, 7 8 localization, 78 negative co-operativity, 85 resolution, 75-79 specificity of auxin binding, 72-75 Membrane binding of abscisic acid, 71, 8 4 auxins, 71 gibberellic acid, 71 morphactins, 71, 72 naphthylphthalarnic acid, 71-72, 75 Methods of investigating cell wall synthesis, 9 0 3,Methyleneoxindole (3-MO) as reactive intermediate in IAA action, 6 0 inhibition of reduction by NAA and 2,4-D, 6 0 Mucor rouxii, in vitro chitin synthesis, 139-139

N J Jablonski diagram, 4 L Lignification evolution, 201, 206-207 in early vascular plants, 179-1 80 mechanical support, 177-1 81 tensile forces generated during transpiration, 180-1 81

1,Naphthylacetic acid (NAA) binding b y maize coleoptile membranes inhibition by diazonium salts of CAPA or chloramben, 80-82 formation of aspartate conjugates in pea, 55, 56 inhibition of 3-MO reduction, 60 NAA-I4C binding t o membranes of maize, 7 2 Nitella, orientation of cellulose, 1 10, 111

2 38

SUBJECT INDEX

Nitrogen assimilation and pH regula- Penicillium verruculosum, /3-1,2 glution, 193, 198 canase-hydrolysis of cell organelle Non-cellulosic components of cell fractions, 123, 124 walls Petunia hybrida pollen tubes, X-ray composition of primary wall in diffraction studies of cellulose, 125 sycamore, 93, 1 18 Phaeophyceae, gas bladders, 191 enzymic fractionation of cells, 92, Phaseolus, emission spectrum, 23 93 Phenolics, excretion, 207 extraction from whole cells, 92, 93 Phenylalanine ammonia-lyase, in lignin structure of primary wall of sycabiosynthesis, 206 more, 94-95 Photosynthetic apparatus, similarities Nostoc, emission spectrum, 23 between chlorophytes and land Nucleotide sugar donors plants, 155 in cellulose synthesis, 136-137 Photosynthetic pigments of higher in chitin synthesis, 138-139 Algae and plants conversion of radiation, 6-7 in synthesis of noncellulosic materials, 139 fluorescence spectra, 22-29 hypothetical model of distribution of chlorophyll species, 30 orientation of pigments within 0 photosynthetic apparatus, 31-37 special features of the chloroplast Oocystis system, 5-7 orientation of cellulose, 105-106, Pisu m 107,108,109,110,111 site of synthesis of cell wall comcell fractionation studies, 114, ponents, 102, 103 115, 116,117, 119,120 Orientation of cellulose structure of cellulose, 96 colchicine experiments, 105, 106, % sugar radio composition of cell 108, 109, 110 and cell wall fractions after I4Cin Chaetomorpha, 105 glucose labelling, 120-121 in Cladophora, 105 symplastic phloem unloading, 197 in Nitella, 110, 11 1 transport of synthesized materials, in Oocystis solitaria, 105, 106, 129, 132,133 107,108, 109,110,111 Plasmalemma in Porteriochromonas stipitata, identification from cell fraction105, 106 ation studies, 1 12, 1 17 Orientation of pigments within photosynthesis of cell wall components, synthetic apparatus 99, 100,102,103, 104,105 determination of orientation using Plasma membrane ATPase activation polarized light, 3 1-37 by IAA, 5 8 , 6 0 in Chlamydomonas, 33 Plasmodesmata, structure and function in Chlorella, 34 in multicellular chlorophyta, 158, in Mesotaenium, 33 160, 161 Oryza, 0 2 dependence in angiosperms, Pleurochrysis scherffelii 207 site of synthesis of cell wall components, 99 structure of cellulose, 98 P Poikilohy dry Paleozoic, atmospheric 0 2 concentraand production of dessicationtion, 208 resistant spores, 199 Parenchymatous tissue, measurement in evolution of vascular land of specific conductance, 175, 176 plants, 199

