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Advances in

BOTANICAL RESEARCH VOLUME 11

Advances in

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

Department of Plant Biology, University of Birmingham, Birmingham, England

Editorial Board H. W. WOOLHOUSE W. D. P. STEWART

W. G . CHALONER E. A. C. MAcROBBIE

John Innes Institute, Norwich, England Department of Biological Sciences, The University, Dundee, Scotland Department of Botany, Bedford College, Regent’s Park, London, England Department of Botany, University of Cambridge, Cambridge, England

Advances in

BOTANICAL RESEARCH Edited by

J. A. CALLOW Department of Plant Biology University of Birmingham Birmingham, England

H. W. WOOLHOUSE John Innes Institute Norwich, England

VOLUME 11

1985

ACADEMIC PRESS (Harcourt Brace Jovanovich, Publishers) London Orlando San Diego New York Toronto Montreal Sydney Tokyo

COPYRIGHT 8 1985, BY ACADEMIC PRESS INC.(LONDON) LTD. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY. RECORDING. OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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CONTENTS CONTRIBUTORS TO VOLUME 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PREFACE.. . ........ .... ...

..

vii ix

.................................... ............................................. Principles of Laser Light Scattering. . . . .........

3 7

Laser Light Scattering in Biological Research M. W. STEER, J. M. PICTON, AND J. C. EARNSHAW 1.

111.

1V. V. VI. VI1.

Introduction

Laser Doppler Microscopy . . . . . . . . . . . Biological Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prospects ......... ....................................... Appendix-Notation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

37 62 64 65

Transport and Fixation of Inorganic Carbon by Marine Algae N. W. KERBY AND J. A. RAVEN I. 11. 111.

IV . V. VI. VII. VIII. IX . X.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inorganic Carbon System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Transport of Inorganic Carbon between the Medium and Marine Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carbon Fixation in Marine Algae.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ribulose-l,5-Bisphosphate Carboxylase/Oxygenase (RUBISCO) . . . . . . . The Occurrence of RuBPo Activity, and of the PCOC, in ................................ Marine Algae. . . arine Algae.. . . . . . . . . . . . . . . . . . . . P-Carboxylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C, Metabolism in the Phaeophyceae .............................. .................. ... Conclusions ...................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cell Wall Storage Carbohydrates in Seeds-Biochemistry the Seed “Gums” and “Hemicelluloses”

71 72 75 85 88 94 101

109 114 116 118

of

J. S. GRANT REID I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Structures of Cell Wall Storage Carbohydrates in Seeds . . . . . . . . . . . . . .

125 126

vi

CONTENTS

III. Formation and Postgerminative Catabolism ......................... IV . Considerations of Biological Function ............................. V . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

132 148 152 153

Welwitschia mirabilis-New Aspects in the Biology of an Old Plant D . J . VON WILLERT 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Osmoregulation and Chemical Composition ........................ 111. Water Economy and Water Uptake ............................... IV . Photosynthesis and Carbon Balance ............................... V . Energy Balance ............................................... V1 . Concluding Remarks ........................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AUTHOR INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

157

162 172

179 188

189 189 193 203

CONTRIBUTORS TO VOLUME 11 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

J. C. EARNSHAW (l), Department of Pure and Applied Physics, The Queen’s University of Belfast, Belfast BT7 lNN, Northern Ireland N. W. KERBY (71), A. F. R. C . Research Group on Cyanobacteria, Department of Biological Sciences, The University, Dundee DDI 4HN, Scotland J. M. PICTON (l), Department of Botany, The Queen’s University of Belfast, Belfast BT7 INN, Northern Ireland J. A. RAVEN (71), Department of Biological Sciences, The University, Dundee DD1 4HN, Scotland J. S . GRANT REID (125), Department of Biological Science, University of Stirling, Stirling FK9 4LA, Scotland M. W. STEER (l), Department of Botany, The Queen’s University of Belfast, Belfast BT7 INN, Northern Ireland D. J. VON WILLERT (157), Institut fur Angewandte Botanik, Universitat Munster, D-4400 Munster, Federal Republic of Germany

