No title

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

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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

vii

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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

1

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

4

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 (<36 pm diameter) magnetic beads were taken up by phagocytosis into the strands. These were subjected to magnetic fields of varying strength and

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

5

direction and their movements recorded by videomicroscopy for subsequent measurement. From these studies it was concluded that the cytoplasm behaves as a viscoelastic material beyond its yield point. Fluorescein-labeled proteins have been injected into fibroblast cells and their movement followed by the method of fluorescence recovery after photobleaching (Wojcieszyn et al., 1981). This showed that diffusion of the injected labeled proteins (68 and 160 kDa, respectively) was reduced to about one-seventieth (1.0 X lo-* cm2 sec-I) of that in water, similar to that expected in a 61% sucrose solution (about 70 X l o p 2 poise). The same method was used to study the movement of a native cellular protein, actin (42 kDa), injected into cells of amoebas (Wang ef al., 1982). Diffusion of this protein in the cytoplasm was found to be between one-half and one-eighth of that in aqueous solution, significantly faster than the diffusion found in the small fibroblast cells described above. This difference was ascribed to a difference in cytoplasmic organization between the two cell types, particularly the absence of extensive microfibrillar networks in the large amoeba cells. Similar levels of diffusion were found by Paine et al. (1975) in another type of large cell, oocytes of Rana. Their method involved the use of autoradiography to study the movement of injected radioactive dextrans (5- 150 kDa). They suggested that the reduction of diffusion within the cell, compared with aqueous media, was due to a high internal viscosity and collisions between the diffusing molecules and cytoplasmic structures. Studies of in vitro gel structures (Sellen, 1983) have shown that ions and small molecules can diffuse freely, while the movement of larger molecules is depressed by the gel network, leading to a reduction in their apparent diffusion coefficients. Cinemicrography of sieve tubes was used to record the motions of starch grains by Barclay and Johnson (1982). Frame-by-frame analysis of the film enabled an estimate to be made of the diffusion coefficient of the starch grains and hence the viscosity of the sieve tubes. The values obtained were lower than expected for this size of particle suspended in 20% sucrose, the presumed content of the sieve tube. Nuclear magnetic resonance techniques have provided much valuable information on motions in living cells (Hazelwood, 1979; Finch, 1979). These have established much lower viscosities than those found by Lehman and Pollard (1965), generally in the range 3-10 X l o p 2 poise. They have also shown that the self-diffusion rate of water is less than that observed in bulk water. Tanner (1983) recorded a range from 1.2 to 6.3 X l o p 6 cm2 sec- I (compared with 2.4 X cm2 sec- I for pure water) but concluded that these were no different from comparable solutions of similar polymers and, therefore, that there was no evidence of a special “biological structure” effect. This range of self-diffusion values for water yields viscosities in the range of 20-38 centipoise (cP). NMR analysis requires a relatively large sample, -300 mm3, so it is quite unable to make unambiguous measurements of individual cells or part of cells. Present evidence therefore appears to suggest that diffusive motions inside

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M. W. STEER ET AL.

living cells are quite different from those found in bulk solutions. There is, however, a remarkable paucity of information on the physical conditions inside living cells and the precise effects that these have on the behavior of the various cytoplasmic components. B . MOTILITY

Motility is a phenomenon uniquely characteristic of biological material. Numerous organisms exhibit motility. Many of these (e.g., bacteria, protists, male gametes) are microscopic in dimensions and swim in an aqueous medium. There is considerable biological interest in the study of these motile unicellular organisms (e.g., Brokaw and Gibbons, 1975). They possess a wide range of structural specializationsthat confer motility on the cell; they exhibit a corresponding range of swimming activities and most are able to adjust their swimming behavior to stimuli received from the external medium, such as heat, light, partial pressures of carbon dioxide and oxygen, nutrients, and toxins. An understanding of the biochemistry and physiology of the swimming processes is dependent on a thorough knowledge of the swimming motions, which are often complex and difficult to observe microscopically. In addition to the motility of individual cells and organisms, many cells exhibit continuous directed movements of the cytoplasm, known as cytoplasmic streaming (Jahn and Bovee, 1969). This is most clearly seen in large, vacuolate plant cells (Allen and Allen, 1978a) such as onion epidermis, Tradescantia stamen hairs, Elodea leaves, and internodal cells of Nitella and Chara. Typically these cells exhibit streaming rates in the range 5-100 pm sec- l . Related cytoplasmic movements occur in the tip extensions of nerve cells, the advancing plasmodia1 front of Physarum (Wohlfarth-Bottermann, 1983), and in the amoeboid movements of such cells as phagocytes, Amoeba (Allen and Allen, 1978b), and various protists. Streaming in living cells can be observed microscopically, using the motion of the resolvable particles as a guide to the underlying cytoplasmic flow. This has its drawbacks, as there is no guarantee that such particles travel at the same rate as the bulk flow, a view supported by the observation that larger particles travel more slowly than smaller ones. Direct measurement of the flow rate is difficult, leading to the use of cinemicrographic methods for recording, followed by a frame-by-frame analysis of particle movement, Such observations have provided much valuable information on the responses of flow to various internal and external conditions (e.g., Tominaga and Tazawa, 1981). It is clear from electron microscopic and biochemical evidence that the active components of cytoplasmic streaming are below the level of resolution of light microscope systems. Streaming certainly involves the activities of microfilaments (7-nm-diameter fibrils) which may beat, oscillate (Fig. I ) , or rotate (Jarosch and Foissner, 1982). In so doing they may generate a flow either on their

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

7

Fig. 1 . Diagram showing structural arrangement of the outer layers of a Nitella internodal cell. The chloroplasts remain stationary in the center adjacent to the cell wall. Cytoplasmic streaming occurs in the endoplasm as a result of the activities of numerous endoplasmic filaments. (Reproduced from Allen, 1974.)

own or through associations with a cell membrane system, such as the endoplasmic reticulum. While there have been many attempts to understand cytoplasmic streaming by studying living cells and isolated parts of the cytoplasm (Nagai and Hayama, 1979; Kamitsubo, 1980), it is apparent that the basic problem lies in the inability of conventional optical systems to characterize the motions of the structures generating the flow. 111. PRINCIPLES OF LASER LIGHT SCATTERING A. PROPERTIES OF LASER BEAMS

Light beams from lasers are very intense, highly coherent, and monochromatic and are usually plane polarized. None of these characteristics is essential for applications to dynamic light scattering; successful experiments with conventional light sources have been reported (Jakeman et al., 1976). The use of lasers in dynamic light scattering experiments is, however, so universal that one of the alternative names for the technique is laser light scattering. The most significant advantage (in this field) of laser light beams is their high intensity. The laser power is concentrated into a narrow beam which diverges very slowly as it travels away from the laser and also into a very narrow range of optical frequencies. By contrast, light from a thermal source (e.g., an incandes-

8

M. W. STEER E T A L .

cent lamp), when similarly collimated and restricted in frequency, would be of very low intensity. The light output by a laser is highly coherent and monochromatic. A coherent beam can be considered as one in which the light waves form a single continuous sinusoidal oscillation. In practice the laser output maintains such an oscillation for a period of time, after which the phase of the oscillation alters randomly. The characteristic time period over which the laser maintains its oscillation is called the “coherence time” of the beam. Lasers are characterized by much longer coherence times than other light sources. If the coherence time were infinite (i.e., if the wave phase never changed) the light would have a unique frequency. The random shifts in phase lead to a broadening in the spectrum of the light; a laser does not produce light of a single frequency. However, for the types of experiment to be considered here the laser acts as i f it produced petj5ectly monochromatic light (Cummins and Swinney, 1970). Thus frequency shifts less than the width of the laser spectrum can be resolved. Even modest lasers can produce light beams which are plane polarized to a high degree. This may be useful in optical system design and also permits the detection of depolarized light scattered by optically anisotropic particles. The light beam from a laser is very well defined, usually being stable in direction and in the spatial divergence about that direction. The profile of intensity across the beam is characteristic of the particular mode in which the laser is operated (Kogelnik and Li, 1966). The fundamental (TEM,,) mode has a Gaussian intensity profile. Such a defined beam propagates through optical systems in an entirely predictable fashion, retaining the Gaussian profile. The known intensity distribution at the focus of a lens can define the illuminated volume in a laser light scattering system. For heterogeneous biological samples this may be most useful in permitting the selection of a relatively homogeneous sample volume. B . BASIC SCATTERING CONSIDERATIONS

A typical scattering experiment is shown in Fig. 2. A laser beam is focused into the sample volume and light is scattered at all angles. This scattered light is detected by a photomultiplier which can be placed at an angle 8 with respect to the incident beam. The detector responds to the time-averaged intensity of the light as it cannot follow the optical frequency. In fact, a photomultiplier produces an output comprising responses to individual detections of photons (with probability governed by the incident intensity). Depending on the requirements of the subsequent electronic processing, the detector output may be retained as digital photodetection events or may be smoothed into an analog output photocurrent. The former option, indicated in Fig. 2 by the amplifieridiscriminator combination, is now favored, with the availability of fast photon counting systems. If the scattered light is projected onto a distant screen, the pattern formed

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

-v k0

9

SAMPLE

n

LENS

SIGNAL ANALYZER

DISK

AMP

Fig. 2. A typical scattering experiment. The signal analyzer may be a spectrum analyzer working in the frequency domain or a correlator in the time domain.

appears to be granular on a very fine scale. This granularity (or speckle) is due to interference between light scattered by different particles. As the scattering particles move, the intensity of light at a single point in the speckle pattern will vary as the phase differences between the different light waves interfering at that point change. The motion of the scattering particles is reflected in the time variation of the intensity of a given speckle. Changes in a single speckle (or at most a few) can be detected by placing a pinhole in front of the detector (Fig. 2). The scattering volume, or volume of specimen which is sampled, is defined first by the focusing of the incident beam and second by the lens and slit system selecting that portion of the scattered light falling on the photomultiplier. If this scattering volume is made so small that only a few particles which scatter light lie within it, undesirable effects may arise due to number fluctuations (see p. 15). The incident light beam and the scattered light at angle 8 are characterized by wave vectors k, and k, (Fig. 2). The two beams lie in a plane-the scattering plane. The wave vectors have essentially equal magnitude (k = 27~/X)as the scattering causes only infinitesimal changes in wavelength. The change in direction of the light on scattering is expressed through the scattering vector: q

=

k, - k,

(1)

which has magnitude q = 2k0 sin(W2) = (4~n,,/X) sin(8/2)

(2)

(no is the refractive index of the scattering medium) and is in the direction of the bisector of the angle of scattering, 8. It is usual to orient the plane of polarization of the laser beam perpendicular to the scattering plane just defined. In many experiments the scattering plane is horizontal and a vertically polarized laser beam will be correctly oriented. In

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M. W. STEER ET AL.

TABLE I CW Laser Wavelengths Laser type He-Ne Ar+ Krf

He-Cd Dye lasers

Wavelengths available (nm) 632.8 528.7, 514.5, 496.5, 488.0, 476.5, etc. 752.5, 676.4, 647. I , etc. 442, 325 425-725 tunable with various dyes

other cases the laser may be mounted on its side or an optical device (such as a half-wave plate) may be used to rotate the plane of polarization of the light. A wide variety of lasers providing continuous wave (CW) output beams of stable amplitude at a variety of wavelengths is available (Table I). Apart from considerations of cost and convenience, various factors may influence the selection of a laser. Shorter wavelengths will increase the scattering intensity (by l/h4, see Section III,C) and could take advantage of greater sensitivity of photomultipliers to light of shorter wavelength. However, the absorption spectrum of biological specimens should be considered also-the concentration of power in highly focused beams can cause unwelcome perturbation of the samples. We have observed that even a highly attenuated beam from a He-Ne laser when focused upon the cytoplasm of Amoeba proteus causes the organism to move away. In certain circumstances the variable wavelengths available with dye lasers could be useful; such lasers have successfully been applied to laser light scattering work (Jones and Johnson, 1976). C. CONVENTIONAL LIGHT SCATTERING

Light scattering only occurs when optical heterogeneities are present in a medium; a beam of light will pass undisturbed through a medium which is absolutely homogeneous. For pure fluids or molecular solutions thermal fluctuations in density provide sufficiently large heterogeneities so that scattering always occurs. In the present context, variations in refractive index (or, more exactly, dielectric constant) occur due to the presence of particulate matter in the scattering medium. The variation in the intensity of light scattered at different angles has long been used to characterize scattering particles. While this is not of concern here, it is useful to emphasize some relevant aspects of conventional light scattering. Consider a beam of light of intensity I,, polarized perpendicular to the scattering plane, incident upon a dilute solution of a single species of small scattering particles (of volume V and of refractive index n ) immersed in a solvent of refractive index no. The scattered intensity at a distance d from the sample is

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

11

where A is the wavelength of light and there are N particles in the scattering volume. The scattered intensity for small particles (
12

M. W. STEER ET AL.

- a

---------:

0 2

2

I b

l

,

,

4

3

5

7

6

CT

r

T

,

,

,

,

,

,

,

l

-

0-

+/-b

L

\I

-8 -10

'

Fig. 3. The intensity of light scattered by a sphere as a function of the size parameter a. (= 2 ~ r n ~ r l hNote ) . the logarithmic scale of intensity. (a) Intensities scattered at various angles: 180" (solid line), 130' (long dash), 90" (medium dash) and 50" (short dash) for light polarized perpendicular to the scattering plane; (b) intensities scattered at 90" for light polarized perpendicular (solid line) and parallel (dash) to the scattering plane.

D . DYNAMIC LIGHT SCATTERING

The previous section was concerned with scattering from static particles; we now turn to consider moving particles. In this situation the above considerations concerning the scattered intensity still apply. The motions affect the spectrum of the scattered light, which is broadened or shifted in frequency depending upon the motions involved.

13

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

The basic relation governing the frequency change (Am) of light scattered by a particle moving with velocity v (a vector quantity having direction as well as magnitude) is the Doppler shift equation: Aw

=

q-v

(4)

where q is the scattering vector defined in Eq. (1). Expanding Eq. (4), the single frequency shift produced by particles moving with a unique velocity is

Aw = 27 Af

=

(4n n

VIA)

sin(812) cos

+

(5)

where c$ is the angle between the velocity vector and the scattering vector. This explicitly shows that light scattering can only detect that component of the velocity which is parallel to q. By suitably selecting the direction of q (via the experimental geometry) it is possible to select or eliminate specific components of directed motion. If 4 is either 0" or 180", the Doppler shift A o has the same magnitude, but it has opposite signs for the two cases. Experimentally it is possible to measure the sign of Aw, permitting separation of motion in opposite directions. Note also how Ao varies with the scattering angle 8, being maximum 300

/'

/

30"

1

0.20

' 3 0 ° Fig. 4. Polar graphs of the intensity (plotted as I,/a3)scattered by particles of different sizes (a= 0.6, 1.0, 2.0, 3.0, and 4.0).

M. W. STEER ET AL..

14

for back scattering (0 = 180") and zero for 0 = 0". The frequency shifts involved are low, compared to the optical frequency. Using light of A = 633 nm from a He-Ne laser and assuming flow in an aqueous medium, the maximum Doppler shift (0 = 180", 4 = 0") is 4.20 Hz for a velocity of 1 p,m sec- (compare with the laser frequency of 4.7 X lOI4 Hz). Clearly if several particles having different velocities act as scatters (e.g., involving the velocity distribution across a flow profile) the spectrum of the scattered light will contain all the appropriate Doppler frequencies. Measurement of this spectrum provides information on the probability distribution of velocities within the sample. This area of laser Doppler velocimetry has been highly developed in the engineering context. The problems of adapting these techniques to the biological milieu have been reviewed by one of us (Earnshaw, 1983a). Various biologically interesting applications are discussed below. Should a given particle not maintain a steady velocity but exhibit random fluctuations in velocity, this will be reflected in the spectrum. The specific motions involved will govern the exact form of the spectrum. Thus Brownian motion (or diffusion) leads to a broadened spectrum centered on the original laser frequency (coo), the broadening being related to the diffusion coefficient of the species of particle involved. In this case the frequency spectrum S(w) has the Lorentzian form (see Fig. 7)

r '(")

R E-

cc (co -

coo)2

+ r2

(6)

FERE Nc E POINT

Fig. 5. The optical path differences for light scattered by a particle at I j and light reaching the detector via the origin.

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

15

which is centered on the laser frequency o,,and has half width at half of the maximum height (linewidth) I?, where

r = Dq2

(7)

The diffusion coefficient I) is related to the hydrodynamic particle radius rH (i.e., the radius of the particle plus its associated water) and the viscosity q of the suspending fluid by the Stokes-Einstein relation D=-

kT 6nq

(8)

'H

( k is the Boltzmann constant and T the absolute temperature). Combining this with Eq. 7 (and using h = 633 nm), we find for aqueous solutions (q = 1 cP)

where rH is in micrometers. Thus particles of radius 0.1 pm will yield r of 1501 sec - at a scattering angle of 180", and lower values if the angle 8 is less than 180".

If diffusion is superimposed on directed motion-as in electrophoretic light scattering (e.g., Ware, 1983)-the spectrum will be centered at the Doppler shifted frequency (wo Am) and diffusively broadened More complex motions will lead to more complex spectra. Thus Berg6 et al. (1967) studied light scattered by motile spermatozoa and showed that particles moving faster than purely diffusive motions led to excess broadening of the spectrum. The form of the spectrum was then correlated with the distribution of swimming speeds (randomly oriented) of the spermatozoa. In various studies, many complicating factors have been taken into account. These include, for the case of diffusion, the effects of (1) rotation of nonspherical particles, (2) polydispersity among the scattering particles, (3) interparticle interactions, and (4) multiple scattering in very dense suspensions. Some of these effects will be returned to later. One subtle effect which may arise in applications to biological samples is worth mentioning. If the average number of particles within the scattering volume is not large, then random variations in that number will cause appreciable fluctuations in the intensity of scattered light at the detector. These number fluctuations will, at the least, disturb the results expected from other processes (Schaeffer and Berne, 1972). Thus attempts to limit the scattering volume in studies of heterogeneous biological specimens (using, for example, laser Doppler microscope systems) must be tempered by the concomitant reduction in number of scattering particles.

+

(r).

16

M. W. STEER ET AL. E. OPTICAL MIXING SPECTROSCOPY

The spectrum of light scattered by moving particles cannot be resolved by conventional spectroscopic methods. Novel techniques have been devised to analyze these narrow spectra. To appreciate the capabilities and limitations of these methods it is necessary to have some understanding of the underlying principles. This section introduces these principles at a very basic level. Two approaches are used. One involves “hand-waving’’ arguments almost exclusively, and those to whom mathematical formulas are quite repugnant may turn to Section 111,E,2, Informal Approach, and press on. Unfortunately, like most physical arguments, the ideas are most clearly expressed in mathematical terms. The following subsection thus sketches the principles as simply as seems consistent with some degree of precision, to introduce readers to more rigorous treatments which they may encounter elsewhere (Pusey and Vaughan, 1975; Earnshaw and Steer, 1983; Chen and Hallett, 1982). It is hoped that the subsequent informal approach may illuminate the arid equations.