239

SUBJECT INDEX

Polarization of Chlarnydomonas, 33 of Chlorella, 34,36 of Mesotaenium, 33 use of plane polarized light in determination of orientation of pigments, 31-37 Polyvinylpyrrolidone, use as a cryoprotective agent, 20 Porteriochromonas stipitata, orientation of cellulose, 105, 106 Prasinophyceae, ancestors of land plants, 155 Progenitors of vascular land plants, 155-162 ‘Proton pump’ hypothesis in rapid cell elongation, 58, 60 Psilophyton, ratio of water movement through xylem and parenchyma, 176,177 P. princeps, tracheids, 169 PVP see polyvinylpyrrolidone R Resonance, 6 Rhizopus arrhizus, p-1,3 glucanasehydrolysis of cell organell fractions, 123, 124 Rhynia fossil evidence for existence of homiohydry, 203 ratio of water movement through xylem and parenchyma, 176 R. Gwynne- Vaughanii, reconstruction from fossil evidence, 167 R. major O2 supply to subterranean organs, 209 ratio of internal and external areas, 189 structure of axis, 167 structure of phloem, 169 structure of tracheids, 180 Ribosomes binding of benzyladenine, 65 binding of cytokinins, 65 Ribulose diphosphate carboxylase, affinity for C 0 2 , 208 Ricinus emission spectrum, 13 spectral distortions, 13

R. communis sieve plate structure, 194, 195 stem structure, 178 xylem structure, 172, 173 Root pressure, transport of solutes in xylem, 192 S

Scatchard analysis of auxin binding data, 71-72 Scenedesrnus obliquus, site of synthesis of cell wall components, 104 S. pannonicus, site of synthesis of cell wall components, 104 Secondary plant products excretion, 198 in relation to vascular plant evolution, 206-207 Silurian algal fossil record, 162 land plant fossil record, 162-163 Sites of synthesis of cell wall components golgi apparatus, 99, 100, 101, 119 in Chaetomorpha melagmiurn, 102 in Chrysochromulina chiton, 99 in Oocystis apiculata, 102, 103 in Pleurochrysis scherffelii, 99 in Scenedesmus pannonicus, 104 in S. obliquus, 104 in Zea mays, 104 plasmalemma, 99, 100, 102, 103, 104,105 Spectral distortions chlorophyll concentration, 10-1 2 destruction of organelles, 12-13 freezing techniques, 10, 13, 14-21 the effect of scatter, 10, 11 Spinach, emission spectrum, 12, 23, 25,39 Sporopollenin in Wenlockian aerial spores, 199 synthesis in chlorophytes and land plants, 155 Stomata control of water loss, 182-1 86 evolution, 190 guard cell osmotic pressure, 191 homiohydry, 202, 203 Stomata1 resistance in vascular plants, 184

240

SUBJECT INDEX

Streptomyces QMB814 sp, -1,4 glucanase-hydrolysis of cell organelle fractions, 123, 124 Sucrose, use as a cryoprotective agent, 20 Symplastic water movement, specific conductivity, 176

T Tracheid structure, in fossil vascular plants, 180 Transfer cells, for symplast-apoplast solute exchanges, 197-19 8 Transport of dissolved solutes excretion, 198-199 phloem, 193-197 symplast and apoplast, 197-19 8 xylem, 192-1 93 Transport of synthesised cell wall materials determination of site of synthesis, 125, 126,127 .effect of cyclohexamide on transport in animal tissue, 128 effect of glycolytic inhibitors, 129 effect of inhibitors of oxidative phosphorylation on transport, 129 effect of respiratory inhibitors on transport, 129, 130, 131 physiology of secretion in animal cells, 128, 135 physiology of secretion in plant cells, 129, 130, 131, 132, 133, 134,135 rate of transport, 126, 127, 128 Trimerophytales, fossil record and structure, 162, 163 Triticum, sucrose flux in the symplast during phloem loading, 193

V Valoniu, structure of cellulose, 97 Vascular plants, fossil structures, 162-169

Vicia, sucrose flux in the symplast during phloem loading, 193 V. faba, leaf structure, 182 W

Water transport in plants apoplastic movement, 175 cell t o cell movement, 175 hydraulic conductivity, 174 pathway and resistances, 172-174 symplastic movement, 176

X Xerophytes, cuticular resistance, 185 Xylem and liquid-phase water transport significance of lignification, 177-1 81 transpirational flux of water, 170-1 72 as a low resistance pathway for mass flow, 171-177 Z Zea mays cell fractionation studies, 11 8, 119, 120 polysaccharide content of golgi bodies, 119, 120 polysaccharide production of extracellular slime, 1 19-120, 121 site of synthesis of cell wall components, 99-1 00, 126 percentage radiocomposition of cell and cell wall fractions, 120, 121 transport of synthesized materials, 133 Zosterophyllum fossil structure, 162 ratio of water movement through xylem and parenchyma, 176, 177 stomata1 structure, 166