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PREFACE The individual volumes of Advances in Botanical Research have traditionally presented articles of very diverse subject matter. While some reviewers have felt that this has detracted from the appeal of this series, others have commented on the value of this approach in permitting the publication of articles that do not necessarily fit easily into other review publications. The present volume continues this emphasis on diversity although readers may be interested to know in advance that future volumes of a more thematic nature are actively being considered. In this volume Steer et al. discuss the exciting possibilities of laser Doppler microscopy for biological research at the cellular level, as, for example, in the characterization of cell particles and the study of their interaction in membranes. This technique may not be widely appreciated in the biological community and while much of the treatment is mathematically rigorous and physical, this should not deter the reader from considering the value of this approach in biology. There can be no clearer form of encouragement than that generously expressed in the final sentence of this article. Two contributions are broadly concerned with plant biochemistry. The nature of carboxylation mechanisms in plants appears to be an enduring theme and it seemed to the Editors that the situation in algae was ready for critical evaluation, which Kerby and Raven do admirably. While previous volumes have included discussions of seed storage proteins, many seeds are characterized by specific carbohydrate polymers in their cell walls and Reid’s article treats these various polymers for the first time as a botanically coherent group of substances and attempts to explore their biological (function) significance. The last article is von Willert’s interesting account of the biology of that most unusual plant, Welwitschia. von Willert’s approach is primarily physiological and the reader would do well to bear in mind the unique difficulties faced by the scientist in attempting to do controlled, replicated physiological experiments on this strange plant. J . A . Callow H. W. Woolhouse

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Laser Light Scattering in Biological Research

M. W. STEER, J. M. PICTON, and J . C. EARNSHAW" Department of Botany "Department of Pure and Applied Physics The Queen's University of Belfast Belfast, Northern Ireland

1. Introduction ......................... .... 11. Biological Motion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Diffusion . . . . . . . . . . . . . ..................................... B. Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... 111. Principles of Laser Light Scattering . . . . . ........... . A. Properties of Laser Beams . . . . . . . . . . ........... B. Basic Scattering Considerations ........................ C. Conventional Light Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... D. Dynamic Light Scattering . . . . . . . . . . . E. Optical Mixing Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Interpretation of Data . . . ........................................... ... IV. Laser Doppler Microscopy ............................... A. Instrument Design . . . . . . . . . . . ..................... B. Standard Test Systems . . . . . . . ..................... V. Biological Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

....................................... B. Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

....................................... ....................................... VII. Appendix-Notation .................... ........... .......... ........................................................

1 3 3 6

7 7

8 10 12 16 25 28 28 33 37 37 45 48 62 64 65

I. INTRODUCTION The earliest observations in cell biology were made at the light microscope level on living cells. The visual impact on these early observers of the teeming activity exhibited by cells has since been experienced by every student of introductory ADVANCES IN BOTANICAL RESEARCH. VOL I I

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Copyright 0 1985 hy Acadrrnki Press Ini (London) Ltd All right5 of reproduction in any form rcierved lSBN0-I?005911 8

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M. W. STEER E T A L .

biology courses. Cell biology has, of course, moved on from such simple, yet effective, methods to the range of molecular, biochemical and ultrastructural techniques used today. These succeed because they concentrate on one aspect of the cell system: a particular function, a group of reactions, an aspect of structure. Such techniques have enabled cell biologists to gather an impressive range of information about the structure and function of cells and their components. There is now an increasing interest in trying to assemble this information into a complete picture of living cells. Living systems are dominated by dynamic events, from the level of molecules to whole cells and organisms. These dynamic events occur over time scales ranging from those of chemical reactions to whole cell movements. Study of these events, either in vitro or in vivo, requires a technique that can sample over very short time scales and can do so without disrupting or interfering with either the course of the reactions or the freedom of movement of the components and without causing injury to living systems. Laser light scattering is such a technique; it has been used extensively in the physical sciences and engineering, and over the past 10 years has proved to be of increasing value to the biologist. As with any technique, it requires an understanding of the theoretical principles, the practical and instrumental requirements, and the limitations which apply to the interpretation of the results. Here we have attempted to review these topics and the recent advances that have been made by the application of this technique to problems in biological research. Hence we hope that biologists faced with similar problems will be able to judge the potential value of the technique for their particular purposes and pursue their interest further through the references provided. The name of the technique, laser light scattering, clearly identifies the principal phenomenon on which it rests, the scattering of light. Light incident upon an optically heterogeneous medium will be scattered. In the context of this article the medium will be aqueous and the heterogeneities will range in size from cells or organelles to macromolecules. If the heterogeneities, or particles, are in motion they will impart Doppler shifts to the wavelength of the scattered light. Thus measurements of the wavelengths present in the spectrum of the scattered light will yield information concerning the motions occurring within the sample. Lasers produce an intense beam of a single wavelength, and so their use considerably simplifies the analysis of the scattered light. The motions of interest in biological studies are slow, and correspond to Doppler shifts which are very small (of the order of a few kiloHertz) compared to the frequency of the incident light beam (about 10IJ Hz). Consequently conventional spectroscopic techniques are useless; they have quite insufficient resolving power. Optical mixing techniques must be used. For example, the scattered light may be mixed with light from the original laser beam. The detector, a photomultiplier, responds to the combined field, and gives an output signal displaying a beat signal at the frequency difference between the scattered and reference light. This beat frequency may subsequently be analyzed by electronic means