I . Formal Approach The scattered light which falls upon the detector has an intensity I , that is proportional to its amplitude (E,, called the electric field) squared I,

0:

E, E,*

All of the particles ( N in number) lying within the scattering volume at a given instant contribute light to this scattered field. The total scattered field at time t is the sum of the individual contributions at that instant. If a particle is at position rj at time c we can describe its contribution to the scattered field as Ej(rj, t). Then the total E, at time t is

The contribution Ej(rj, t ) will have an amplitude (the scattering amplitude) which depends upon the nature of the particle and will also depend upon its position rj. This position dependence arises from the different path lengths followed by light scattered from the particle at position rj and by light scattered by an (imaginary) particle at a reference position (Fig. 5). The path difference produces a phase difference C$j:

C$j

=

(k, - ks).rj = q-r.J

(12)

If we consider identical particles (with equal scattering amplitudes A ) , Eq. (1 I ) can be expanded to show the phase terms 4:

E, ( t ) = Z A j

&‘j

e%‘

(13)

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

17

The final factor reflects the oscillation of the optical wave at the frequency of the light, wo. At this stage it is instructive to evaluate the scattered intensity at the detector, I,. Substituting Eq. (13) into Eq. (lo), we find that I, depends upon the instantaneous positions (rj) of all the particles in the scattering volume: ePi+k = A' C Z eiq.Crj - )k' I, = E, E,* = C Z Aj A, (14) j

k

j

k

Here we sum over all the particles twice (once for contributions to E, and once for Es*), causing interference terms to appear (involving rj - rJ. It is this interference between light scattered by all the particles which produces the random speckle pattern referred to in Section III,B above. If the particles move, the interference terms will change, causing the intensity I, to vary with time. The variation of the intensity at a given point in the speckle patterns reflects the motions of the particles. This time variation of intensity gives rise to a broadening (and/or a frequency shift) in the spectrum of the scattered light. If an optical spectrometer could be used, it would measure the optical spectrum, So(w). This is mathematically related (by Fourier transformation) to the autocorrelation function of the scattered electric field. This measures how quickly the field loses all similarity to its value at a given instant. In principle, if we measure either the spectrum or the correlation function, we can infer the other. The fundamental correlation function, known as the first-order (or field) autocorrelation function G(')(T), is defined by the product of the fields at time t and at a later time t T:

+

G(')(T)= (E, ( t ) E,* (t +

7))

(15)

where the angle brackets indicate averaging the product for many values of t (time averaging). The value of G(')(T)equals I, at T = 0 and then usually falls off in some way with increasing delay time until it is zero at t = ~0 when all similarity in the field has vanished. If the time characterizing this decay is Tcsec, the corresponding optical spectrum will be broadened about the laser frequency wo by about ( 1 /T=) Hz. If the correlation function were independent of delay time (when the scattered intensity would be constant) the spectrum would be a spike or 6 function at the frequency wo. Now the frequency differences involved in laser light scattering are, as already noted, too low to permit S,(w) to be measured directly. Neither can the field autocorrelation function be measured directly, as optical detectors respond to the intensity of light falling upon them. We can, however, form the intensity (or second-order) correlation function by autocorrelating the detector output:

C ( ~ ) (= T )(Is($

+ 7))

(16) This is related to the power spectrum S,(w)-the power of the detector output as a function of frequency. We will see directly that from this second-order correlation function we can usually recover the fundamental first-order function. I,(t

18

M. W. STEER E T A L .

The field and the intensity correlation functions, G(I)(T)and G ( 2 ) ( ~have ), shapes which are determined by the motions of the scattering particles. If we increase I s by, for example, increasing the intensity of the laser beam falling on the scattering medium we do not affect this shape, although we will increase G ( 2 ) ( by ~ ) Is2. It is convenient to remove the arbitrariness in the scale of these correlation functions by normalizing them. The normalized functions are written as g(I)(T) and g ( 2 ) ( T ) , defined by g(")(T)

= [G(")('T)] / [Gcl)(0)]"

(17)

where n = 1 or 2 as appropriate. In many cases, g(*)(T) can be interpreted to yield g(l)(~).Provided that many scatterers are present in the scattering volume, the so-called Siegert relation holds, g'2'(T)

= 1 f 1g'1)(T)12

(18)

This permits the optical spectrum to be recovered from postdetection signal processing. Notice that in forming the intensity correlation function we lose all knowledge of the optical frequency wo. Equation (14) shows that the scattered intensity does not depend upon wo. Consequently, experiments involving the detection of the scattered light alone cannot detect Doppler frequency shifts. The role played by movement of the scattering particles in the correlation functions is easily demonstrated. We can substitute the expression for the scattered electric field [Eq. (13)] into the field correlation function [Eq. (15)], obtaining

Consider a dilute solution of identical particles having equal scattering amplitude A . The positions of different particles will be quite uncorrelated, so that in the double sum in Eq. (19) those terms involving one particle at time t and another at time f T will average to zero. Finally, provided the solution does not change with time we can set t = 0 to simplify the equations. In these circumstances the field correlation function becomes

+

G(~)(T =)N

A2 (eiq.[r(T) -r(0)1)

(20)

showing that the correlation functions are determined by the motion of individual particles. For the case where the scattering particles are diffusing, Eq. (20) can be shown to lead to

G(~)(T =)NA2 e-DY2T

(21)

which is the Fourier transform of the optical spectrum obtained for this case [Eq. ( 6 ) ] ,The normalized field correlation function, g(I)(T), is e-D92T. As already

19

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

noted, we actually measure the intensity autocorrelation function; using the Siegert relation [Eq. (lS)], we see that g(2)(7) = 1

+ e-2DdT

(22)

In a real experiment, various experimental factors (geometrical and coherence) cause the exponential term to be multiplied by a factor (y) less than unity. In some experiments it is necessary to measure a Doppler frequency shift-to measure flow rates, for example. Experiments involving detecting the scattered light alone cannot measure a frequency shift. If the detector output is to contain beats at the Doppler frequency A o , then the detector must receive some light at the original laser frequency wo as well as the scattered (and Doppler shifted) light at frequency oo+ A o . This mixing can be arranged by directing a portion of the original laser beam to fall upon the detector without having been scattered by moving particles. Various such heterodyne arrangements have been devised (e.g., Fig. 6 ) . In some biological cases light will be scattered by static parts of the specimen; such unshifted light may provide a suitable reference beam. In this heterodyne case the detected intensity is f ( t ) = [E,

+ E,(t)lz = I , + 2E,Es(t) + l , ( t )

(23)

where I , is the constant intensity of the reference light at the detector and I , is the fluctuating intensity due to scattering from moving particles. The autocorrelation function of this intensity has various terms, including a constant term (due to I R 2 ) , the usual self-beat term (due to Is2), and a mixing term [due to ERE&) E,E,(t + 7)).This latter cross-beat term reflects A o directly. The full expression for the intensity correlation function is g(2)(T) = 1

+ (Zs/I)2 1 g,'*)(~) -112 + 2(1,/1) g,(')(T) cos AWT

(24)

1'

n ND FILTER FILTER

SPECTRUM ANALYZER P ( W )

PHOTON CORRELATOR G ( f )

Fig. 6 . A schematic heterodyne experimental system. A laser beam is focused upon the scattering volume; light scattered by moving particles is detected in any direction other than that of the incident beam. A portion of the original beam is mixed with the scattered light at the detector (PM). Beam splitters (BS) divide and recombine beams and neutral density (ND) filters attenuate the reference beam.

M.W.STEER ET AL.

20

where the average values of the intensities 1, and 1 are used and where g,*(')(7)is the correlation function of the scattered field alone. Such experiments permit Aw to be measured-the Doppler shift is just the period of the cosine term in Eq. (24). 2 . Informal Approach A rather less formal approach to laser light scattering which may be helpful is based on the approach of Forrester (1961), as adapted by Cummins (1974). These qualitative ideas are illustrated in Fig. 7. For generality we will consider particles undergoing uniform translation superimposed on free diffusion. The scattered light will be Doppler shifted in frequency and will also be spread in frequency about this shift, due to the diffusion. We may wish to measure the Doppler shift, the diffusive broadening, or both. The optical spectrum of the scattered light &(o)Jwill be, in the present case, a Lorentzian centered on the Doppler shift frequency (see Fig. 7A). If the scattered light alone is detected (using, for example, an experimental arrangement as in Fig. 2), the various frequency components of the spectrum can

wo

w0+40

wo

+

50

wo

+

60

FREQUENCY ( k H z ) ,

(kHz1

TIME (msec)

Fig. 7. Light beating spectroscopy: (A) A Lorentzian optical spectrum centered at Ao = 5 x 104 sec-' and having linewidth = I x lo3 sec-' (note wo on frequency scale); (B) the Lorentzian self-beat power spectrum (note the linewidth is 2r sec- I ) ; (C) the exponential correlation function derived from the spectrum of B (mean decay time is 1/(2r)sec); (D)a heterodyne spectrum derived from A by mixing a reference beam of frequency wo with the scattered light; (E)the correlation function corresponding to the spectrum of D.

LASER LIGHT SCATT'ERING IN BIOLOGICAL RESEARCH

21

be regarded as mixing together at the detector, giving rise to such beat frequencies as w in Fig. 7A. No beats occur at the frequency Aw. The frequency spectrum of the detector output power [the power spectrum, Sp(w)] is sketched in Fig. 7B. It retains the Lorentzian form of the optical spectrum but is centered at zero frequency, and it is doubled in width to 2r sec-I. The power spectrum is related to the intensity correlation function [ s ( ~ ) ( T ) of ] the scattered light [Eq. (16)]. This correlation function, in the present case, is an exponential function of mean lifetime 1/(2r) as as in Fig. 7C. It is clear from Fig. 7B and 7C that detection of the scattered light alonecalled self-beating or intensity fluctuation spectroscopy-cannot measure the Doppler shift Aw. Also, the intensity correlation function is related to the square of the field correlation function [via the Siegert relation, Eq. (18)l. The field correlation function would be an exponential mean lifetime l/r sec-I [cf. Eq. (21)l. To measure the Doppler shift, Am, we add to the scattered light falling upon the detector part of the original laser beam at the laser frequency wo (the reference beam). In this heterodyne case, as well as the self-beating of the scattered light, beats occur between the reference beam and the scattered light. The power spectrum of the detector output will resemble Fig. 7D. We still have the self-beat peak, centered at zero frequency and of width 2 r , but a new feature, the Doppler peak, appears. This reproduces the optical spectrum, being centered at Aw and having width r. The correlation function corresponding to the power spectrum of Fig. 7D is sketched in Fig. 7E. It comprises as exponentially damped cosine wave (deriving from the Doppler peak) set upon an underlying exponential background (corresponding to the self-beat peak). The wider a spectral peak, the faster the decay of the correlation function. The damped cosine wave thus decays more slowly (1 than the underlying exponential [ 1 i(2r)l. A significant difference in experimental practice between spectrum analysismeasurement of S,(w)-and photon correlation-measurement of G ( 2 ) ( ~ ) must be pointed out. In heterodyne systems, if the spectrum is measured the Doppler peak is clearly separated from the self-beat peak (provided the Doppler shift is sufficiently large). However, if the correlation function is measured the oscillatory function which is related to the Doppler peak is directly superimposed upon the decay corresponding to the self-beat peak. If both portions are significant (as in Fig. 7E), the data analysis becomes quite complex. These complications can be avoided by ensuring either that the intensity of the reference light I , is negligible (when the self-beat term is dominant) or that I , is very much greater than the scattered intensity I , (when the self-beat term is negligible). Oliver (1974) has shown that I , should be about 30 times as strong as I , to ensure satisfactory heterodyne operation. Figure 8 shows two measured heterodyne correlation functions. In one case only diffusive motion was present and the correlation function simply decays exponentially. The other case corresponded to diffusive motion plus overall

/r)

M. W . STEER ET AL.

22

B

A b

\+ \

1 +t

1

0.60

1

1

1.20

,

1.80 2.10 TIME ( t ) (msec)

Fig. 8. Experimentally observed correlation functions: (A) corresponding to diffusive motion alone; (B) corresponding to diffusion superposed on an overall flow.

translation; the damped cosine wave lies upon a flat background as the optimum conditions just described were used.

3. Photon Correlation The photomultiplier output is a signal containing various frequencies related to the motions of particles in the sample. These frequencies can be described in terms of spectra or correlation functions, to the measurement of which we can now turn. The most efficient instruments involve photon correlation, but other systems may be useful. In the frequency domain, the simplest scheme would be to send the photomultiplier output through a narrow band-pass filter which only passes signals of frequencies within a range w to o + So.By scanning the filter frequency w over the range of interest, the power spectrum S,(o) of the detector output can be measured. The frequency range covered by such spectrum analyzers can be varied by changing the step size Sw, as usually a fixed number of frequency steps are provided. Typically a range up to 1 MHz may be covered. Such scanning spectrum analyzers have been widely used. Apart from the inefficiency of the process (only one frequency is sampled at a time), there is the possibility that the spectrum may change over the time during which the frequency is being scanned. This may be a more significant problem for biological specimens. Various more rapid processors for spectrum analysis are available (Oliver, 198 la). The method of estimating the intensity or photon-counting correlation function G ( 2 ) ( is ~ )illustrated in Fig. 9. The data shown in this figure have been simulated using the scheme described by Hughes et al. (1973). A light beam of constant average intensity is incident upon a detector. The instantaneous intensity is a

C

T

c

I

-

10

n ( t )n 3 3 3 6 6 610 5 3 6 2 1 9 2 9 1 15 3 6 3 610 3 2 9 215 2 9 3 6 5 3 6 6 2 151 9 4 3 5 3 3 6 2 1 3 1 3 3 6 1

I

1

2 2 3 1 1

1 5 2 2101 3 L 5 2 i 6 z m z 3 4 3 1 1 0 1 6 2 6 4

2 6 2

5 1 9 1 5~. 3

3 1

2

1

2 3 6

5 2

2

3

70

n

t

2

5

2

1

2 I .

3

2

1

1

1

1

2

10

2

3

2

6

1 1

I

2 1

2 7

. 1

1

2

2 I

3 I

n

60

1

2

3 2 3

1 2

2

~

3215

1 3 6 3 1 9

1 3 3

3

1

2 1 3 2 3 1 3 6

3 2 3 4 6 1 6 1 6 4 2 3 2 6 1 6 1

6 2 2 3 1 2 8 4 6

n

I 3 3

2 1 6 1 2 3 1 6

2 3

n n

40TIME (t)50

(t+ T ) 2 1 2 2 1 L 2 1 4 2 2 2 1 1 2 6

2 15 1 3 610 I 4 5 1

n

30

20

2

4

1

1 IS 2 5 3 5 1 1 3 1 3 1 1

1

4

LHI

2102 4 5 2 2 2101

I 2 3 1

1

6

2 3

1

-!

Fig. 9. The intensity of scattered light (I,)as a function of time (tin units of sample time) gives the instantaneous probability of detecting a photon. The number of photon detections within a sample time at time r is n ( t ) . At each time f , the individual products n(f)n(t+ T) are shown, the delay time T running vertically for each time t. These products are summed horizontally (over 50 sample times) to give the correlation function g ( 2 )(7)shown as X. Also plotted and 10,OM) samples (0). are correlation functions accumulated over loo0 samples (0)

24

M. W. STEER E T A L .

random function of time Z(t). Photodetection events occur at various times, with probability proportional to l(t). A counter counts the number of photodetections, n(t), occurring within a sample time (7') at time t. The photon correlation function G ( 2 ) ( at ~ )delay time T is estimated by forming the product n(t) n(t T ) and summing these products over a large number of sample times. Figure 9 shows such products for 50 sample times ( t ) and 29 delay times (7). The resulting correlation functions can be normalized.

+

g ( 2 ) ( T ) = [G(2)(T)]/[G(~) (0)12 = N

X n(t) n(t + [X n(t)I2

T)

Finally, Fig. 9 shows the photon correlation function summed over the 50 samples shown and normalized. For comparison, correlation functions summed over 1000 and 10,000 samples are shown. The smoothing effect of averaging over many samples is evident. When possible, experiments should involve long data collection times (>lo6 sample times) to reduce the random errors in the observed correlation functions. The scheme described is that of a full correlator; the electronic processing involved is sketched in Fig. 10. Photodetector output pulses are counted over the sample time T by a 4-bit counter (counting from 0 to 15). At the end of the sample time the contents of this counter are transferred to the first store of a shift register, the contents of the shift register stores being shifted one position along. During the next sample time, the count n(0) multiplies the contents of all the following shift register stores. The products n(t) n(t - T ) are added to the contents of the various channel memories (note that T is in increments of 7'). It is computationally convenient to remember n(t - T ) rather than to wait for n(t + T ) ; the correlation function is not affected, as it is symmetrical about t = 0. Such correlators use the signal very efficiently, computing many channels (e.g., 256) simultaneously. The time scale covered is governed by the clock period, sample times between 100 nsec and 1 sec commonly being provided. As will appear below, it is often vital to determine the asymptotic value of the observed correlation function at large T (the "background"). To help in this, it is common to be able to introduce a large delay between certain of the correlator data channels (e.g., 64 or 256 times the sample time 7') to permit measurements at large delays. Early photon correlators involved multiplying n(t) by a clipped version n,(t T ) , defined as zero if n(t - T) 5 k (the clip level) and as unity if n(t - T ) >k. This scheme is computationally very simple (involving only addition) and, provided k = ri, gives a good approximation to the photon correlation function. However, in studies of living biological specimens, we have found that fi tends to fluctuate, making the maintenance of the correct clip levels impossible. In such circumstances the observed correlation functions become very distorted. While anatysis in the frequency or the time domains has usually been taken to

25

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

SAMPLE TIME CLOCK

SET SHIFT REGISTER

COUNTER

MULTlPLI ERS

I l l l l i STORES

Fig. 10. The essential functioning of a multibit photon correlator.

yield equivalent information, it has recently been shown that in some circumstances the two techniques may be complementary. Volochine (1983) and Fujime et af. (1983) have been concerned with situations where diffusive behavior involving a spread of linewidth values (r)occurs. Both groups point out the reciprocal relationship between spectra and correlation functions; while the correlation function is sensitive to faster processes (larger r),the spectrum is more sensitive to slower processes. Thus the average values deduced for r from the two techniques differ. Investigations using both methods yield more information than either alone. F. INTERPRETATION OF DATA

The extraction of meaningful information from experimental data is not separable from the experimental procedure. Details of that procedure (sample time T , precision of measurements, etc.) will directly govern the information available from the data. We have seen that photon correlation will yield data on the field correlation function, g(')(.r), which is the Fourier transform of the optical spectrum. This derives from the dynamic processes occurmg in the sample; the task of data interpretation is to extract information describing these processes from the observed g(*)(~). To attempt this there must exist a physical model of the processes

26

M . W. STEER E T A L .

which is amenable to expression as a mathematical function. This function will usually involve one or more variables (parameters), the values of which can be varied to make the functions "fit" the data points. The word fit is used here in the sense of least-squares fitting. The parameter values are varied to seek the minimum of the sum of squared deviations

s= 2

[g(')(Tj)- f l . X ,

Tj)I2

(26)

.i

over all the delay times observed (all T~).The term in the square brackets is the deviation between a given data point--g(')(Tj)-and the functionfevaluated for the same delay time and a particular parameter value x . This procedure will almost invariably involve the problem of nonlinear least-squares fitting. Many relevant computer procedures have been published and most computing centers will have libraries of suitable routines. To take a concrete example, consider the diffusion of a single molecular species (of unique molecular weight). In this case the field correlation function is g(l)(~) z

e-rT

(27)

where r = Dq2. This equation can be fitted to the observed data by varying (Y and Provided the experiment has been properly carried out, accurate values of r may be determined. The following provisos are essential: (1) the sample times T~ must be suitably chosen; (2) the background of the unnormalized intensity correlation function must be precisely known; and (3) the data must be of suitable precision. If the sample comprises two different molecular species, the model involves four parameters:

r.

g(l)(T) =

a I e-1'17

+ aZ e-rzT

(28)

where a,and a2 reflect the intensities scattered by the two populations. As the number of parameters in the model increases in this way, the programs required to analyze the data become more complex and the precision of determination of the parameters falls. For the particular example mentioned, Provencher (1976) has published details of an analysis procedure (DISCRETE) which is completely automatic, requires no a priori information, and is freely available. When the distribution of particle sizes becomes continuous, the problem of determining the form of the distribution becomes very intractable. If P ( r ) is the probability distribution of the particle radii, this must be weighted by the intensity scattered by particles of different size to yield the distribution of diffusional linebroadening, cx(T). Then

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

27

where we should note that a(T) may have several peaks, corresponding to different populations of particles. The general problem of inferring a(r)from measurements of the correlation function has been discussed by Chu (1983). Very sophisticated mathematical techniques must be used. Again Provencher (1982) has made available a powerful program (CONTIN). Similarly, if light scattered by particles experiencing translational flow with unique velocity v is detected in a heterodyne arrangement, the correlation function reflects a single Doppler frequency: gf2)(7) = A

+ B cos(Aw.r)

(30)

This can easily be fitted to the data to yield the velocity v (or its component parallel to 9 ) . Such a situation is rather unrealistic, as all flows display spatial variation of velocity. In any real experiment, extraction of the distribution of particle velocities occurring in the sample volume is not straightforward and sophisticated mathematical analyses (e.g., Davies et a/., 1980) are necessary. However, in nearly all cases a rough estimate of the average velocity involved in very easily obtained from the period of the oscillatory correlation function (or from the frequency of the Doppler peak). The two physical models discussed are of the simplest; complications may easily occur. In the case of diffusional motion, nonspherical scatterers may undergo rotational diffusion, the scattering particles may interact (impeding free diffusion), or in dense suspensions light may be scattered by more than one particle in crossing the scattering volume. Some of these effects-such as rotational diffusion-are well understood and may be incorporated in the model. Others, such as multiple scattering, are only now being investigated rigorously, so that attempts to allow for them are not yet firmly grounded. With physical systems it is often possible, then, to specify a suitable mathematical model. This can then be fitted to the data. It is worth bearing in mind that a computer will willingly fit almost any model to any data; the human experimenter must examine the data and the alleged best fit function critically. In particular the residuals [g(')(Tj) should be examined closely to detect any trends or runs. For a perfect fit the residuals should be randomly distributed about zero. Other models may fit the data; the fit may not be unique. Oliver (198I b) has an interesting discussion of a combined experimental and interpretation procedure designed to ensure uniqueness of the fit. It is not always possible to do this rigorously, but given data of adequate precision it may be possible to choose between a few possible simple models. With biological systems (particularly in vivo) it is not so easy to devise a suitable mathematical description of the processes involved. Nor, often, is it possible to acquire experimental data of sufficient precision to ensure a unique choice of model. The data may fluctuate due to biological variability or the processes may be changing with time, demanding brief experiments (with poor statistics) to avoid averaging out significant variations. In such circumstances it

28

M. W . STEER ET AL.

may well be that the data can only be demonstrated to be consistent with a model (or, perhaps, several models). The parameter values deduced using such models can only be regarded as plausible. In designing such models of biological processes, external information will be used; the more that is available, the more the ambiguities can be reduced. The model may lead to other experiments, the results of which may assist in rejection of unsuitable models. It may be possible, by exercizing experimental ingenuity, to reduce the number of processes which contribute to the observed spectra or correlation functions. Thus, if diffusion is to be studied in the presence of directed flow, a suitable experimental geometry would set q perpendicular to v, completely removing the Doppler shift from the detected spectrum of the scattered light. If such simplification of the model is possible, it is always advisable. In particular it may be possible to study individual processes before embracing the full complexity of the entire system. Thus, in the above example, an experiment to determine the diffusion coefficient of the flowing particles would permit diffusive effects to be allowed for exactly in subsequent studies of the Doppler spectrum itself.

