Advances in Botanical Research, Volume 5

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Advances in

BOTANICAL RESEARCH VOLUME 5

This Page Intentionally Left Blank

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).


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


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


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


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


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


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.


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


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


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


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


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).


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


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).


*

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


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-


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
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).


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.


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).


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.


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


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


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

53 54 54 56 58 59 59 60 61 71

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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


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

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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.


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


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.


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


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


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


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).


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


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

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