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

3

which may involve recovery of the original frequency spectrum of the scattered light (spectrum analysis), or, more conveniently, we can measure the time autocorrelation function of the detector output. Many reviews, texts, and conference proceedings have been published since the first experiments in optical mixing spectroscopy in the mid-1960s. An excellent and elementary account of the physical techniques is to be found in the review of Pusey and Vaughan (1975); more extensive and detailed coverage will be found in the proceedings of a series of NATO Advanced Study Institutes (Cummins and Pike, 1974, 1977; Chen et a l . , 1981). The applications of laser light scattering to biochemical and biological problems have been reviewed by Carlson (1975), Bloomfield (1981), and Chu ( I 979). A further NATO Advanced Study Institute (Earnshaw and Steer, 1983) was directly concerned with the interface between the physical techniques and biological problems addressed in the present article. Nearly all of the references given above demand fairly substantial levels of mathematical ability; here we have striven to provide a very basic introduction to this literature. This article opens with a brief discussion of the motions encountered in biological systems and of the conventional methods for recording them. The principles of laser light scattering are considered in the next section. Here, as already indicated, we have attempted to provide an account that will be comprehensible to the nonspecialist. The commercial availability of the necessary instrumentation and computer software has placed this technique at the disposal of many biologists, and we believe that this section should enable the reader to undertake routine observations and analyses. The application of these light scattering techniques to microscopic samples, living cells for example, is discussed in the section on laser Doppler microscopy. This section concentrates on design aspects and the testing of such instruments. Biological applications of laser light scattering are brought together in a single section and described in approximately increasing order of complexity of the biological system and of the correlation functions obtained from them. Hence this section starts with the characterization of homogeneous protein solutions in vitro and progresses first to more complex solutions undergoing dynamic changes, then to motions of cellular structures and model membranes, and finally to the motility of whole cells and flow of extracellular fluids. Many of the in vitro applications have been discussed extensively elsewhere, so we have been selective, attempting to indicate the range of studies undertaken while concentrating on the cytoplasmic and whole cell levels. Finally we assess the future for this technique in biological research and attempt to identify those areas where we think it will lead to substantial advances. 11. BIOLOGICAL MOTION A. DIFFUSION

Diffusion processes exert a fundamental influence on the behavior of molecules and ions in solution. Within cells diffusion represents the most primitive type of

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M.W. STEER ET AL.

transport system, yet one that is vital to the initiation of all chemical reactions in the cytoplasm. Water in the cell (Drost-Hansen and Clegg, 1979) serves not only as a solvent for diffusing molecules and ions, but also as a crucial determinant in the structural folding of macromolecules, such as proteins, and in the organization of lipids forming the all-important plasma and cellular membranes. The movement of particles in the cytoplasm of normal living cells is not dominated by Brownian motions. The particles will undergo these motions only if the cells are physically damaged or killed. This implies that the living cytoplasm cannot be a simple suspension of the cell components. Two possible explanations for this reduced mobility of cellular particles have been proposed, but it is likely that a combination of both will be required to account for all the dynamic characteristics of living cytoplasm. The first possibility is that the cell components may exist attached to an extensive network of filaments and fibers within the cytoplasm, which limits their freedom of movement (Wolosewick and Porter, 1979; Small, 1981). The second is that water molecules form specific associations with hydrophilic macromolecules and solutes in the cell, so that a proportion of the total cell water is “bound” (Clegg, 1979). This bound water may influence the activity of adjacent water molecules, forming “multilayered” water (Ling, 1979a,b). The outcome envisaged is that only a small proportion of cytoplasmic water could behave freely as “bulk” water (Fulton, 1982) and be capable of acting as a suspending medium for free particle motion. These proposals are by no means universally accepted. The theoretical basis used for the analysis of the nuclear magnetic resonance (NMR) data leading to interpretations favoring the existence of multilayered water have been criticized (Villa et al., 1983; Borghi et a / . , 1983). Also, observations on the hydration and solution of protein molecules suggest that the bound water molecules on the surface of the protein do not have unique conformations; they can be accommodated into the bulk phase water without the formation of an additional bounding layer of specially oriented water molecules (see review by Rupley et d . ,1983). Diffusion of components within cells is therefore of considerable interest, both at the level of individual molecules and at that of whole cell structures. Diffusion coefficients of cellular components depend on the viscosity of the cytoplasm (Pollard, 1979). According to Lehman and Pollard (1965) the larger a molecule the greater will be the viscosity that it will experience in the cytoplasm. Their estimates of viscosity were based on experiments with extruded bacterial cytoplasm. The estimates ranged from 1- 10 poise, compared with a value of 1 x lo-* poise for water under standard conditions. At the present time conventional methods of assessing viscosity and diffusion coefficients in living cells are far from satisfactory. Gross physical properties of the cytoplasm of Physarum coenocytic plasmodia1 strands have been investigated in situ by an ingenious adaptation of the falling ball viscometer (Sato et al., 1983). Small (