IV. LASER DOPPLER MICROSCOPY A. INSTRUMENT DESIGN

1 . Introduction The previous section has set out in some detail the potential of laser light scattering techniques for the detection of particle motion. The advantage of these techniques, especially from a biological viewpoint, is their ability to detect motions down to the molecular level while subjecting the specimen to a minimum of disturbance during the observations. Thus, the techniques are especially suited to the study of living systems; however, a disadvantage is that they are unable to cope very effectively with heterogeneous specimens. Biological specimens are, by their very nature, exceptionally heterogeneous compared with the nonliving world outside. Also, this heterogeneity is present on a submicroscopic scale, so that gross general observations contribute little to an understanding of particular cell activities. These problems force us to make use of laser beams focused to a narrow waist (
LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

29

focusing the incident laser beam into the specimen, and collecting the scattered laser beam from both static and moving parts of the specimen. Various arrangements based on this single-beam principle, and on a more complex double-beam model, have been designed and built (Cochrane and Earnshaw, 1978b; reviewed by Johnson and Ross, 1983). Here we will concentrate on only two, both of which appear to be useful to biologists. Other possible solutions to the design problems will be discussed later. The two designs described in detail here are our “basic” design and one designed by Johnson (1982, 1983; Johnson and Ross, 1983) which is more complex.

2. Basic Laser Doppler Microscope Our present laser Doppler microscope has evolved from the design described previously (Earnshaw and Steer, 1979a,b). It is based on a Zeiss Universal microscope fitted with an epiillumination objective carrier (Fig. 11). The standard microscope phototube is mounted in place of the binoculars, so that the specimen may be viewed through the eyepiece and photographed as required. A viewing telescope is mounted in an adjustable vertical tube, so that the back focal plane of the objective can be focused onto the plane of a pinhole aperture

Kl

P. M . BF P2

--

CORREL ATOR

C’L2

A E Y EPI ECE

J

c3

POL

v 2

//

BFPl

------

U

ND LASER

I

Fig. 1 I . Optical path of laser Doppler microscope. ND, neutral density filters; h/2, half-wave plate; BFPI , back focal plane of objective lens L,;h/4, quarter-wave plate; POL, polarizer; BFP2, aperture and image plane of telescope lens Lz, viewing BFPI; P.M., photomultiplier; AMP, amplifier; DISK, discriminator.

30

M. W. STEER ET AL.

(typically 100 pm diameter). This pinhole is mounted on the front of the photomultiplier detector tube and the whole assembly bolted to a micrometer-driven x-y translation stage, so that the aperture can be accurately and reproducibly located at any part of the back focal plane. This arrangement allows detection of light scattered at an angle to the vertical axis, and so avoids direct reflections of the unscattered incident beam. A He-Ne laser is mounted on an optical bench; both ends of the laser can be moved vertically and horizontally to permit the light beam to be centered on and made parallel to the optical axis of the microscope. The light beam, which can be attenuated by neutral density filters, passes into the epiillumination system of the microscope. Both the microscope and the optical bench are isolated from building vibrations by being mounted on a heavy (270 kg) polished granite slab. The instruments are attached to the slab by double-sided adhesive tape to prevent accidental displacement. The slab is supported on three automobile inner tubes inflated just sufficiently to lift it from the bench. A mounting arrangement of this type is absolutely essential to provide protection from external vibrations. The photomultiplier is shielded from light scattered by the objective lens or other optical elements by exploiting the plane polarization of the laser beam. A polarization analyzer (Fig. 11) is placed above the epiillumination system and the plane of polarization of the laser beam (initially vertically oriented) is rotated [using the half-wave (hi2) plate] until the undesired reflections are suppressed, when the plane of polarization is perpendicular to the analyzer. A quarter-wave plate (X/4) is then placed between the objective and the specimen and rotated until the optical axis is at 45" to the plane of polarization of the incident light. In this position plane-polarized light passing down through the h/4 plate, being back scattered or reflected in the specimen, reemerges through the X / 4 plate linearly polarized in a direction at right angles to the incident beam. It is thus parallel to the analyzer and passes through to the detector aperture. The specimen can be mounted on a standard microscope slide, or in any viewing chamber designed to maintain an adequate aqueous environment for the specimen. For many studies it is adequate to use a low numerical aperture lens, preferably with as long a working distance as possible. This design only permits the detection of diffusive motions in the specimen. Detection of translation flow requires a further modification, since, with 180" back scattering (as used here), the scattering vector (9) is parallel to the laser beam and thus perpendicular to the microscope stage. To detect flow, the velocity involved must have a component parallel to the scattering vector. We have overcome this problem by supporting the microscope slide at an angle to the horizontal plane with a micrometer-driven wedge (Earnshaw and Steer, 1979b). Depending on the working distance of the objective lens and the design of the lens mounting, it is possible to support the slide at about 30" to the horizontal plane. Detection of Doppler shifts with this single beam arrangement depends on mixing light scattered from moving parts of the specimen with light scattered

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

31

from static regions (the reference beam). This does not provide a particularly efficient control of the amount of reference beam arriving at the detector. The microscope described here has been tested extensively (Section IV,B) and successfully used to examine a range of biological materials. Adequate count rates can be obtained from small biological specimens using beam diameters at the focused waist of 4-10 km. We suspect that larger specimens (e.g., Amoeba cells) lead to diffuse scattering of the incident beam so that scattered light is detected from a larger volume of cytoplasm than expected. This problem does not seem to arise in the examination of very thin specimens (e.g., mammalian tissue culture cells growing on glass coverslips).

3. Johnson's Laser Doppler Microscope A laser Doppler microscope has been built in Aberdeen by Johnson (Johnson, 1982, 1983; Johnson and Ross, 1983). This microscope incorporates a number of features which provide excellent control over the placement of the detector aperture in the back focal plane, and allows accurate deflection of the incident beam so that translational flow can be detected in the specimen plane (Fig. 12).

PHOTOMULTIPLIER

to view diffraction

aperture in plane d diffraction pattern, to select angle of scatter Bertrand lens

altenuator \

canalvze r

.

~

,u~;-oit,pr

X45 water I rnmer s Ion

lens

-IIT r

1

law concre le base on ai r bags

GE---II

1

-8

Fig 12. Laser Doppler microscope designed and built by Johnson. (Reproduced from Johnson, 1982 )

32

M. W. STEER ETAL. I I

pm tube

I

, aperture to select angle of I

I scatter

A lens

'

cover slip

flow I

Fig 13. Optical arrangement for detection of translational flow in Johnson's microscope The half-silvered mirror is tilted so that the incident beam is off axis The scattered beam that retraces the incident beam path is selected by the aperture in front of the photomultiplier (pm) tube. (Reproduced from Johnson, 1983.)

A Bertrand lens is used to form an image of the back focal plane of the objective lens. A viewing prism can be inserted to view this image and so allow placement of one of a number of apertures, held on a revolving disk, in any position in this image plane, so defining the scattered light detected by the photomultiplier. In practice a diffraction pattern, generated from a replica of a diffraction grating placed in the specimen plane, is used to define the angle of scattered light arising at the photomultiplier, by placing an aperture over a given diffraction order spot. This microscope will record diffusive movements in the specimen. Translational flows are detected by offsetting the incident beam entering the back of the objective lens (Fig. 13) by tilting the half-silvered mirror. The main beam diffraction spot is observed in the back focal plane and moved to the position of one of the higher order spots previously selected with an aperture. This exactly defines the angle at which the beam enters the specimen. Again it should be emphasized that this single beam instrument will only detect a Doppler shift in frequency if the scattered light is mixed with reference beam light from a static part of the specimen. This instrument has been successfully calibrated against standard systems for diffusion and translational motion and used for biological investigations of sieve tubes (Johnson, 1983; Johnson and Ross, 1983). 4 . Future Designs

It is apparent from the above descriptions that commercial light microscopes are far from suitable starting points for the construction of a laser Doppler micro-

LASER LIGHT SCATTERlNG IN BIOLOGICAL RESEARCH

33

scope. They require extensive modification, but yet, while giving excellent viewing facilities for observing the specimen, they still fail to provide the necessary flexibility for the laser beam systems. Various approaches have been tried to improve these systems, for example bringing the incident laser beam up through the condenser lens (Herbert and Acton, 1979), with little real improvement. It is clear from Section 111 that there are many occasions when it is advantageous to examine a specimen over a wide range of scattering angles. Clearly new laser Doppler microscope designs should attempt to return to the basic scattering system shown in Fig. 2. One solution to this would be to abandon the use of commercial light microscope bodies and mount all lenses, etc., on optical benches, so that the specimen can be observed as required while allowing adequate access of the incident and scattered laser beams. Some instruments of this type have already been built and tested (e.g., Mishina et a / . , 1975). B . STANDARD TEST SYSTEMS

Before embarking upon the examination of biological specimens, it is advisable to study a few well-defined physical systems in which only a limited number of specific motions are occurring. Typical test systems are described below, some of the results presented being derived from the testing of our laser Doppler microscope. These tests were designed, as far as possible, to simulate the types of experiment which might be envisaged for biological work. For example, experiments were deliberately kept short; the statistical errors on the resulting data are indicative of the problems which may arise in biological studies. Initial tests of our microscope system were carried out on aqueous suspensions of polystyrene latex microspheres. Such microspheres are supplied by the manufacturer as allegedly highly monodisperse populations (but see below). Various sizes of spheres were studied in separate experiments. Self-beat correlation functions were measured by placing the detector pinhole at a point in the back focal plane which was illuminated only by light scattered by diffusing particles. The observed correlation functions were exponential in character and were analyzed using the program DISCRETE (Provencher, 1976). The program uniformly fitted the data with a best solution comprising a single exponential component (up to three components were permitted). The values deduced for r for spheres of various sizes are shown in Fig. 14, together with the values predicted for aqueous suspensions (and A = 633 nm). Given the brief experimental duration (typically 100 sec), the errors shown (the statistical fitting errors found by DISCRETE) are quite acceptable. In experiments when the angle of scattering was varied by scanning the detector pinhole across the back focal plane, the observed values of r did not, within the errors, vary. This accords with the slight variation of sin2(8/2) over the range (180 t 15)". The only situation in which heterodyning occurred in these scans

M.W.STEER E T A L .

34

1o

-~ 3

-

2

0

In

Q)

L

1

0

0.2

0.4

0.6

DIAMETER

0.8

1.0

Ip m

Fig. 14. Values of 'I determined for polystyrene microspheres of various sizes. The expected values are shown as a continuous line. The error on r for spheres of diameter 0.176 pm was smaller than the plotted point.

was very close to 8 = 180°, when specular reflection of the laser beam by the microscope slide provided a reference beam. In a second series of tests using polystyrene microspheres, mixtures of two different sizes of spheres were prepared. From dilutions of the stock suspensions of spheres of the two different sizes, mixtures were prepared with volumetric ratios of 1 : 1 and 1 : 5 . No attempt was made to ascertain the absolute concentrations involved. These two mixtures were then examined with the laser Doppler microscope to estimate the relative contributions to the correlation functions of the two components [a,and a2 of Eq. (28)]. The correlation functions (e.g., Fig. 15) were again analyzed using DISCRETE. Two exponential components gave the best fit, as expected, and the r values found by the program were compatible with those expected for the sphere size used. In some cases (where a component had small relative amplitude) the errors on r were large. For mixed solutions involving widely differing r values the correlator sample time was selected so that the slower exponential decay was well determined; otherwise the determination of the r of the faster component would be inaccurate (cf. Chu et al., 1982). The coefficients a Iand a2will be determined by the relative numbers of particles of each type present in the scattering volume and by the intensity of

35

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

0.51

I

I

I

I

I

1

I

I

1

I

TIME (msec) Fig. 15. A typical correlation function observed for a suspension of microspheres of diameters 0.091 pm and 0.797 pm, the volumetric contributions being in the ratio 1 : 1. The function is of multiexponential form.

light scattered by the two types of particle. As discussed in Section IILC, a relatively small number of large spheres can dominate the detected intensity. The individual values of a,and 0 1 ~yielded the ratio R = 0 1 , / a 2 .The changes in the ratio on passing from the 1 : 1 mixture to the 1 : 5 mixtures were found (Table 11) for three different mixtures. The ratio should change by a factor of 0.2. Given the brevity of the experiments in these tests, the results are compatible with expectation. These tests suggest that particle diffusion can be reliably monitored using our laser Doppler microscope, and further that the relative amplitudes of different components of the correlation functions can be connected with relative particle numbers (weighted by the appropriate scattered intensities). It is known that the standard polystyrene microspheres are not as homogeneous in size as is claimed by the manufacturer (e.g., Chu er al., 1979). While TABLE I1 Relative Concentrations of Particle Populations Particle sizes (pm)

Volumetric ratio

fll

0.091

+ 0.176

I:1 I :5

0.239 0.283

k k

0.077 0.047

0.627 0.262

0.091

+ 0.312

1:I

0.139 0.248

& ?

0.055 0.010

0.493 0.253

0.4116 0.488

k

0.0050 0.2540

1:5

0.091

+ 0.797

I:I

1 :5

R = a,/u

fl2

* 0.010

0.0442

R(l:l)/R(l:5)

5

0.080

0.380 1.08

0.013

o,353

2

* 0.014

0.009

0.28 k 0.1 I 0.981 2 0.066

o,29

5

0.047

1.620 0.036 11.05 2 1.7

* 0.047

* 0.0069

* 0.26 *

~

o.087

o,12

o,147 rt o.023

36

M. W . STEER ET AL

the breadth of the distribution of particle sizes is small enough that DISCRETE will fit a single exponential to correlation functions observed for particles of one nominal size, more sophisticated analyses can extract their size distribution. Various approaches exist and the reader is referred to Chu (1983) for a review. Here we show the results of one such approach (Fig. 16) for particles of nominal size 0.091 pm (Earnshaw and Lavery, 1982). The particles are smaller than the nominal size (d = 0.083 pm) and the standard deviation of the size distribution (u = 10.1 nm) is much greater than suggested by the manufacturer. Data for such complex analyses must be very precise and this work was not undertaken with the laser Doppler microscope. Measurement of flow velocities requires that the velocity vector should have a component parallel to the scattering vector (9) and that a heterodyne arrangement be used. As noted above, a heterodyne signal is only achieved with our laser Doppler microscope for scattering angles very close to 180". In this case q is parallel to the incident laser beam (i.e., vertically downward) and we have used a

I1

-

10

-

9-

/\\ I \

Fig. 16. The distribution of particle radii of microspheres of nominal diameter 91 nm. The solid line derives from light scattering data, the dashed line being a smooth curve through the distribution determined by electron microscopy. (Adapted from Earnshaw and Lavery, 1982.)

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

37

sloping microscope stage to perceive velocity components within the specimen and parallel to the slide. This arrangement is somewhat inconvenient and Johnson's (1982) system of varying the angle of incidence of the laser beam so that q has a component parallel to velocities in the plane of the specimen has advantages. The ability to measure flow has been demonstrated for both systems; Johnson showed that the velocity derived from light scattering compared well with results from other methods. V. BIOLOGICAL APPLICATIONS A . PARTICLES

1 . Particle Characterization a . In Vitro Studies. Light scattering techniques have achieved considerable success in the estimation of the size and shape of particles from their translational and rotational diffusion coefficients. The techniques are applicable to a very wide range of particle sizes from small proteins to large viruses and bacteria (see Bloomfield, 1981, for a recent review). As this method for determining physical parameters is by far the most rapid, convenient, and accurate available, it has been used routinely for very many in vitro studies. These will not be reviewed here, apart from reiterating some of the points made in earlier sections. Purpose-built commercial instruments are available for particle characterization by light scattering, usually incorporating microprocessors with appropriate software. The main criterion in specimen preparation is that the particle suspensions should be as pure (homogeneous) and dust free as possible. Suspensions of two or more components can, as discussed previously, be analyzed, but this considerably increases the complexity of the data interpretation and decreases the accuracy of the final answer. The calculation of size and diffusion coefficient requires an estimate of the local viscosity of the suspending medium. In some cases this may be impossible, due to the interaction of the components with the water phase and the presence of variable amounts of other small molecules. A method of determining absolute diffusion coefficients independently of viscosity values has been devised by Hwang and Cummins (1982) for rod-shaped particles. In a study of collagen monomers they measured the ratio of the translational (DT) and rotational (DR) diffusion coefficients at 90" scattering angle, and used this to obtain a length estimate of the particle. The local viscosity was then determined from the particle dimensions and diffusion coefficients. Vesicles represent a larger and more complex particle type that has been characterized by laser light scattering. An early study by Siege1 et al. (1978) described the effect of ionic environment on the surface charge of chromaffin granules. Differences in surface charge result in different rates of migration in electrophoretic fields, giving different Doppler shifts of the scattered laser beam.

38

M. W . STEER E T A L .

An extensive review of this technique has been prepared by Ware (1983). Studies on vesicles of a rather different nature have been undertaken by Yu (1983). These are vesicles that form as a result of osmotic swelling of retinal rod disc membranes when they are dispersed into suitable media. Light scattering has been used to probe various structural and physiological properties of these vesicles in a continuing study designed to further understand the in vivo activities of rod membranes. b. Viruses. Particle characterization of the in vivu biological situation inevitably involves the analysis of complex heterogeneous suspensions, but this is not necessarily the case with virus particles. Indeed purified suspensions of some virus particles are of such uniform size that they are preferable to the more commonly used latex bead suspensions for instrument calibration (Nieuwenhuysen and Clauwaert, 1978). The study of viruses has, in turn, benefited from the application of light scattering techniques (for example, see Bloomfield et al., 1982). These are especially useful for the study of labile viruses and their substructures. For example, the lengths of the “spikes” of influenza virus particles has been successfully estimated by comparing the sizes of intact particles with those of particles without spikes (Kharitonenkov et al., 1978). c. In Vivo Studies. The behavior of particles inside living cells is of considerable biological interest. Studies of living cells by laser light scattering are complicated by the heterogeneity of particle sizes present and by the lack of information on the local viscosity to which the particles are subjected (see Section 11,A). The problems of heterogeneity can be reduced by selectively studying parts of cells containing a limited number of component types. Interpretation of data from such studies is greatly assisted by a detailed structural knowledge of the size and number density of the components present. The level of data analysis will be determined by the type of information that is required. Estimation of absolute parameters is difficult; however, the problems are less formidable if one is only interested in comparing cytoplasmic behavior under different conditions and establishing the relative levels of motion present in each case (Steer, 1983). There have been several reports of the detection of diffusive motions in cells by laser light scattering methods. Piddington and Sattelle (1975) recorded an increase in motions within stimulated nerve ganglia. Johnson (1982, 1983) describes observations on particle motions within plant sieve tubes made with a laser Doppler microscope (Section IV,A,3). These particles appeared to correspond to starch grains and an analysis on this basis showed that their motions were less than expected for particles of this size suspended in a 20% sucrose solution, which is the fluid medium usually encountered in these cells. Other studies, to be described in a later section (V,C,4), have used light scattering to study cytoplasmic streaming. In doing so some of the investigators have also recorded, and commented upon, the existence of diffusive motions in their specimens; however, no attempt has been made to analyze them further. We have undertaken a light scattering study of particle diffusion in growing pollen

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

39

Fig. 17. Longitudinal section through the tip of a pollen tube of Tradescantia virginiana. Note the high concentration of vesicles (V) at the tip and the mitochondria (M)behind this region. The paucity of ribosomes (R) and abundance of granular material (which may result from microfilaments) is characteristic of the cytoplasm at the tip. X26.000.

tubes (Steer et ul., 1984). These studies, although far from complete, serve to illustrate some of the problems encountered in such work and possible approaches to their solution. In our investigations the laser Doppler microscope has been employed to locate the position of a relatively small (4 p,m diameter) laser beam within the tip region of pollen tubes (Fig. 17) and to collect the scattered light. This system has several advantages: the pollen tubes are small (7-8 p,m diameter), highly active cells which react rapidly to changes in external conditions. The pollen grains, which are readily available throughout the year, germinate to form tubes more than 100 p,m long in 10 min, which continue to grow at about 20 k m min-I.

M. W.STEER E T A L

40

This growth is accompanied by fusion of secretory vesicles with the plasma membrane at the tube tip (Fig. 17). The necessary structural background has been provided by extensive quantitative electron microscopic studies, which have also yielded some dynamic information. Of particular interest to the present work was the finding that approximately 5000 vesicles min-’ fuse with the tip (Picton and Steer, 1981). The laser Doppler microscope (Section IV,A) was set up to examine diffusion; that is, with the specimen flat on the microscope stage and the pinhole in the back focal plane set to collect back-scattered light at about 170”. A typical correlation function from this system is shown in Fig. 18. The function appears to have the exponential form typical of diffusion broadening of the spectrum. Assuming that this is the correct interpretation, we have analyzed the data with the routines DISCRETE and CONTIN (Provencher, 1976, 1982). These have detected three major components contributing to the signal. Typical sets of results are given in Table 111. Gamma (I?, labeled “lambda” by Provencher), is proportional to the diffusion coefficient of the particle, D (Section III,D), and the relative signal level, a,depends upon relative number density of the particle. The individual component values of r exhibit considerable variation (compare different runs in Table III). This variation occurs both within the same pollen tube and between different tubes. There is a more consistent relationship, however, between the r values for the different components within

10

20

1

1

L

30

40

50

TIME (rnsec)

I 60

Fig. 18. Normalized correlation function from the tip region of a pollen tube. The circles represent data points and the line is that of the best fit solution found by the CONTIN program. Diameter of scattering volume, 4 pm;detector aperture set at 170” (back scatter); experimental duration 4.0 sec.

41

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

TABLE Ill CONTIN Analysis of Correlation Functions from Pollen Tubes Component 1

Correlator run number 7 1 17 13 65 6

rl[ l-,iz

29k20 1124.5 7.024.0 3.723.0 2.1k1.2

a,h

(cP)

0.36 5.7 0.65 14 0.25 23 0.25 46 0.52 72

Component 2

r2 820280 320250 185-r-40 74k-16 47.6210 74.25k30

Component 3 rl

a2

(CP)

0.04 2.0 0.027 4.7 0.05 8.1 0.089 20 0.14 32 0.12 20

r3

15,000*2,000 1,900k250 1,200?200 548 k 70 2,080k320 1,666k300

a3

<0.01 <0.01 0.023 0.076 0.07 0.09

r

The errors quoted for values are the width of the peak at half-height. a values are the relative signal strength of each component within each run; they cannot be compared as absolute values between runs. q values are calculated on the assumption that the hydrodynamic radius (YH) of component I is I .O pm and of component 2 is 0.1 p n .

each run; this also extends to the a. values. These values have to be viewed against the background of the data analysis programs, which are unable to determine accurately the r and a. values of the faster components from the data provided. From a consideration of the size and number of the structural components present in pollen tubes, and the viscosity values recorded by others from living cells (Section II,A), it may be suggested that r, arises from mitochondria and r2 from secretory vesicles, while r3 is related to a much smaller component, possibly ribosomes. This view is further supported by the observation that the signal levels (a2) for r2are relatively higher in correlation functions recorded at the extreme tip, where vesicle numbers are greatest (Fig. 17). If the r values and particle sizes (from electron microscopy) are used to calculate local (intrinsic) viscosity values, it is found that viscosities of 2-72 CP are present (Table 111). These values lie within the broad range already believed to occur in living cells (see Section 11,A). The mitochondria experience higher effective viscosities than the vesicles; however, the ratio between their respective viscosities remains remarkably constant despite changes in their absolute values. The slight change that does occur in this ratio is related to the level of the average cytoplasmic viscosity. The difference between the two falls slightly (from a ratio of about 3 to 2.3) in going from the lowest viscosity levels to the highest. Such a consistent pattern would be expected from a system such as the pollen tube, where the relative numbers and sizes of components remain fairly constant. It can, of course, be argued that the allocation of r values to these cellular components is arbitrary. Why should r, not represent diffusion of vesicles'?Apart from

42

M. W. STEER ETAL.

the increased signal levels found for the r2component from vesicle-rich areas mentioned earlier, this would mean that the local viscosities would have to be considerably higher, 75-750 CP for secretory vesicles and 25-500 CP for ribosomes (if these are assumed to be represented by the r2component). Such high viscosity conditions were encountered by Lehman and Pollard (1965) in extruded cytoplasm from bacterial cells, but they have not been reported from living cytoplasm. Even if these proposals are accepted, one question still remains unanswered: why does the overall cytoplasmic viscosity show so much variation? The growing tube tips are believed to be supported by a microfibrillar network against the osmotic pressure of the tube contents (Picton and Steer, 1982). Extension of the tube is accomplished by a controlled weakening of this network allowing the tip to extend forwards, with new plasma membrane and cell wall material provided by vesicle fusion. Observation of growing tubes shows that this extension process is not smooth and continuous, but proceeds as a series of surges, interrupted by less active periods. It seems likely that the surges would involve the generation of low viscosity conditions, as the microfibrillar network weakened, and the intervening less active period would correspond to high viscosity conditions as the network was reestablished. This would explain the wide variation in the sets of r values recorded from pollen tubes. Initial studies have demonstrated that successive correlation functions from the same tip, each collected over a period of 4.0 sec and separated by a correlator resetting time of 5 sec, yield I?, values that exhibit smooth changes rather than random variation. Also, correlation functions from tubes growing under supraoptimal levels of Ca2+ ion concentration frequently give low r, values, consistent with the high viscosity conditions that might be expected to predominate in such tubes (Picton and Steer, 1983). Clearly further investigations are required to confirm or refute the various proposals made. In their present state they demonstrate the way in which information derived from various sources can be used to impose restrictions on the interpretation of light scattering data and lead to the formulation of further experiments to test particular aspects of the system.

2 . Particle Interaction Light scattering techniques are especially suited to the study of time-dependent changes at a molecular level. Physical changes occurring in the components of a reaction can be followed on a time scale of a few seconds without interfering with the course of the reaction. An early illustration of the time resolution possible with these techniques is provided by a study of the dependence of casein precipitation on concentration, in which polymerization reactions were followed over a total time span of less than 3 sec (Parker and Dalgleish, 1977). Quite complex reactions can be studied in this way and the observations tested against theoretical models. A number of biologically significant processes have been

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

43

followed by these techniques, and some of these will be included here to illustrate the range of systems studied and the type of results obtained. Collagen fibril formation has been studied by Gelman and Piez (1980). They used laser light scattering followed by spectrum analysis to study the monomer (cf. Section V,A, 1,a) and the intermediate steps in fibril formation. As expected, the first phase (step 1) of assembly was accompanied by a rapid fall in the diffusion coefficient (Fig. 19), however, during the next period, step 2, no changes in this parameter could be detected, although it was known that changes essential for the final aggregation were taking place. The failure to observe a decline in diffusion coefficient during step 2 was considered to be due either to the insensitivity of this parameter to further increases in length, or to the molecules undergoing flexing motions, in which case segmental diffusion effects, independent of overall length, would dominate. Light scattering studies on assembly-disassembly reactions of microtubules were initiated by Sattelle in Cambridge. Reversible temperature-dependent cycles were monitored by light scattering of preparations from bovine brain (Sattelle et al., 1977). A detailed comparison of dogfish and bovine microtubule monomer protein (tubulin) showed that the translational diffusion coefficient of the bovine material was significantly lower than that from dogfish (Palmer and Sattelle, 1981). Although this difference was explicable in terms of the presence of a significant 35-S (ring) component in the 6-S (dimer) preparation from cattle, the value for dogfish dirner was still only about a quarter of the theoretical value

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44

M. W. STEER ETAL.

for a pure dimer solution. Model fitting, assuming the presence of previously undetected ring components in the dogfish preparation, suggested that there was less than one ring present per 250 dimers. Polymerization of the respective tubulins by incubation at increasing temperature was followed by monitoring the fall in diffusion coefficient (Fig. 20). It was shown that, in agreement with previous observations, the tubulin from dogfish, a poikilothermic species, polymerized at a lower temperature than that from warm-blooded cattle. Further studies of the bovine tubulin have been directed to a more detailed examination of the aggregation process (Sattelle et al., 1982). A more complex polymerization event, the aggregation of the protein fibrinogen to form fibrin chains in the clotting of blood, has been the subject of several light scattering studies. A recent review by Cummins (1983) outlines the main conclusions of work by Wiltzius et ul. (1982). These have shown that, contrary to earlier proposals, the rod-shaped fibrinogen molecules aggregate end to end to form dimers in the earliest stages of polymerization. It is only subsequently, when side-by-side aggregation of fibrin occurs, that staggered overlaps are found within the aggregates. Dissociation-association phenomena cannot be studied readily in living cells

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LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

45

due to the heterogeneous nature of the cell contents. A notable exception to this statement is the study of actin-myosin interactions in muscle cells. This appears to be a potentially attractive system, since the isolated components can be studied and characterized in v i m before attempting to resolve their dynamic activity in vivo. Light scattering studies of the components in vitro have indeed revealed much new evidence, especially on the degree of flexibility of the macromolecules concerned and on their interactions (Carlson, 1983). The myosin molecules were shown to consist of a rather rigid tail (light meromyosin) flexibly linked to the heavy meromyosin head. Also the role of tropomyosin in reducing the flexibility of the actin filaments was confirmed. As a climax to these in vitro studies, cross-bridge cycling was studied under conditions of declining ATP availability. However, application of these methods to the in vivo situation was less satisfactory, possibly because the light scattering signals expected from the moving myosin molecules are masked by other dynamic events in the muscle, or perhaps because the models for such motions are imperfect. Nevertheless, this paper by Carlson (1983) is a mine of valuable information on theoretical and practical aspects of the study of proteins in solution by light scattering methods. There have been claims that cross-bridge cycling can be observed in living muscle; for example, Fan et al. (1983) have published data from Limulus muscle and have carried out several experiments to verify their interpretation. B. MEMBRANES

Biological membranes separate the cell from the external environment and provide the boundaries of the internal cellular compartments. They also provide a supporting matrix for the siting and structural arrangement of many enzyme systems. The structural integrity of these membranes is determined by the interactions of the lipid components with each other and with the surrounding aqueous media. Hence the physical properties of membranes are of considerable biological importance. For instance, the membrane fluidity, which has been widely studied, governs the permeability of the membrane. Laser light scattering has been used to study the physical properties of membrane systems; to date only work on model membranes has been reported. Studies of simple model systems are a necessary prelude to the application of any new technique to complex biomembranes. This field has recently been reviewed (Earnshaw, 1983b) and so only a brief summary will be given here. The thermal agitation of molecules in the fluids adjacent to the membrane produces corrugations of the membrane at the molecular level. These corrugations (called capillary waves) scatter light quite effectively, the scattered intensity rising as the membrane tension is reduced. The spectrum of the scattered light reflects the propagation and decay of the interfacial capillary waves. In general this depends upon four membrane parameters: the membrane tension and compressibility and two separate interfacial viscosities. Various other methods

46

M. W. STEER ET AL.

for assessing some of these properties exist, but all involve perturbation of the membrane or the insertion of molecular probes. Laser light scattering is essentially a nonperturbative technique. The spectrum of the scattered light may be calculated in terms of the properties of the fluid interface. Early experiments on the surfaces of pure liquids (Langevin, 1974; Byrne and Earnshaw, 1979a) concentrated on verifying the correctness of these calculations. Light at various scattering angles (i.e., scattering vector q) was analyzed. Theoretical predictions of the dependences of the frequency and linewidth of the scattered light upon q were verified. As a general comment, experimental checks of such q dependence always form a useful test of the validity of the model used in data interpretation. Subsequently various studies of fluid surfaces supporting monolayers of amphiphilic molecules have been reported (e.g., Byrne and Earnshaw, 1979b; Langevin and Griesmar, 1980; Hkd and Neuman, 1981). These studies have all been somewhat flawed in that the approximate model used in data interpretation was inadequate to permit extraction of the four relevant physical parameters of the monolayer. These difficulties can be overcome by making observations at several different scattering angles (Eamshaw, 1982) or by using a more appropriate model (Earnshaw, 1983~).In view of these reservations about the monolayer studies, they will not be discussed further. Bimolecular lipid membranes formed from glycerol monooleate (GMO) have been studied by Crilly and Earnshaw (1983a,b). Such black films are very thin, and thus scatter very little light. However, the data interpretation is much easier than for monolayers, offsetting these experimental difficulties. The observed frequency and linewidth of the scattered light directly yield the interfacial tension and viscosity of the membrane. The membrane viscosity involved here is a shear viscosity acting transverse to the plane of the membrane; it is not the viscosity measured by molecular probe techniques such as fluorescence photobleaching recovery. Bilayers formed from solutions of GMO in n-decane, which retain significant quantities of solvent, yielded tension values which were in good agreement with values determined for bulk lipid solutions and which decreased with increasing lipid concentration in a manner compatible with other data. Similarly, solventfree GMO membranes had tensions about 1 dyn cm-', in agreement with accepted values. The viscosity of these latter membranes was substantially greater than zero (in contrast to the bilayers including solvent). The incorporation of cholesterol into GMO membranes caused both the interfacial tension and the viscosity to rise (Fig. 2 1). Saturation occurred at cholesterol concentrations similar to those corresponding to saturation of other membrane properties. The light scattering methods have recently been extended (Fig. 22) to studies of the thermotropic phase transitions of bilayers (Crawford and Earnshaw , 1984). These transitions have an obvious biological significance. This area promises to be a most rewarding application of laser light scattering. The tem-

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48

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perature dependence of the surface properties will permit thermodynamic analysis of the basic molecular interactions involved. Changes in these interactions following chemical or physical modification of the membranes would permit greater insight into the molecular processes governing membrane structure and lead to important advances in our understanding of biological membranes. C. MOTILITY

1. Introduction Characterization of the swimming motions of microscopic organisms is often difficult or impossible using conventional light microscopy. This is due mainly to the narrow field of view and small depth of focus of the optical system and partly to the restricted depth of medium that can be accommodated in most instruments. It is, therefore, only possible to follow the activity of a single organism at a time, making it very difficult to obtain an overall estimate of the behavior of a motile population. Laser light scattering enables the mean swimming parameters to be derived for a population of cells that are free to express the full range of their characteristic motions. At a cellular level motility is closely linked to cell function. In many cases our knowledge of the underlying mechanisms is still very poor. This is a result of the

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

49

difficulties involved in identifying the actual structures responsible for the motive processes, and the need for accurate characterization of the motions taking place. Light scattering methods have the advantage of being able to detect the motions of particles invisible to light microscopists, and also of simultaneously recording the activities of large numbers of moving particles. The first attempts to use this technique to detect biological motility were made with bacterial cells. Their well-characterized “random walk” pattern of motion was used by Nossal et al. (197 1) to develop a theoretical basis for the interpretation of the data. They concluded that the correlation functions obtained gave a good estimate of the average behavior of the cells. Their theories have subsequently been the basis of many motility studies in bacteria and other motile cells. However, two basic assumptions were made: namely that the scattering particles were spherical and that they moved in straight lines over short distances. Both of these are inapplicable to many motile cells, but the problems can be reduced by collecting the data at small scattering angles where the contributions from cell shape and rotational motion are minimized. Subsequent workers have used a variety of experimental and theoretical approaches to improve data interpretation and the information obtainable. Parameters determined from bacterial cells include translational and rotational velocities as well as cell size and shape. Mean walk length can be determined from low-density homogeneous suspensions using a technique known as number fluctuation spectroscopy, by which the movements of individual bacteria into and out of the sample volume can be detected. A considerable amount of effort has been put into the study of the migration of chemotactic bands using light scattering, where the high density of bacteria involved makes conventional light microscopic studies difficult. Although the majority of motility studies using light scattering have concentrated on bacterial motion, they have resulted in only minimal gains in biologically important information. The value of this bacterial work lies in the development of appropriate light scattering methods and in the theoretical interpretation of the results. Detailed discussion of this work will not be undertaken, as an excellent, comprehensive review is available elsewhere (Chen and Hallett, 1982). The value of applying light scattering methods to the study of motility will be illustrated by discussion of work involving spermatozoa and unicellular algae which relies on the theoretical framework developed for bacteria. Other important areas that will be included are studies of cytoplasnlic streaming, in which conventional methods of examination are unable to characterize the propulsive motions, and also investigations of blood flow, where determination of flow profiles has clinical applications. 2 . Sperm Motilio In recent years there has been considerable interest in using laser light scattering techniques for the study of spermatozoa1 motility. Estimations of sperm motility

50

M.W . STEER ET AL.

are required routinely for animal breeding, and also assist in studies of human fertility. Conventional methods for such estimations are either very subjective, as in the case of microscopical observations, or very time consuming, as with cinematographic analysis. After Berg6 et al. (1967) demonstrated that the spectrum of laser light scattered from spermatozoa was influenced by the motility of the sample, hopes were raised that laser light scattering might provide both a rapid and an objective method for assessing sperm motility. The first studies were based on the theoretical predictions of Nossal(l97 1) for the frequency spectra and electric field autocorrelation functions that would be obtained from hypothetical swimming speed distributions. Most workers aimed to produce a swimming speed distribution for their samples, as this would provide a good assessment of the overall motility of the spermatozoa. However, their conditions of measurement and the theoretical bases of their approaches tended to vary, and as a result various groups obtained different additional parameters. For example, Cooke et al. (1976), using self-beat analysis in the time domain, were able to determine the fraction of swimming cells in a sample of bovine sperm, as well as a swimming speed distribution. Shimizu and Matsumoto (1977) obtained swimming speeds for both pig and abalone sperm with an approach that indicated that the effects of multiple scattering, rotation, and Brownian motion were negligible in their system. However, using the depolarized component of their scattered light, they obtained a distribution of flagellar rotational velocities which was in good agreement with that measured by light microscopy. Investigations of human spermatozoa have been carried out principally by members of the Saclay group in France (Dubois et al., 1975; Jouannet et al., 1977) who compared the motility parameters obtained by laser light scattering with those obtained by conventional microscopy from the same samples. Using heterodyne detection and analysis in the frequency domain, they were able to determine the characteristic velocity (peak of velocity distribution), the density of the sperm population, and the percentage of this that was motile. They concluded that the method gave an objective and precise method of motility analysis, and their equation for the distribution of spermatozoa swimming speeds has generally been used by subsequent workers. Members of the Saclay group have extended their investigations to the study of sperm in undiluted semen and in cervical mucus (Volochine and Bosq-Rolland, 1979). They have also been studying the kinetics of immobilization of antisperm antibodies, and can characterize objectively the activity of a serum at a given dilution. Their interest in sperm motility within cervical mucus has led to light scattering studies of the mucus itself. It is well known that there is a cyclical variation in the ability of sperm to penetrate cervical mucus, which is believed to be a result of changes in its molecular structure. The laser studies have detected oscillations in the mucus and have also provided support for Odeblad’s (1968)

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

51

theory that the fibrillar network of cervical mucus increases around the time of ovulation. Human sperm have also been studied by Finsy et al. ( 1979) but, in contrast to the previous group, they used self-beat detection and analysis in the time domain. They were able to determine mean sperm velocity (reproducible to 5%) and the fraction of motile sperm (reproducible to 10%). Steiner er a / . (1977) have extended the technique to a pharmacological application by demonstrating the stimulatory effects of kallikrein on human spermatozoa. Using heterodyne detection they followed the continuous stimulation of swimming speed over a 6-hr period. In all the above studies the basic assumption is made that the scattering particles are point scatters, moving in short, straight trajectories of constant velocity. Independently, both Craig et a1 (1979) and Harvey and Woolford (1980) pointed out that such assumptions are not really valid for spermatozoa, particularly in the case of bovine sperm which are far from spherical. Harvey and Woolford (1980) concluded that neither the swimming speed distribution nor the proportion of motile cells in a sample can be measured by laser light scattering, unless orientational effects have been included in the data analysis. They suggest that, at low scattering angles, the similarity of velocities inferred using the simple point scattering analysis to those from other techniques had been fortuitous. Following the extension of Nossal’s theory by Chen et al. (1977) by treating Escherichia coli as a prolate ellipsoid, Craig et al. (1979) carried out a much more rigorous analysis of the motion of bull sperm. These were assumed to be oblate ellipsoids with the center of mass moving along a helical trajectory. Velocity and rotational distributions were also included in their analysis. The calculated electric field autocorrelation functions agreed well with those obtained experimentally. Woolford and Harvey (1982) approached the problem differently and transferred their attentions to the slower part of the spectral decay (detected using longer sample times). They suggested that in the regions of fast decay, intensity fluctuations caused by the sperm heads would mask any Doppler beat present as a result of sperm motility. However, the proportion of nonmotile sperm (another important parameter in terms of artificial insemination) could be obtained from the slow region of the decay. They concluded that the relative amplitudes of the components of the autocorrelation function yield potentially valuable information about the relative number of dead cells, but that the picture is complicated by the disturbance of dead cells by swimming cells. The difficulties in the interpretation of light scattering data from spermatozoa arise not only from their complex motion but also from their shape. By choosing to study Asterias sperm, Herpigny and Boon (1979) have obtained a more homogeneous population (a very high percentage of motile cells) and also a sperm of

52

M. W . STEER E T A L .

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Fig. 23. Normalized correlation functions obtained from living Asterias spermatozoa at scattering angles of 90" (0)and 50" (0) compared to that obtained from dead cells (+) at 90" scattering. The upper and lower time scales refer to dead and living cells respectively. (Reproduced from Herpigny and Boon, 1979.)

spherical shape. Interpretation of the data (Fig. 23) suggests a large contribution from off-axis motion (i.e., rotation and oscillation) at detection angles greater than 20". At small scattering angles translational motion is more important. There is still considerable interest in refining the objective light scattering methods to yield, directly and accurately, the swimming speed distribution. Recently Wilson and Harvey (1983) have shown that signal complexities arising from spermatozoal shape can be considerably reduced by using an experimental design involving two laser beams. The beams are focused a small distance apart in the specimen so that particle transit times between them can be measured. Analysis of cross-correlation between the detected signals from the two beams indicates that the effect of spermatozoal asymmetry on the swimming speed distribution obtained is minimal. These various attempts to ascertain spermatozoal motility using laser light scattering suggest a fair degree of optimism for the technique as a whole, particularly as it is easily automated (Earnshaw et al., 1983). There is still a considerable need for careful analysis of the experimental data before the technique can be extended from one that gives a general assessment of sample motility to a method capable of accurately characterizing the various types of motion exhibited by spermatozoa. However, in many fertility and pharmacological studies such precision is not really necessary, so that in many cases the currently avail-

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

53

able light scattering techniques could be applied successfully without further refinement.

3. Algal Motility The motile cells of unicellular algae have also been the subject of light scattering studies, and they exhibit various complex motions, somewhat like spermatozoa. The first investigations in this field were carried out by Ascoli et al. (1978a,b) working with Euglena grucilis. This organism has an ellipsoid shape and a complex rototranslatory motion (Fig. 24) so that the theoretical models for point scatterers are not readily applicable. Ascoli et ul. (1978a) have attempted to simplify the problem by carefully selecting the experimental conditions and detection methods, so that the number of motility factors contributing to the measured signal at any one time is reduced. Unidirectional motion is induced in their samples by the application of an alternating electric field. When the translational velocity is oriented perpendicular to the scattering plane, rotational velocities and flagellar beat frequencies can be measured by using self-beat detection and a suitable detector position. Alternatively, with reorientation of the swimming direction to allow detection of the Doppler shifts, the swimming speed distribution can be determined using heterodyne detection at small scattering angles. These techniques have been used to investigate the effect of several different factors on Euglena motility. In cells

A

Fig. 24. (A) Schematic drawing of Euglena gracilis motion. (B) The path traced out by the anterior end of the cell on a plane perpendicular to the direction of motion. (Reproduced from Ascoli et a/., 1978a.)

54

M . W. STEER ET AL.

50

Hz Fig. 25. Spectra showing the movement of the Doppler peak to higher frequencies as a result of the increase in swimming speeds of dark-adapted Euglena after transfer to the light. The sharp peaks at 50 Hz are an artifact of the detection system. (Reproduced from Ascoli et al., 1978a.)

exposed to white light after dark adaption, an increase in mean swimming speed (Fig. 25) and in the frequencies of body rotation and flagellar beat could be detected over a 30-min period (Ascoli et nl., 1978a). Increasing the strength of the orienting electric field was also found to stimulate motility (Ascoli et a l . , 1978b). However, comparison with the motility changes induced by increasing the temperature of the sample led to the conclusion that the cells are stimulated

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

55

by the warming effect of the electric field. The effect of pH and light intensity on Euglena motility have also been studied (Ascoli and Frediani, 1980) and light scattering techniques have now been extended to investigate phototaxis in Haemococcus (Ascoli and Frediani, 1983). Another alga that has been studied by laser light scattering is Chlamydomonas. This alga exhibits a more simple motion than Euglena in that it does not include a rotational component. Theoretical models of Chlamydomonas motion and the expected patterns of light scattering have been deduced from cinematographic studies (Racey et al., 1981). Comparison of experimental autocorrelation functions with the theoretical predictions indicates that, after calibration, the light scattering system provides a rapid method of determining mean progressive swimming speed in Chlamydomonas. Motile unicellular algae have attracted less interest as a subject for light scattering studies than the more topical spermatozoa. However, as the structure and motions of unicellular algae are generally not as complex as those of sperm cells, the data obtained should be more easily interpretable. Comparison of the theoretical approaches used in the two systems (Craig el al., 1983) therefore may represent a promising way forward. 4 . Cytoplasmic Streaming The phenomenon of cytoplasmic streaming in plant cells has been intensively studied over many years, and yet our understanding of its underlying mechanisms is still extremely limited. Elucidation of the molecular processes involved requires both identification of the structural components generating the flow and characterization of the precise motions taking place. It is in this second area that laser light scattering techniques could assist considerably. Despite the many possible advantages of the method, its application to the study of cytoplasmic streaming has been somewhat limited to date. Investigations have been restricted to the fresh water algae Nitella and Chara and the slime mold Physarum. There has been a brief study of Elodea. The detection of cytoplasmic streaming by light scattering was first reported by Piddington (1 974), and since then several groups have taken an interest in the field. Mustacich and Ware (1974, 1976, 1977a) have made a considerable contribution with their work on Nitellaflexilis. They were able to detect a well-defined peak in the frequency spectrum obtained from a streaming cell (Fig. 26), from which they derived a mean streaming velocity of 72 pm sec- at 25.3"C. This peak was not present in spectra recorded from nonstreaming cells. Analysis of data collected over a range of scattering angles indicated that the majority of cytoplasmic particles, regardless of size, were travelling with approximately the same velocity. This was also concluded in work by Sattelle et al. (1979), again working with Nitellaflexilis, in which they determined a mean streaming velocity of 50-60 pm sec- I . Like Sattelle et al. (1979), Langley et al. (1976) recorded intensity autocorrelation functions and found a mean streaming velocity

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56

M. W. STEER ET AL.

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of 60 pm sec - at room temperature in Nitellu opaca. The only other algal species to have been the subject of light scattering studies is Cham corallina. In this species Sattelle and Buchan (1976) obtained a clearly defined frequency peak which corresponded to a velocity of 46.7 pm sec- at 20°C (Fig. 27). This agrees well with their own light microscopic measurements of streaming velocity. The determination of translational cytoplasmic streaming velocities, however, does not add greatly to our knowledge of the process as a whole, except possibly to confirm previous light microscopic measurements. It is the detection of the more complex motions of the streaming particles that might be of more value. However, Mustacich and Ware (1974, 1976) report that except at high scattering angles, where the motion of small scatterers is preferentially detected, their data indicate that diffusion is not contributing greatly to the velocity profiles recorded in Nitella cells. This contrasts with the conclusions of Sattelle ef ul. (1979), who claimed that they could distinguish Brownian motion and vectorial streaming as separate components in their correlation functions. Mustacich and Ware (1977a) have tried to identify the source of low frequency components in their spectra. They eliminated the translational Doppler peaks from the spectra by working at 0" scattering angle, and the remaining low frequency decay they found to be dependent on both the occurrence of streaming and the presence of chloroplasts. They suggested that at least some of their low frequency component resulted from the stationary chloroplasts periodically masking the streaming particles of the endoplasm. However, Sattelle et al.

57

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

(1 979) could detect no differences in the correlation functions obtained from normal cells compared to cells from which chloroplasts had been removed. Laser light scattering may also be used to determine the flow profile in the cells. By comparing the spectra obtained from polystyrene spheres flowing in a 20-pm channel with those obtained from Nitellu, Mustacich and Ware (1976) concluded that the flow profile in the cells lies somewhere between a parabolic profile and a plug flow. Much of the information gained from light scattering studies of algal cells, however, merely supports observations previously made using light microscopy. For example, Mustacich and Ware (1976) were able to confirm the linear dependence of streaming velocity on temperature, which has been observed microscopically on several occasions in the Characeae (e.g., Tazawa, 1968; Pickard, 1974). Their observation that temperatures in excess of 34" irreversibly damage the streaming mechanism had originally been reported by Lambers (1925). Mustacich and Ware (1976) have also used light scattering to look at photoinhibition of streaming and have assessed the effect of various chemicals on the velocity distributions detected (Mustacich and Ware, 1977a). Streaming ceased comM ATP, and with M pletely in cells within 30 min of treatment with

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58

M.W.STEER ET AL.

ATP an increase in the low-frequency component of the spectrum could be detected, although streaming itself was not inhibited. During cytochalasin B inhibition of streaming, the Doppler peak tended to move to lower frequencies, indicating a decrease in streaming velocities, but there were no other changes in the spectral shape. These studies on characean algae have proved valuable to those wishing to explore theoretical models for the generation of the motive force in streaming. The limits on streaming velocities in cells, obtained from the velocity histograms published in the papers cited above, have been used by Nothnagel and Webb (1982) to set the criteria which need to be met by an acceptable model for actidmyosin-generated cytoplasmic flow. Of the three models they considered, only the interaction of actin cables with myosin molecules attached to fibrous or membranous network could generate the observed rates of cytoplasmic streaming. The advantages offered by these giant cells have been fully exploited by Sheetz and Spudich (1983). They sliced open the cells and pinned them out, exposing a flat bed (-0.3 X 4 cm) of chloroplasts covered with cables (0.2 pm diameter) of actin filaments all polarized in the same direction. They successfully visualized movement of myosin molecules along the cables by attaching them to fluorescent beads and making multiple photographic exposures. However, it should be possible to detect and accurately quantify such movements directly and continuously using appropriate light scattering techniques, even in the absence of the marker beads. The other major subject for light scattering studies of cytoplasmic streaming has been Physarurn polycephalurn. The rapid streaming of the endoplasm in this species should be easily detectable by light scattering, although the flow is complicated by periodic changes in streaming velocity and direction. Again Mustacich and Ware (1977b) have been the primary investigators. Frequency spectra obtained at periods of maximum flow do not exhibit clearly defined peaks (Fig. 28) but indicate the presence of a wide range of streaming velocities. From such spectra Mustacich and Ware deduced a median streaming velocity of 0.65 mm sec- These authors put considerable emphasis on the fact that they could detect particles moving with velocities in excess of 3 mm sec-I. Such high velocities had not been previously reported from light microscopic observations, and they suggested that they were contributed by particles too small to be detected visually or else they represented the motions of contractile proteins or filaments. It is also possible that these high velocities are caused by rotation of the streaming particles. However, the relevant portions of their spectra are of small amplitude and may not merit such emphasis. Newton et al. (1977) measured maximum flow rates in Physarum of 0.9 mm sec and could also detect differences in the maximum progressive and recessive streaming velocities. By continuously recording correlation functions

LASER LIGHT SCATTERING IN BIOLOGICAL RESEARCH

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1000

Frequency ( H I ) Fig. 28. A typical spectrum obtained from a Physarum vein showing the range of velocities present at a time of maximum flow. Actual velocities (mm sec-1) may be calculated by multiplying the frequency in Hz by 0.00361. (Reproduced from Mustacich and Ware, 1977b.3

from the same vein, they were able to follow the “beating” phenomenon previously reported by Kamiya (1959); i.e., periodic changes in the maximum velocity over many streaming cycles. Mustacich and Ware (1977b) have also attempted to interpret their data in terms of the flow profile occurring in the plasmodia] veins. The simplest flow pattern that would be consistent with their experimental data is a hyperbolic one. This is an interesting conclusion, as it contrasts with the observations of Kamiya (1950a,b) who reported that the flow profile in Physarum was in the form of a flattened parabola. However Kamiya, for observational ease, drew his conclusions from the motions occurring at the slowest parts of the streaming cycle, while Mustacich and Ware (1977b) usually worked at maximum streaming velocities. The obvious conclusion, that the flow profile changes throughout the streaming cycle, is supported by additional observations made by Mustacich and Ware (1977b) at lower streaming velocities. The effect of increased temperature on streaming in Physarum has also been studied by Mustacich and Ware (1977b). They reported that the number of streaming direction reversals per minute increases linearly with temperature, but they could not detect any overall increase in streaming velocity. An observation made by Mustacich and Ware ( I 977b) which is possibly important in terms of the streaming mechanism is the detection of velocities in the vein transverse to the main streaming direction. These were found to oscillate in phase with the longitudinal velocities and were almost as large. Mustacich and Ware suggested that such velocities were the result of wall undulations and

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interior invaginations or possibly caused by contracting filaments. Transverse velocities in Physurum viens have also been detected by Picton (unpublished observations) using laser light scattering. However the velocities detected were much lower: up to about 90 Fm sec- with the majority of particles travelling at 30 pm sec-' or less. Such velocities were interpreted as being the result of tumbling motions of the streaming particles. The only light scattering study of streaming in higher plant cells is that of Earnshaw (1977), using Elodea. Because of the slower streaming velocities involved, the contribution to the signal by nontranslational motions of the particles becomes substantially greater. This makes interpretation of the data much more difficult. Comparison of the correlation functions from normal cells with those obtained from cells treated with glutaraldehyde, Ca2+, or Mg2+ ions, led Eamshaw to conclude that the tumbling motion of chloroplasts made a significant contribution to the detected signal. It can be seen, therefore, that little, if any, new information about cytoplasmic streaming has been obtained using light scattering techniques. However, the method should not immediately be dismissed. The various types of particle motion involved in cytoplasmic streaming can now be detected by a rapid, objective technique and identified as individual components contributing to the recorded data. It is in the interpretation of these data that the difficulties lie, and in particular, the identification of those components attributable to the more complex motions. However, it is these motions which are least easily characterized by conventional microscopy. Advancement of this field requires not only a better understanding of the physical techniques being used, but also better application of the wealth of biological knowledge already available. For example, the structure and physical characteristics of a Physurum vein have been very well established, and important developments have been made in the experimental handling of the organism (Wohlfarth-Bottermann, 1983). More valuable interpretation of the light scattering data might therefore be achieved by combining recent biological advances with light scattering techniques. Each streaming system, however, requires individual treatment and analysis. There must be a substantial investment in time and energy by the investigator, who should also have considerable understanding of the system in a biological context. This has not generally been the case to date. 5 . Blood Flow

The characterization of blood flow profiles has many clinical applications and the possibility of using a rapid, noninvasive technique has obvious attractions. The ease with which translational velocity distributions can be obtained by laser light scattering makes the method apparently ideal for such studies, and initial investigations were fairly successful. Interpretation of the light scattering data is based on theoretical principles

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developed from in vitro studies like those of Kreid and Goldstein (1971) who obtained velocity profiles for red blood cell ghosts flowing in narrow synthetic channels. In vivo studies have generally been combined with light microscopic observations (e.g., Einav et al., 1975a,b; Mishina et al., 1975; Feke and Riva, 1978), and fiber optics have also been employed (Tanaka and Benedek, 1975). Such studies, however, did not usually take into account any of the complex motions of the flowing red blood cells themselves, such as rotation and bending of individual cells (Goldsmith and Marlow, 1972) or the interactions between cells (Goldsmith and Marlow, 1979). Other factors influencing the detected signal, and the extraction of flow profiles from it, include diffraction, multiple scattering, and problems introduced by averaging over too large a portion of the flow profile (Cochrane and Earnshaw, 1978a). These various difficulties and complexities led to a general pessimism as to the potential usefulness of the technique (Born et al., 1978). Recently there has been more rigorous investigation of the various parameters involved in blood flow and the theoretical implications for the light scattering data obtained. Although such studies cannot eliminate the problems, they can help to indicate under what circumstances meaningful data may be collected. In the case of isolated veins (Cochrane et al., 1981) it has been suggested that selection of veins less than 400 pm in diameter will reduce problems of alignment, multiple scattering, and velocity gradient averaging, while the knowledge that wall curvature and the presence of fatty tissue disrupt the scattering signal should assist in the selection of suitable veins for study. The interpretation of data obtained from microvascularized tissue has also been put on a firmer theoretical basis. Bonner and Nossal(1981) have shown that if the diffuse nature of the light incident on the moving cells and the expected predominance of lowangle scattering produced are taken into account, then the power spectrum detected does reflect the behavior of the flowing red blood cells. Although in terms of the theoretical understanding of the data obtained limitations do exist, they do not necessarily detract from the potential usefulness of the method, and light scattering has now been used in several medical applications. For example, Hamilton et al. (1982) have used laser Doppler velocimetry to detect changes and defects in the microcirculation of human skin, which can become impaired, and requires accurate assessment during treatment. Nilsson et al. (1982) have constructed a practical laser Doppler flow meter suitable for clinical use. They have assessed its performance in a variety of applications including measurement of blood flow in skin, testis, and bone marrow and also in allergy testing. Although absolute quantitative measurements were impossible, they found that relative changes in blood flow were easily detected. The application of light scattering methods to the study of blood flow is, therefore, a good illustration of the different levels of expectation for the technique from the physical and biological viewpoints. Difficulties in terms of the

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detailed theoretical interpretation of the data need not prevent the method from being put to. use successfully in the study of a biological problem. VI. PROSPECTS We will now bring together and emphasize those aspects of laser light scattering that are most relevant to the biologist, and identify those areas of biology that are most likely to benefit from an application of these techniques in the future. The advantages of this technique are clear: laser light scattering provides a sensitive and nondestructive probe of particle motion. The delicacy and sensitiveness of such light beams are perhaps best illustrated by reference to the work on membranes (Section V,B), where the motions of the fragile bilayer have been accurately monitored over long periods of time. The disadvantages are equally clearly defined: the complexity of the experimental data and its untangling can be beyond the ability of the most skilled minds. Given that data interpretation is the main limitation of this technique, it is apparent that this consideration should be given priority in the design of any experiment. Even before a laser or correlator is switched on, the experimenter should have a clear idea as to the type of data that will be collected. Preferably this idea should be in the form of a mathematical model, from which appropriate correlation functions can be predicted. This approach is not, of course, uncommon among physical scientists, who are well used to drawing graphs (based on theory) first and plotting experimental observations subsequently, but may be somewhat startling to a biologist, who is used to plotting data points first and deciding where to draw the line later. The biologist is frequently unable to approach this ideal, and so it is appropriate to consider alternative strategies. These have the common aim of attempting to reduce to a minimum the number of alternative interpretations of the data. Thus, estimates of the size and number of the various types of particle in the scattering volume provide useful information on the relative signal levels to be expected from each component in the total scattered light field. Some knowledge of the types and amplitudes of the various particle motions present is of value in interpreting decay constants and periodicities in the correlation functions. Any information on biochemical reactions within the system, or on physiological responses of the system, is also of value. If these processes result in changes within the sample, either with time or due to altered conditions, then the interpretation of the correlation functions obtained should be consistent with this information. The prime consideration is to reduce to a minimum the number of particle types making major contributions to the correlation function. This can be achieved by various means. The sample can be extensively purified so that it is monodisperse. The size of the scattering volume can be reduced, as in the case of the laser Doppler microscope, so reducing the number of particle types in the

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incident beam. The appropriate experimental geometry, such as scattering angle, can be chosen so that contributions from unwanted components are suppressed relative to those of interest. This leads directly to a useful experimental check of the interpretation placed on the data. If observations are made at a number of different scattering angles, then the predicted dependence of the fitted parameters on the scattering angle should be confirmed by the experimental data. Despite these caveats, the biologist may make considerable progress by sidestepping a rigorous analysis of the data and using the technique as a probe to detect changes in the sample without a full understanding of the nature of the parameters which are being observed. This point was emphasized at a recent meeting (Earnshaw and Steer, 1983) as a viable alternative to a rigorous analysis of the data. Studies on in v i m preparations of biological macromolecules are already reaping the benefits of light scattering techniques. The recent progress that has been made in the more complex fields of macromolecular motion and interaction has proved that these techniques are very effective in this field, and we expect that there will be a considerable expansion of such work in the future. The living, or in vivo, level of biology is the most complex and poses the greatest problems in terms of heterogeneity of the sample and subsequent data analysis. Here we see the crisp edges of analysis of data from simpler systems becoming blurred, a price to pay for progress in difficult situations at the present time. The establishment of sufficient understanding of sperm motility has, for example, produced an objective method of assessment of immediate value in the medical and veterinary fields. Detection of blood flows in delicate and injured tissue justifies the scale of the assumptions made in interpreting the data. These studies may be improved upon; more rigorous analyses are being developed so that, for example, the motions of sperm or algal cells may be accurately defined for the incorporation into an integrated picture of the structures and mechanisms that impart motion to the cell. The cellular level is relatively unexplored at the present time. The various initial studies have shown that both translational and diffusional motions can be detected in living cells, but the full value of the data obtained cannot be extracted, through lack of appropriate biological information. These studies represent a significant improvement over other methods of investigating living systems, and the prospects for the successful analysis of carefully selected protoplasmic activities are quite good. Many, if not all, of the successful biological studies using laser light scattering reported in this article have been made possible by the involvement, either directly or indirectly, of physical scientists fully conversant with the technique. It seems possible that, for relatively simple applications, this need not be the case in the future. This view stems from the availability of detailed discussions of various aspects of the technique and the availability of commercial instruments and software programs. These should be sufficient to enable the biologist to

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make considerable progress before seeking help and guidance from those established in the field, although such assistance is always generously given. VII. APPENDIX-NOTATION

The notation used in this paper is defined below. Some symbols are used with two different meanings, but the sense should be clear from the context. Readers should be aware that there is no standard notation in the field of laser light scattering; a partial listing of alternatives is to be found in Earnshaw and Steer (1983). A

Scattering amplitude of particle Translational diffusion coefficient d Distance of detector from scattering volume E, Electric field of scattered light Ej(ri, t ) Scattered electric field at a time t due to particle at r, Measured field autocorrelation function Measured intensity autocorelation function Normalized field autocorrelation function Normalized intensity autocorrelation function Total detected intensity of light Intensity of laser beam incident upon scattering volume Reference beam intensity Intensity of scattered light (1) Boltzmann's contant (2) Clip level in clipping correlator Wave vector of incident beam Wave vector of scattered light Number of particles within the scattering volume Refractive index of scattering particles Refractive index of suspending medium Number of photon detections over sample time at time t Mean rate (per sample time) of photon detections Angle-dependent scattering form factor Scattering vector (b- k,) Particle radius Hydrodynamic radius of particle Position of jth scattering particle Sum of squared deviations Spectrum of frequencies w Optical spectrum of light Power spectrum of detector output (1) Absolute temperature (2) Sample time Time Velocity vector General parameter in fitting procedure (1) Particle size parameter = 2 ~ r r n ~ / h (2) Relative contribution of particle species to g(')(T)

D

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Linewidth (or decay time) associated with diffusing particles A coherence factor Frequency shift (Hz) Circular frequency shift (27rAf, sec- 1 ) Dynamic viscosity of liquid Angle of scattering Wavelength of light Delay time in correlation Decay time of correlation function ( I ) Optical phase term (2) Angle between q and v Phase difference due to scattering at r, Frequency (2mf, sec - 1) Frequency of laser light

ACKNOWLEDGMENTS We are grateful to the Science and Engineering Research Council and the Medical Research Council for financial assistance and to our research students for their contributions to various aspects of this work.

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Transport and Fixation of Inorganic Carbon by Marine Algae

N . W. KERBY* and J. A. RAVEN? * A . F. R . C . Research Group on Cyanobacteria

* fDepartment of Biological

Sciences

The University Dundee, Scotland

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inorganic Carbon System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Transport of Inorganic Carbon between the Medium and Marine Algae . Carbon Fixation in Marine Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ribulose- I ,S-Bisphosphate Carboxylase/Oxygenase (RUBISCO) ............. A. Enzyme Extraction from Marine Algae . . . . . . . . . . . . . . . . . . ............. B. Properties of RUBISCO from Marine Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. The Occurrence of RuBPo Activity, and of the PCOC, in Marine Algae .......... VII. Carbon Isotope Disc ............. VIII. P-Carboxylases . . . .......... ............. IX. C4 Metabolism in th .............. X. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . ................................................ 11. 111. IV. V.

71 72

88 88 YO 94 101 109 114 116 118

I. INTRODUCTION Carbon dioxide is the source of cell carbon during photoautotrophic growth of algae. Light energy is converted into the chemical energy of ATP’ and NADPH,, IAbbreviations used are the following: ADP, (ATP), adenosine 5’-di(tri)phosphate;CDP, (CTP), cytidine 5’-di(tri)phosphate;CDP, (GTP), guanosine 5’-di(tri)phosphate;GOT, glutamate oxaloacerate transaminasc; IDP, (ITP), inosine 5’-di(tri)phoaphate; MDH, malate dehydrogenase; NADPH, ADVANCES IN BOTANICAL RESEARCH. VOL. I I

71

Copyright 0 1985 by Academic Press, Inc (London) Lld. All righla of reproduction in any form reserved. ISBN 0-12-0059l I-X

72

N. W. KERBY A N D J . A. RAVEN

most of which is used to convert CO, into reduced carbon compounds. Our understanding of the pathway of carbon metabolism in marine algae has been aided by the use of carbon isotopes. In 1952 Steeman Nielsen introduced the “C-14 technique” for measuring primary productivity in aquatic systems. This technique is, however, not without its problems (Carpenter and Lively, 1980; Morris, 1980; Peterson, 1980). Many of the problems and uncertainties can be attributed to the fact that measurements are made in the absence of precise information on the transport and subsequent assimilation of inorganic carbon. Additionally, the use of carbon isotopes has helped elucidate the pathway of carbon assimilation in a number of marine algae, and has shown that the photosynthetic carbon reduction cycle (PCRC) is operational with ribulose- 1,5-bisphosphate carboxylase/oxygenase (RUBISCO) as the carboxylase. However, other early labeled products of photosynthesis, in particular C, acids, have given rise to the notion that certain marine algae may have a C, type of photosynthesis similar to that in specialized angiosperms. This notion has been reinforced by the finding that certain marine algae have gas exchange characteristics, when grown under air-equilibrated CO, concentrations, similar to those of C,-type plants. As will be seen later, these attributes can be explained in terms of anaplerotic P-carboxylations operating parallel to that of RUBISCO and CO, “concentrating mechanisms” which lead to a suppression of ribulose bisphosphate oxygenase (RuBPo) activity. During recent years certain advances have been made and various enzyme activities have been demonstrated in extracts of marine algae. Furthermore, certain enzymes have been purified but the number so far purified is extremely low. For these reasons the contribution to our basic understanding of the processes involved in plant carbon assimilation by marine algae is comparatively small. Marine algae have interesting characteristics relating to the transport and fixation of inorganic carbon and it is our aim to summarize some of the more recent findings. It is not the intention of this article to consider the accumulated end products of photosynthesis (for reviews see Craigie, 1963; Kremer and Kirst, 1982). Aspects of carbon metabolism have been considered in microalgae by Morris (1980) and in macroalgae by Kremer (198 lc) and Willenbrink (1982). Ion transport has been reviewed by Raven (1970, 1980, 1984) as has photorespiration by Tolbert (1974) and Raven and Beardall (1981). 11. INORGANIC CARBON SYSTEM

Table I shows some of the properties of the inorganic carbon system in seawater which are relevant to the assimilation of inorganic carbon by marine algae and reduced nicotinamide adenine dinucleotide phosphate; OAA,oxaloacetic acid; PC, pyruvate carboxylase; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PEPCTrP, phosphoenolpyruvate carboxytransphosphorylase; Py, pyruvate; RuBP, ribulose-1,5-bisphosphate;RuBPc, ribulose-1,5-bisphosphatecarboxylase; RuBPo, ribulose-1.5-bisphosphate oxygenase.

73

CARBON METABOLISM IN MARINE ALGAE

TABLE I Some Attributes of Carbon Dioxide Relevant to Their Interaction with Marine Algaea ~~

Parameter CO, in equilibrium with 35 Pa CO, in the gas phasehmol m - 3 pK,,c of carbonic acid

pKa," of carbonic acid

DHCOc/m2 sec -

~

Value in freshwaterh 22.43 (5) 15.95 (15) 11.90 (25) 9.27 (35)

~~

Value in seawaterb (35700 salinity) 18.75 (5) 13.46 (15) 10.18 (25) 8.06 (35)

(5) (15) (25) (35)

6.11 6.05 6.00 5.97

(5)

10.55 (5) 10.43 (15) 10.33 (25) 10.25 (35)

9.34 9.23 9.10 8.95

(5)

6.52 6.42 6.35 6.31

Reference Skirrow (1975)

Skirrow (1975)

(15) (25) (35)

Skirrow (1975)

(15) (25) (35)

0.953X 10-9 (0) I .94X 10-9 (25) 2.18 x 10 - 9 (30)

Kigoshi and Hashitani

0.521 X 10-9 (0) 1.09X 10-9 (25) 1.28X (30)

Kigoshi and Hashitani

0.414x10-9 (0) 0.804~ 10-9

Kigoshi and Hashitani

( 1963)

( 1963)

( 1963)

(25) 0.974X 10-9 (30)

Rate constante for hydration of CO, (CO, + H,O H,CO,)/sec - 1 Rate constante for hydration of CO, (CO, + H,O 4 H,CO,)/m, mol-1 sec-1 Rate constante for hydration of CO, (CO, + OH - -+ HCO, - )/m3 mol- I sec - I

0.037 (25)

8.5 (25)

0.037 (25)

Edsall (1969)

9.4X 1 0 - 7 (25)

Edsall (1969)

14.1 (25)

Edsall (1969)

(continued)

74

N. W. KERBY AND J. A. RAVEN

TABLE 1 (Continued)

Value in seawaterb (35%0 salinity)

Value in freshwaterb

Parameter Rate constante for dehydration of H,CO, (H,CO, -+ CO, +

14 ( 2 5 )

8 (25)

Reference Gutknecht et al. (1 977)

H,O)/sec - 1 Rate constant‘ for dehydroxylation of HCO, (HC0,-

-+

OH-)/sec-

CO,

15X 10-4 (25)

19X 10-4 (25)

Walker et al. ( 1980)

+

I ~

b

Modified from Table 5.1 of Raven (1984). Values in parentheses indicate degrees Celsius The pK. values quoted are

[‘Hco~j pKa~= -loglo [C02

+ H2C03]

d The self-diffusion coefficient of C 0 2 in the gas phase 1.04X 10W5 m2 sec- I at 0°C (Radford, 1964). The values are some four orders of magnitude greater than those in aqueous solution, where seawater and freshwater values are very similar. Alternative approaches to the aqueous-phase diffusion coefficients of HC03- and C0:(and of H2C03) may be found in Rackham (1966) and in Walker et ul. (1980). The influence of the isotopic composition of inorganic carbon on diffusion coefficients of inorganic carbon species in the aqueous phase and (for carbon dioxide) in the gas phase has been discussed by O’Leary (1981) and Farquhar et al. (1982). The reactions of carbonic acid in solution are k HC03- + H + kt-3 (2032- + H + H 2 0 + COz H2C03 k-2 k-3 OH- + COz HC03-* The ionic reactions with rate Constants k + 2 , k - 2 . k + 3 , and k - 3 are very rapid (diffusion limited). The reactions denoted by k + 1 , k - k + 4 . and k - 4 have rate constants which are shown in the table; note that the reaction with rate constant k + can have rate constants in units of sec- I or of m3molsec - I depending on the view adopted concerning the units of water activity [compare Gutknecht er al. (1977) with Walker el a / . (1980)l.

cyanobacteria. We note that the air-equilibrium CO, concentration in seawater is lower than that in freshwater; in both media the solubility increases at lower temperatures. The pK,, is some 0.4 units lower, and the pKa2is some 1.2 units lower, in seawater than in freshwater. This means that, at a given pH, the CO,:HCO,- and the HC0,- : CO,,- ratios are lower in seawater than in freshwater. The rate constants for these reactions must, of course, also be different in seawater than in freshwater inasmuch as the equilibrium constants reflect the ratio of rate constants. The ionic reactions (HC0,- + H+ H,CO,; HC0,-) are rapid, while the hydrationldehydration and CO,*- + H+ hydroxylation/dehydroxylationreactions are slow (Table I). This latter observa-

CARBON METABOLISM IN MARINE ALGAE

75

tion is significant in relation to the rates at which dissolved HC0,- can be converted to CO, within diffusive range of a submerged plant, and the rate at which atmospheric CO, can be converted to HC0,- in the apoplastic and capillary film water of emersed algae (e.g., intertidal algae at low tide). Within the cells (cytosol and/or chloroplasts) of eukaryotic marine algae there is the enzyme carbonic anhydrase which catalyzes the hydratioddehydration reactions (Graham, 1982; Raven and Glidewell, 1981; Graham and Smillie, 1976). This enzyme is important in converting HC0,-, which has been transported into the cell or the stroma, into CO,, the species which is used in the RuBPc reaction of RUBISCO, and, presumably, in converting CO, which has entered the cells (or been produced by decarboxylation reactions: Raven, 1972) into HC0,- , the substrate for a number of carboxylases found in marine algae (Section VIII). membrane system with an outer, “leaky” membrane which is freely permeable to compounds of low molecular weight, a narrow (-20 nm) intermembrane space, and a “tight” (nonleaky) inner membrane. The Dinophyta have one, and the Phaeophyta, Bacillariophyta, Prymnesophyta, Chrysophyta, and Cryptophyta have two, additional membranes round their plastids, giving a total of three in the Dinophyta and four in the other divisions. The permeability properties of these extra membranes is poorly characterized. 111. THE TRANSPORT OF INORGANIC CARBON BETWEEN THE MEDIUM AND MARINE ALGAE

The pathway which inorganic carbon takes from the medium to the site of RUBISCO activity (in the cytosol of cyanobacteria, and the chloroplast stroma of eukaryotes: Section IV) involves, for a submerged eukaryotic alga, the unstirred layer (boundary layer) at the surface of the plant, the cell wall, the plasmalemma, the cytosol, the plastid envelope membranes, and the stroma. The chloroplast envelope of the Chlorophyta and Rhodophyta consists of the “classic” twomembrane system with an outer, “leaky” membrane which is freely permeable to compounds of low molecular weight, a narrow (-20 nm) intermembrane space, and a “tight” (nonleaky) inner membrane. The Dinophyta have one, and the Phaeophyta, Bacillariophyta, Prymnesophyta, Chrysophyta, and Cryptophyta have two, additional membranes round their plastids, giving a total of three in the Dinophyta and four in the other divisions. The permeability properties of these extra membranes is poorly characterized. The cyanobacterial pathway of inorganic carbon transfer comprises, for a submerged organism, the boundary layer, the gram-negative outer membrane, the rest of the cell wall, the plasmalemma (cytoplasmic membrane), and the cytosol. The outer membrane is relatively resistant to permeation by lipid-solution processes, but has nonspecific permeability to compounds of low molecular weight conferred on it by the presence of proteinaceous pores (Nikaido and Rosenberg, 1981).

76

N. W . KERBY AND J . A. RAVEN

For marine algae which are exposed to air, the pathway is modified in that the inorganic carbon source is the carbon dioxide in air rather than the carbon dioxide-bicarbonate system of seawater. The carbon path from bulk air comprises the unstirred (boundary) layer of air at the plant surface, the capillary film of water at the plant surface, and the rest of the pathway described above for the submerged plants. The boundary layer (unstirred layer) is, operationally, the fluid layer in which inorganic carbon movement normal to the plant surface must be by diffusion rather than by mass flow. In practice, of course, there is not a sharp boundary between the well-stirred bulk medium of uniform composition and the unstirred boundary layer in which inorganic carbon movement to the plant surface is by diffusion only. Our idealized linear decrease in inorganic carbon concentration corresponds to the concentration gradient (C, - C,)/6 given by the Fick's law [Eq. (I)]: J = D (C, - C,)/6

where J

= steady-state inorganic carbon flux from the bulk phase to the outer

surface of the cell wall in mol m P 2 secdiffusion coefficient for the relevant species of inorganic carbon in m2 sec C, = inorganic carbon species concentration in the bulk phase in mol mP3 C , = inorganic carbon species concentration at the outer surface of the cell wall during steady-state photosynthesis in mol m6 = unstirred layer thickness in m

D

=

However, this algebraic artifice is useful in defining an effective unstirred layer thickness which is helpful in comparison of unstirred layer limitations on the rate of photosynthesis with other limitations in one organism, and for comparisons between organisms. Transport across the cell wall is by diffusion, albeit with the likelihood of a lower value for D in a Fick's law (Eq. 1) analysis. Transport across membranes can be by lipid solution (for CO,), or by a mediated process (for HC0,- and, questionably, CO,). The former process can be analyzed by Fick's law, with appropriate factors for the distribution coefficients for CO, between the aqueous and lipid phases (which can be incorporated into C, and Cw), and for D (the diffusion coefficient of CO, in membrane lipid), and 6 (membrane thickness). The latter process generally obeys saturation kinetics, with the flux (mol rn-, sec-I) at a given inorganic carbon concentration available to the porter being analyzable into terms for porter density (mol porter m-,), the maximum specific reaction rate of the porter [mol inorganic C moved (mol porter)- I sec- 'I, and the half-saturation constant ( K , , 2 )of the porter (mol m-3). We may accordingly combine the Briggs-Haldane/Michaelis-Menten [Eq. (2)] with the relationship

CARBON METABOLISM IN MARINE ALGAE

77

between maximum flux, maximum specific reaction rate, and areal density of porters [Eq. (3)] to give an expression for the achieved flux in terms of halfsaturation constant, the porter density, the porter maximum specific reaction rate, and the substrate concentration [Eq. (4)]. For a mediated transport reaction, the Briggs-Haldane/Michaelis-Menten relationship can be written v = ( V C ) / ( C+ K,,,) where v

=

(2)

achieved flux in mol m P 2 sec-1

C = substrate concentration at which the flux v is achieved in mol m-3

V = maximum flux which the membrane can catalyze at substrate saturation in mol m-2 sec The relationship between the specific reaction rate of a porter at substrate saturation [S/mol substrate (mol porter)- sec- '1, the porter areal density (A/mol porter m-,), and the transmembrane flux at substrate saturation (V) is given by Eq. (3):

'

V

=

SA

(3)

Substituting for the expansion of V [Eq. (3)] into Eq. (2) gives Eq. (4): v

=

(SAC)/(C

+ K,,,)

(4)

For a given value of C, the magnitude of v will be conditioned by genotypic and phenotypic influences on K,,, (e.g., phenotypic influences of the availability of cosubstrates for primary or secondary active transport), S, and A (e.g., genotypic or phenotypic influences on the quantity of porter per m2; phenotypic on/off influences on the activity of porters). The upper limit on A in Eq. (3) is set by the mass of protein which the membrane can accommodate, the fraction of this which is taken up by other proteins, and the molecular weight of the porter under consideration. The value of S is constrained inter alia, by rate/efficiency considerations which limit specific reaction rates for active transport proteins to upper limits of the order of 100-1000 sec- at 25°C (see Raven, 1980, 1984). This, with relatively high areal densities of 10 nmol porter m-2, accounts for V values for active porters of 1-10 pmol m P 2 sec-' (Raven, 1980, 1984; Raven and Smith, 1980). Transport of inorganic carbon in the cytosol and stroma is essentially diffusive [Eq. (l)] but with the added possibility that the inorganic carbon species may associate with other diffusible molecular species, e.g., with proteins via the relatively nonspecific associations of carbamino complexes or the more specific associations with carbonic anhydrase as enzyme-substrate complexes (Raven and Glidewell, 1981; Lorimer, 1983; Beardall and Raven, 1981). The role of these complexes in inorganic carbon transport is constrained by the kinetics of

78

N. W. KERBY AND J. A. RAVEN

association and dissociation of inorganic carbon species, the concentration of the protein, and the diffusion coefficient of the complex. The transport of inorganic carbon normal to the plant surface is thus dependent on both chemical reactions and diffusion. The chemical kinetics can be expressed in terms of Eq. (4), with v and A being expressed in terms of an element of cytosol or stroma volume within a small distance (of the order of nanometers) of the source side (for the “on” reaction) or the sink side (for the “off” reaction) of the aqueous diffusion path. The diffusion pathway between the “on” and “off“ reactions can be described by a Fick’s law equation [Eq. (5)] with appropriate values for d (the distance between source and sink/m) D (of the order of 10- I I m2 sec- for protein), C,,, and Ci (concentrations, in mol m-3, of protein-inorganic carbon complex at the source and sink ends of the pathway, respectively) to J (the flux/mol m-, sec- I ) :

’

J

=

(D/d) (C0 - Ci)

(5)

The use of carbonic anhydrase as a catalyst of transport of inorganic carbon in cytosol and stroma phases may be as important as the role of this protein as an enzyme catalyzing the interconversion of carbon dioxide and bicarbonate. The overall activity of carbonic anhydrase in this interconversion of the inorganic carbon species is described by a form of Eq. (4), where A is the quantity of enzyme (in moles) in the phase under consideration expressed on an external plant area basis. The same formulation may be used for the activity of RUBISCO in carbon dioxide fixation [Eq. (4)]. An important point to note (Raven, 1970) is that it is possible to formulate a “chemical permeability” with the same units as a diffusive transport “permeability coefficient” [Plm sec- I = D / S in Eq. ( l ) , and D / d in Eq. ( 5 ) ] .The rate constant on an area basis (= “chemical permeability”) is given by v/C in Eq. (4),with mol m-, sec-’/mol m-3 giving a quotient with the units in m sec-l. The same artifice may be used for mediated transport [Eq. (4)]. Such formulations are useful in comparing limitations on the overall rate of inorganic carbon assimilation; it is important, however, to distinguish between reactions which are “driven” by the mol m - 3 ( = C ) component (e.g., diffusive transport), and those which, while susceptible to analysis in terms of area-based rate constants, are driven by other energy inputs (e.g., light energy, via ATP and NADPH, in the case of the PCRC; and ATP, directly or indirectly, for active transport). Turning to the consideration of the role of transport of inorganic carbon in limiting the overall rate of inorganic carbon assimilation, it will be seen, below, and later in Sections VI and VII that a purely diffusive flux of inorganic carbon from the bulk medium to the site of RUBISCO is probably the exception rather than the rule, in that there is a substantial body of evidence for the occurrence of some active transport process(es) which maintain [CO,], and [CO,]/[O,], higher at the site of RUBISCO activity than diffusive entry of CO, could account for. This is reflected in an observed accumulation of inorganic carbon species within

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the cell to concentrations higher than can be accounted for by diffusive CO, entry from the medium (see below), and by the occurrence of a higher ratio of RuBPc to RuBPo activity in vivo than in vitro kinetics would predict if CO, entry and 0, efflux were by diffusion (Section VI). It is difficult to find a marine alga whose photosynthetic characteristics accord entirely with diffusive carbon dioxide entry, although Sargassum muticum may be such an organism at low external pH (Brown and Tregunna, 1967) even if not at high external pH where HC0,complicates matters (Thomas and Tregunna, 1968). However, the characteristics of inorganic carbon assimilation in Sargassum have not been studied in sufficient detail at low external pH values to permit analysis of the relative significance of diffusive and biochemical limitations on net photosynthesis under carbon-limiting conditions, or whether RUBISCO activity, or some other biochemical process, is limiting photosynthesis at light and carbon dioxide saturation. These two questions are best tackled together (see discussion in Raven, 1984). The dependence of net photosynthesis on carbon dioxide concentration in aquatic (including marine) plants often shows a more abrupt transition from “carbon dioxide limitation” to “carbon dioxide saturation” than the BriggsHaldane/Michaelis-Menten relationship would predict (see Lloyd et al., 1977; Gavis and Fergusson, 1975; Gavis, 1976; Wheeler, 1980; Smith and Walker, 1980; Raven, 1970). This effect does not have a unique explanation. One possible explanation is in terms of a diffusive lirnitatior, in the carbonlimiting portion of the curve, and a biochemical limitation in the carbon-saturated part of the curve (see Blackman and Smith, 191 1; Maskell, 1928; Hill and Whittingham, 1955; Raven, 1970; Smith and Walker, 1980). This concept is often extended to the estimation of apparent K,,, [Eq. (4)] and 6 [Eq. ( l ) ] values on the assumption that a diffusive limitation is responsible for reducing the initial slope (A rate of photosynthesis/A concentration of carbon dioxide) and increasing the abruptness of transition to saturation (Hill and Whittingham, 1975; Raven, 1970; Smith and Walker, 1980; cf. Chartier, 1970; Jones, 1973). However, the data could also be explained by a transition from one limiting biochemical reaction (e.g., RUBISCO) at low carbon dioxide concentrations to another (e.g., NADPH and/or ATP production by thylakoid reactions) at high carbon dioxide saturation (Caemmerer and Farquhar, 1981). In terms of RuBPc activity, limitation by carbon dioxide is replaced by limitation by RuBP, whose regeneration via the PCRC requires photoproduced NADPH and ATP (Caemmerer and Farquhar, 1981). Such an occurrence would negate the analysis of the photosynthesisicarbon dioxide concentration relationship in terms of diffusion path length and K,,, for CO, for the carboxylase. That this latter possibility must be taken seriously is suggested by two cases in which the extractable activity of RUBISCO, assayed under optimal conditions, exceeds the light- and carbon dioxide-saturated rates of in vivo carbon dioxide fixation (Codiumfragile: Cobb and Rott, 1978; Halimeda cylindracea: Akazawa and Osmond, 1976; Downton er a f . , 1976). As will be discussed later (Section V) the majority of RUBISCO

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assays from marine algae have not yielded activities which can account for the in vivo rate of light- and carbon dioxide-saturated photosynthesis. We note that the hypothesis of diffusive limitation under C0,-limiting conditions and RUBISCO limitation under C0,-saturated conditions predicts an in vivo K t 1 2for CO, in excess of the in vitro K , / , for RUBISCO (e.g., Lloyd et al., 1981, for Chondrus crispus). The alternative hypothesis (RUBISCO limitation when CO, is limiting, redox or ATP-synthesizing reactions limiting at CO, saturation) predicts an in vivo K+(co2)lower than that found for RUBISCO in v i m : such an effect is also predicted for the operation of a C0,-concentrating mechanism (e.g., Badger and Andrews, 1982, for a Synechococcus sp.). These considerations mean that the analysis of the photosynthesis/inorganic carbon concentration relationship is by no means easy, even when carbon dioxide is known to be the carbon source entering the cell, and its transport is by diffusion. If it is assumed that diffusion is the sole factor limiting photosynthesis, then an upper limit on the path length for carbon dioxide diffusion can be derived from A photosynthetic rate (mol rn-, sec- I)/A inorganic carbon concentration (mol mP3); this quotient (m sec-I) can be converted to a diffusion path length by assuming a value for D [Eqs. (1) and (4)] for the diffusion path. If we assume a value of 1.7 X l o p 9 m2 sec-l for Dcol at 15°C (see Section II), then a fixation rate of 1 pmol rn-, sec-1 from an external CO, concentration of 13 mmol m-3 (see Raven, 1981) and, ex hypothesis, zero internal CO,, yields a diffusion path length of 22 pm. The path includes boundary layer, cell wall, membranes, cytosol, and stroma. In the boundary layer D = 1.7 X l o p 9 m2 sec-I seems reasonable, since we are just dealing with seawater. The cell wall Dco2 is probably only 0.25-0.5 that in free solution; the effective membrane Dcoz (incorporating the lipid/water partition coefficient) is near 1.7 X m2 sec-I (see Gutknecht et al., 1977); while the effective Dcol in the cytosol and stroma may be the same with the restriction on CO, diffusion in the protein-rich cytosol and stroma being offset by the diffusion of protein-inorganic carbon complexes, and of HC0,- , whose interconversion with CO, is greatly enhanced by carbonic anhydrase (see Raven and Glidewell, 1981). Thus, our assumption of D = 1.7 X 10W9 m2 sec- for the whole pathway is probably not seriously in error. However, such an analysis is worrying in that a significant fraction of our 22-ym path length is occupied, in many macroalgae, by cell wall and cytosol which separate the outer surface of the cell wall from the “mean” location of RUBISCO in plastids (e.g., Borowitzka and Vesk, 1978: McCully, 1968; Colombo and Orgenigo, 1977); this leaves an uncomfortably thin boundary layer of some 10 pm, which is even further decreased if a finite Cchl (CO, concentration at the site of RUBISCO activity) is assumed. Independent estimates of this Cchlare not readily come by; the carbon isotope method suggests values of Cchl of 0.2-0.8 of that in the bulk phase, with a corresponding thinning of the boundary layer to 10 p.m (if Cchl= 0.2 of Cbulk)or a negative value (if Cch, = 0.8 of Cbulk),assuming in both cases a 7-pm internal diffusion path.

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For large algae, even under optimal stirring conditions, a 6 [Eq. (l)] of less than 10 pm is difficult to imagine. Accordingly, the diffusive supply of CO, to an algal thallus from air-equilibrated water probably cannot support a photosynthetic rate of 1 pmol m- sec- l . Unfortunately, Brown and Tregunna (1967) do not give photosynthetic data on an area basis for their Sargassum muticum which appears to be fixing CO, with diffusive entry at low pH; on the assumption that each gram fresh weight of thallus corresponds to 1000 mm2 of surface of thallus (see Seybold and Egle, 1938; Fagerburg el a l . , 1979), a net CO, fixation rate of 0.72 pmol m-, sec-' can be computed, which implies a small total length of aqueous phase diffusion path length (-30 pm). Is our assumption of diffusive CO, entry in error for Sargassum muticum at low pH? A natural situation in which CO, must be the inorganic carbon source entering the cells is that of emersed intertidal seaweeds. Raven et al. (198 1) have shown that illuminated Ascophyllum nodosum in moist air assimilates atmospheric CO, faster than uncatalyzed conversion of CO, to HC0,- could occur outside the plasmalemma even if all of the aqueous phase outside the plasmalemma were involved. However, in this case two factors militate in favor of a substantial diffusive CO, entry; one is the limitation on the thickness of the aqueous unstirred layer outside the thallus: this cannot be thicker than the capillary film. The other is the likely occurrence of a CO, accumulation mechanism in Ascophyllum nodosum which may be located in the plastid envelope; this could permit photosynthesis to proceed with very low free CO, concentrations in the cytosol (see below). The apparent absence of hard data on transport of carbon dioxide in marine algae when carbon dioxide apparently enters cells by diffusion is seen as a quantitative paradise when we turn to cases in which HC0,- transport across membranes is considered. The only unambiguous evidence for HCO, - use, in the sense of HC0,- being the inorganic carbon species which crosses the plasmalemma during net photosynthesis, comes from measurements of rates of photosynthesis in a predominantly HC0,--containing medium (i.e., one which is at least one pH unit above pKa,, and in which CO, is below air equilibrium) which exceed the rates at which the medium can supply CO, by the uncatalyzed dehydroxylation of HC0,- (Section 11). Work on freshwater algae (e.g., Walker er al., 1980; Miller and Colman, 1980a,b) has emphasized this crucial criterion to a greater extent than has that on marine algae. Nevertheless, a constellation of less critical data, some of which can be used to infer inorganic carbon assimilation faster than HC0,- can be converted to CO, in the medium, are available to suggest that the capacity to use HC0,- is quite widespread in marine algae (e.g., Raven, 1970; Kremer, 1981~).The demonstration that HC0,- is the species entering the cell implies the occurrence of a specific porter, in view of the very low PHC03- of unmodified lipid bilayers (see Raven, 1970, 1977; Raven and Glidewell, 1981; Gutknecht et al., 1977). Further, the cytoplasm-negative value of the transplasmalemma electrical potential difference means that even

,

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mediated entry could only yield a very low cytoplasmic [HCO,-] (and hence low [CO,] at pHCy,= 7.4: Raven and Smith, 1980; Smith and Raven, 1979) during net HC0,- influx. Thus active HCO,- influx is required if HC0,entry is to provide a steady-state CO, concentration which can support the observed rate of photosynthesis (see Raven, 1970, for an appraisal of the inverse relationship between rate of nonenergized HCO, - influx and steady-state internal HCO, - concentration). Active HC0,- influx could be powered either by ATP (primary active transport) or by a cotransport mechanism (secondary active transport) (see Raven et al., 1981). In view of the requirement for intracellular pH regulation which requires that approximately one OH- ion be effluxed (or one H+ ion be taken up) per HC0,- transported into the cell in photosynthesis, an “obvious” mode of transport of HC0,- into the cell would be via a 1 H + : 1 HCO,- symporter. However, Beardall and Raven (1981) point out that such a mechanism could not increase the steady-state intracellular CO, concentration to a value higher than that found in the medium, even assuming excess intracellular carbonic anhydrase activity. Since it has been shown (see Raven, 1970) that net photosynthesis by marine algae at high pH can occur at external inorganic carbon concentrations which correspond to external carbon dioxide concentrations lower than the compensation concentration measured at lower external pH values, it must be concluded that a 1 H + : 1 HC0,- symport mechanism is inadequate to support photosynthesis at high external pH values unless some supplementary CO, accumulation mechanism (e.g., at the chloroplast envelope) is functioning. Clearly a 2 H + : 1 HC0,- or an n Na+ : 1 HCO,- symporter, or a primary (ATPdriven) porter, could all be suitable as a means of actively transporting HC0,into cells. In relation to the problems which were mentioned above concerning minimum unstirred layer thicknesses and the supply of CO, in photosynthesis, HC0,transport at the plasmalemma could alleviate many of the restrictions in view of its high concentration in seawater, i.e., -200 times the free CO, concentration. This superiority in terms of the magnitude of concentration gradients which could be used to drive net fluxes between the bulk medium and the plasmalemma is only slightly offset by the rather lower value of DHCo3- than of Dco2 in free solution, and the somewhat lower permeability to anions relative to uncharged molecules of similar molecular weight in algal cell walls (see Raven, 1970, 1974, and Section 11). Thus a “high” rate of photosynthesis by macroalgae in seawater, i.e., 5 pmol rn-, sec-’ (Raven, 1981), could, with a mean DHCO3of m2 sec-I in unstirred layer and cell wall, occur with a steady-state HC0,- concentration at the plasmalemma of 1.6 mol rn-, (compared to 2 mol rn-, in the bulk solution) if the unstirred layer plus internal diffusion path length is 80 pm, i.e., a S of 73 pm if the internal path length is 7 pm. This seems a more plausible S value for a macroalga, although we may note that balanced

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growth with the observed C : N : P ratio of macroalgae may be difficult with such a boundary layer thickness in view of “natural” bulk-phase concentrations of available nitrogen and phosphorus (see Raven, 1981, 1984). Active HCO, - transport at the plasmalemma could thus alleviate problems of CO, supply through thick boundary layers and, at the same time, increase the internal [CO,] and [CO,]/[O,], thereby offsetting some of the problems encountered in maximizing RuBPc activity and minimizing RuBPo activity when even air-equilibrium CO, and 0, concentrations are considered (see Section VI). It is likely that active HCO, - influx underlies much of the C,-like physiology coupled to C, biochemistry which is found in many marine algae (see Section VI). However, it is clear that such C,-like physiology can be exhibited in situations in which HC0,- transport at the plasmalemma is very unlikely, e.g., with emersed intertidal seaweeds such as Ascophyllum nodosum (Raven et a l . , 1981). Here, if the assumed CO, concentration mechanism is to be based on HC0,- influx, it must occur at the plastid envelope; CO, which has crossed the plasmalemma can, in the presence of carbonic anhydrase, be converted to HC0,- as a substrate for this transport process. As in all cases of HC0,- transport as the basis for CO, supply to RUBISCO, carbonic anhydrase activity is needed for the HC0,- to CO, step unless very large internal steady-state HC0,- concentrations are built up to produce the required flux via the uncatalyzed reaction (Kaplan et a l . , 1980). There is, from the data discussed above, evidence that mediated HC0,influx can occur at the plasmalemma of some marine cyanobacteria and algae, and, possibly, also at the chloroplast envelope of algae. Furthermore, the phenomena discussed in Sections VI and VII below are apparently best explained by the operation of a mechanism which gives an increased steady-state [CO,], and [CO,]/[O,] ratio, at the site of RUBISCO activity relative to that which would occur with diffusive influx of CO, or mediated uniport of HC0,-. However, direct evidence for active HC0,- influx in marine algae and cyanobacteria is limited to two reports: that of Zenvirth and Kaplan (1981) for the chlorophycean flagellate Dunafielfa sulina, and that of Badger and Andrews (1982) for the cyanobacterium Synechococcus sp. In both cases an accumulation of inorganic carbon within the cells to a level higher than could be accounted for by CO, diffusion and HCO, - formation according to the “mean” intracellular pH was established by the silicone oil centrifugation technique. In Synechococcus a number of techniques were employed to determine whether CO, or HCO, was the species involved in active transport across the plasmalemma; while it was demonstrated that HCO, - influx could occur, some evidence pointed to a role for CO, entry in contributing to the intracellular inorganic carbon pool. The high intracellular inorganic carbon concentration demonstrated by Badger and Andrews (1982) may, like that shown for the freshwater cyanobacterium Anabaena variubilis (Kaplan et al., 1980), be related to the lack of appreciable carbonic -

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anhydrase activity in these organisms, with a consequent requirement for a high steady-state HCO, - concentration to provide a sufficiently high inorganic carbon flux to the CO, pool, and to maintain this pool at a concentration high enough to suppress RuBPo (see Section VI). No data are available for Synechococcus spp. to indicate whether the activity of the CO, accumulation mechanism is correlated with a more inside negative value of the transplasmalemma electrical potential difference. Such an effect in the freshwater Anabaena variabilis suggested to Kaplan er al. (1982) that primary active HCO,- influx might be occurring. The data on Dunaliella salina (Zenvirth and Kaplan, 1981) are less clear-cut, in that no direct evidence for HC0,- influx at the plasmalemma was presented, and there is the problem with eukaryotic cells that HC0,- active influx at the chloroplast may occur in addition to, or even instead of, HC0,- influx at the plasmalemma. However, the work clearly shows that an inorganic carbon accumulation mechanism is operative. This is very likely to involve active HCO,influx at some membrane (plasmalemma or plastid envelope); a passive, mediated accumulation of HCO, - in the stroma, involving a HCO, - uniporter in the plastid envelope and an actively maintained electrical potential difference, stroma positive to cytoplasm, seems very unlikely on grounds of the “sidedness” (sensu Mitchell, 1979) of membranes (see Raven, 1980, 1984). In the absence of widely applicable methods for isolating pure, functional plasmalemma and plastid (or plastid envelope) preparations from phototrophic marine algae, the use of subcellular fractionation procedures to investigate the location of HCO,- transport in these organisms is not possible at present. An energetic problem with the pumping of HC0,- (or CO,) is that of intrinsic membrane permeability to CO, (Gutknecht el al., 1977) This high lipid-solution permeability is, of course, an advantage if CO, entry is diffusive. However, active inorganic carbon influx can be severely short-circuited by CO, leakage, a phenomenon demonstrated directly in both Dunaliella salina and a Synechococcus sp. (Zenvirth and Kaplan, 1981; Badger and Andrews, 1982) as efflux of inorganic carbon from cells in pulse-chase and light-dark transient procedures. An energetic cost of the operation of the inorganic carbon accumulation mechanism was inferred by Badger and Andrews (1982) on the basis of low quantum yields of photosynthesis in cells adapted to low CO, with the accumulation mechanism induced and operative, as compared to cells grown at high CO, levels without the accumulation mechanism; disposition of this energy cost between net accumulation and countering the leak is not yet possible. Thus, for two marine 0, evolvers, there is direct evidence for inorganic carbon accumulation, complementing the larger body of evidence for freshwater cyanobacteria and chlorophycean algae (see Raven, 1984). This rather slender thread is used in Section VI to hang the observed suppression of RuBPo activity in many marine algae and cyanobacteria onto the large body of indirect evidence for active HCO, - influx in marine algae and cyanobacteria.

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IV. CARBON FIXATION IN MARINE ALGAE The photosynthetic carbon reduction cycle (PCRC) (Fig. 1) first elucidated for some freshwater unicellular members of the Chlorophyceae (Bassham et al., 1954; Bassham and Calvin, 1957), is now generally accepted to be the major pathway of carbon assimilation in marine algae. It should be noted that not all the enzymatic evidence for the cycle has been sought in algae. The PCRC is the only pathway of carbon fixation capable of regenerating substrate to act as the inorganic carbon acceptor, and consumes a major fraction of the ATP and NADPH produced in the light reactions of photosynthesis. Evidence that ribulose- 1,5bisphosphate carboxylase/oxygenase (RUBISCO) catalyzes a major portion of the inorganic carbon fixation in vivo comes from short-term ''C-labeling experiments. Fixation of inorganic carbon by RUBISCO involves a sequential labeling of 3-phosphoglycerate (3-PGA) followed by sugar phosphates and then other products. The validity of such experiments depends on using a sufficiently short time period during steady-state photosynthesis at an ecologically relevant inorganic carbon concentration and pH. Failure to observe one or more of these

Fig. I . The photosynthetic carbon reduction cycle (PCRC). The enzymes of the cycle referred to by numbers are: ( I ) ribulose- I ,5-bisphosphate carboxylase (regulated enzyme), (2) 3-phosphoglycerate kinase, (3) 3-phosphoglyceraldehyde dehydrogenase, NADP-linked (regulated enzyme), (4) phosphotriose isomerase, (5) fructose bisphosphate aldolase, (6) Transketolase, (7) fructose- I ,6bisphosphatase (regulated enzyme), (8) sedoheptulose- 1,7-bisphosphatase (regulated enzyme), (9) phosphopentose epimerase, (10) Phosphoribose isomerase, and ( 1 1) phosphoribulokinase (regulated enzyme). *, Catalytic role of C02.

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strictures can lead to erroneous conclusions on the nature of the carboxylation reaction. Bean and Hassid (1955) were the first to show that 3-PGA was the immediate stable produci of carbon fixation in a marine alga, Iridophyccus~~ccidum, a member of the Rhodophyceae. Despite differences between the major polysaccharides of red algae and land plants the early pattern of 14C labeling was shown to be the same with label accumulating into seduheptulose, glucose, and fructose monophosphates. Short-term labeling of 3-PGA and sugar phosphates has been demonstrated in a number of marine macrophytes (Bidwell et al., 1970; Kremer and Willenbrink, 1972; Willenbrink and Kremer, 1973; Kremer and Kiippers, 1977; Kremer, 1978) and in microphytes (Beardall et al., 1976; Holdsworth and Colbeck, 1976). Further evidence for the operation of the PCRC in marine algae is implied by the demonstration of a number of enzymes (aldolase, triosephosphate dehydrogenase, and fructose-1,6-bisphosphatase)involved in the cycle (Jacobi, 1957, 1962; Yamaguchi et al., 1969; Hellebust et al., 1967; Nizizawa et a l . , 1972, 1977; Kageyama et al., 1979). Not every attempt to demonstrate the presence of PCRC enzymes has been successful in a particular alga; however, the data suggest that these enzymes are found in all algal classes (Raven, 1974). An auxiliary pathway of carbon fixation occurs in certain specialized angiosperms. Kortshak et al. (1957, 1965) found that the first I4C-labeled products of photosynthesis in sugarcane (Saccharurn oflicinarum) leaves were mostly C, organic acids, e.g., malate and aspartate, rather than 3-PGA (traditionally the first stable product of photosynthesis). However, in these C, plants the PCRC remains the sole path for net reductive carbon assimilation. Here carbon is first trapped in oxaloacetate, malate, or aspartate and is subsequently released at the site of reductive assimilation via a decarboxylation step (for reviews see Hatch and Osmond, 1976; Ray and Black, 1979; Coombs, 1979). Fixation into organic acids also occurs in certain succulents and is known as crassulacean acid metabolism (CAM). In these plants a dark acidification occurs as a result of lightindependent carbon fixation into organic acids and a light deacidification with the subsequent release of CO, which is reassimilated by the PCRC (for reviews see Kluge and Ting, 1978; Osmond, 1978; Osmond and Holtum, 1981). A C, pathway of carbon fixation has been implicated in certain marine macroalgae (Joshi and Karekar, 1973; Karekar and Joshi, 1973), since aspartate was the major initial product of I4CO2 incorporation with significant labeling of the PEP and PGA fraction. Phosphoenolpyruvate carboxylase (PEPC), the initial carboxylating enzyme of the C, pathway, was shown to be present in the green alga Enteromorpha tubulosa (Karekar and Joshi, 1973), and it was suggested that the enzymes necessary for the operation of a C, pathway were present in marine algae. However, these authors failed to show the presence of a decarboxylating enzyme required for the reassimilation of CO, via RUBISCO, and the appearance of label in intermediates of the PCRC. Early labeling of malate and aspartate is not sufficient to demonstrate the presence of a C, pathway; the

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subsequent transfer of label from C, to C, intermediates must be established. In the C, pathway 14C0, is first incorporated into the C, position of malate and aspartate and is subsequently transferred to the C, position of 3-PGA via decarboxylation and refixation of CO, by RUBISCO (Johnson and Hatch, 1969). Neither the presence of PEPC nor label in organic or amino acids is a good sole criterion for C, photosynthesis (Ray and Black, 1979). The presence of an active C, decarboxylase is ambiguous since CAM plants also require such a decarboxylase, and carbon isotope discrimination has been, rather ill-advisedly, used as a test for C, photosynthesis in marine algae (see Sections 111 and VII). The notion that marine macroalgae have C, photosynthesis is now not accepted. Short-term light-labeling experiments (2-5 sec) show that phosphorylated compounds account for over 90% of the 14C-labeled products in marine macroalgae belonging to the Chlorophyceae (sensu h o ) , Rhodophyceae, and Phaeophyceae (Kremer and Kuppers, 1977), and that the maximum percentage of label in C, compounds follows that of phosphate esters. RUBISCO has been shown to be the major carboxylating enzyme in these algal groups. The situation in marine microalgae is somewhat more confused. Heavy labeling of amino acids (up to 50%) and intermediates of the TCA cycle as compared to sugar phosphates has been found after short-term labeling experiments (10-30 sec) in the marine diatom Phaeodactylurn tricornutum (Glover eta!. , 1975). In addition, activity of PEPC comparable to that of RUBISCO was shown (Mukerji and Morris, 1976). A C,-like pathway of carbon fixation has been proposed in the diatoms P. tricornutum and Skeletonema costatus (Beardall et a l . , 1976). However, no attempts were made to determine the distribution of label between the carbon atoms of the C, products and the time periods used (10-30 sec) were probably too long to detect the early products of CO, fixation. Parallel studies on P . tricornuturn have shown that the majority of label is in the C, , a-carboxyl group of aspartate, inferring that it has been derived from 3-PGA (Holdsworth and Colbeck, 1976). Controversy also exists as to the enzyme responsible for f3carboxylation in P . tricornutum and in other marine phytoplankton (Glover and Morris, i979) (see Section VIII). The removal of low-molecular-weight compounds (such as ADP) from crude extracts by gel filtration or dialysis would facilitate the determination of the enzyme responsible for light-independent carbon fixation. A number of investigations have been made relating to light-independent carbon fixation in marine macroalgae (Joshi et a l . , 1962, 1974; Craigie, 1963; Karekar and Joshi, 1973; Kremer and Willenbrink, 1972; Akagawa et al., 1972a,b; Willenbrink et a l . , 1975, 1979; Kremer, 1979, 1981a,b). The major low-molecular-weight I4C-labeled assimilates are amino and organic acids. The labeled amino acids include aspartate, glutamate, alanine, and glutamine. Labeling of serine (Kremer, 1979), leucine, and asparagine (Akagawa et al., 1972b) has also been reported. The labeled organic acids are mostly intermediates of the TCA cycle, e.g., malate, citrate, oxoglutarate, fumarate, succinate, and also

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pyruvate. Apparently labeled tartarate has not been detected (Kremer, 1979). Aspartate, along with alanine and citrate, constitute the most strongly labeled compounds in the Phaeophyceae (Akagawa et al., 1972b; Craigie, 1963; Kremer, 1979) while citrate is significantly less strongly labeled in members of the Rhodophyceae. Rates of photosynthetic carbon assimilation in marine algal mdcrophytes are given by Kremer (1981~)and are in the range of 9-30 nmol (g dry wt)- sec equivalent to 1-8 pmol m-2 sec- (calculated for one side of the thallus). Rates vary between species and such factors as photon flux density, spectral quality, inorganic carbon concentration, temperature, region of alga, season, and the developmental stage influence the observed rates. Members of the Phaeophyceae consistently show faster rates of 14C0, fixation in the dark than do members of the Chlorophyceae and Rhodophyceae (Craigie, 1963; Akagawa et al., 1972a; Kremer and Kuppers, 1977; Kremer, 1979). On average, light-independent carbon assimilation amounts to < 1% of the photosynthetic incorporation in the Rhodophyceae, <2% in the Chlorophyceae, and between 2.5 and 16% in members of the Phaeophyceae. Rates are variable between members of the Phaeophyceae and reported rates differ for the same species. Values as high as 16% of the photosynthetic carbon assimilation (Kremer and Kuppers, 1977) and as low as 2.5% (Kerby and Evans, 1983b) have been reported for light-independent carbon assimilation in Ascophyllum nodosurn. Rates will be dependent on such factors as the time of dark preincubation (Craigie, 1963), the developmental stage, temperature, and season (see Section VIJI). Members of the Phaeophyceae, in contrast to members of the Chlorophyceae and Rhodophyceae, have appreciable activities of phosphoenolpyruvate carboxykinase (PEPCK) (Akagawa et al., 1972c; Kremer and Willenbrink, 1972; Weidner and Kuppers, 1973; Kremer and Kuppers, 1977; Kremer, 1980a,b; Kerby and Evans, 1983a,b). This enzyme appears to be the enzyme responsible for light-independent carbon assimilation since other P-carboxylases are absent from members of the Phaeophyceae (see Section VIII). V. RIBULOSE-l,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RUBISCO) A. ENZYME EXTRACTION FROM MARINE ALGAE

Relatively few reports exist on the extraction of active enzymes from marine algae and few attempts have been made to purify these. Problems exist which can result in low protein yields and rapid inactivation of enzymes. Certain algae, particularly macrophytic members of the Phaeophyceae, are very tough and therefore conventional techniques of tissue homogenization are relatively ineffective. Commercial blenders tend to give low protein yields and very viscous homogenates. Grinding in liquid nitrogen produces greater cell breakage and

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provides an inert atmosphere for enzyme extraction. Brown algae contain large quantities of membrane-delimited polyphenols (9- 14% of the dry weight) (Evans and Holligan, 1972) which are ruptured during cell breakage. The polyphenols undergo progressive polymerization in vivo, producing high-molecularweight nondialyzable polyphloroglucinols (Ragan, 1976), and are readily oxidized on extraction, forming phlorotannins. Tannins are well known to inhibit many enzymes. Acid polysaccharides such as alginate, a major component of brown algal cell walls (Hellebust and Haug, 1972), have also been shown to inhibit certain enzymes (Jacobi, 1962). Additionally, denaturation of enzymes by exposure to unfavorable pH can result by liberation of acidic polysaccharides (Marsden et al., 1981). The liberation of phycoerythrin into aqueous extracts of red algae interferes with enzyme assays based on ultraviolet absorption by reduced pyridine nucleotide (Jacobi, 1962). Protective agents include the use of polymers to complex polyphenols, lowmolecular-weight antioxidants and protein stabilizers (Loomis, 1974; Rhodes, 1977). These compounds, together with low temperatures, are required for obtaining active extracts. Detergents (e.g., Triton X-100, Tween 80) have been employed in homogenization media to help rupture membranes due to their surfactant properties (Kremer and Kuppers, 1977; Kerby and Evans, 1981, 1983a,b; Marsden et al., 1981). Tween 80 increases protein yield by 90% in Fucus serratus (Marsden et al., I98 1) and RUBISCO was not detected in extracts from ruptured F . serratus eggs in its absence (Kerby, unpublished data). The inclusion of protease inhibitors [diisopropylphosphofluoridate (DIPF) and p chloromercuribenzoate (PCMB)] can also lead to greater extractable enzyme activities. Reported in vitro activities of RUBISCO are often not sufficient to account for the in vivo rates of carbon assimilation. For example, only 9% of the in vivo rate was observed for in vitro RUBISCO from Laminaria digitata whereas activities of PEPCK in the same extracts accounted for over 60% of the observed rates of dark fixation (Kremer, 1978). A similar situation exists in the diatom Phaeodactylum tricornutum where the maximum activity of RUBISCO was less than 10% of the maximum rate of light-saturated photosynthesis (Mukerji and Morris, 1976). It is not known whether the above authors compared inorganic carbon-saturated rates in vivo with inorganic carbon-saturated rates in vitro. Gray and Kekwick (1973) found that DIPF substantially increased RUBISCO activities and was required to obtain homogenous preparations from the terrestrial flowering plant Phaseolus vulgaris. Proteases may specifically act on RUBISCO (Peoples and Dalling, 1978; Peoples et al., 1979; Wittenbach, 1978, 1979; Kang et al., 1982). Use of DIPF in extracts of Pilayella littoralis substantially increased the RUBISCO activity; however, loss of activity still occurred with time in crude extracts (Kerby, unpublished data). When determining the enzyme responsible for P-carboxylation in crude extracts (see Section VIII) it is essential to free the enzymes from low-molecularweight endogenous compounds by gel filtration or dialysis. This important step

90

N. W. KERBY AND J. A. RAVEN

has often been omitted in the study of the enzymes responsible for light-independent carbon assimilation. B. PROPERTIES OF RUBISCO FROM MARINE ALGAE

RUBISCO is found in all oxygen-evolving organisms, and is a protein of high molecular weight existing as an aggregate of subunits. All eukaryotes have been shown to contain two subunit types, a large subunit (L) with a molecular weight of approximately 55,000 and a small subunit (S) with a molecular weight of approximately 14,000. A model structure for the basic protomer containing eight pairs of L and S has been proposed based on binding studies, molecular weight determinations, X-ray and optical diffraction analyses, and electron microscopy (for reviews see Jensen and Bahr, 1977; Akazawa, 1979). Certain prokaryotes, e.g., Rhodospirillum rubrum, have been reported to contain RUBISCO composed of a large subunit dimer (LJ (Tabita and McFadden, 1974) and that of Thiobacillus intermedius is an octamer of large subunit (L8) (Purohit er al., 1976) (for review see McFadden, 1980). Despite contradictory reports, cyanobacterial RUBISCOs are now thought to contain both subunit types (Akazawa, 1979; Codd, 1984). RUBISCO catalyzes two reactions: (1) the carboxylation of ribulose- 1 3 bisphosphate to give two molecules of 3-PGA [Eq. (6)] and (2) the oxygenation of ribulose- 1,5-bisphosphate to give one molecule of 3-PGA and one molecule of phosphoglycolate [Eq. (711. Ribulose-l,5-bisphosphate+ C 0 2 + H20+ 2 X 3-phosphoglyceric acid (6) Ribulose- 1,5-bisphosphate

+ 0 2 + 3-phosphoglyceric acid + 2-phosphoglycolic acid

(7)

Oxygen is a competitive inhibitor of the carboxylation (with respect to CO,) and also acts as the substrate for the formation of phosphoglycolate (for review see Lorimer, 1981). RUBISCO is activated by CO, and Mg2+ and activation applies to both carboxylase and oxygenase activities. Knowledge of this activation led to improved methods for the assay of the catalytic activities (Lorimer et al., 1976, 1977, 1978a; Lorimer, 1981). RUBISCO is specifically localized in the chloroplast and also occurs in certain inclusions of prokaryotes, e.g., the polyhedral bodies (carboxysomes) of certain bacteria and cyanobacteria (Shively et al., 1973; Codd and Stewart, 1976; Lanaras and Codd, 1981a,b, 1982) and also in the pyrenoids of certain algae (Holdsworth, 1971; Salisbury and Floyd, 1978; Kerby and Evans, 1978, 1981). The role of these inclusions in cyanobacteria and in algal chloroplasts is unclear (see later). RUBISCO activities have been determined in crude extracts from diverse marine algae (Morris and Farrell, 1971; Weidner and Kuppers, 1973; Weidner et al., 1975; Kremer and Kuppers, 1977; Mukerji and Morris, 1976), but few attempts have been made to purify this enzyme from marine algae. RUBISCO

CARBON METABOLISM IN MARINE ALGAE

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has been purified from a marine cyanobacterium Synechococcus (Andrews and Abel, 1981; Andrews et al., 1981), Bryopsis maxima (Yamada et al., 1978) and Halimeda cylindracea (Akazawa and Osmond, 1976) both members of the Chlorophyceae sensu lato, a pennate diatom Cylindrotheca sp. (Bacillariophyceae) (Estep et al., 1978), and from Spatoglossurn pacificurn (Yamada et al., 1979) and Pilayella littoralis (Kerby and Evans, 1978, 1981), both members of the Phaeophyceae. The enzyme from the marine cyanobacterium Synechococcus sp. was found to be significantly smaller than spinach enzyme when compared by pore exclusion techniques (M,430,000) and was shown to have both large (57,000) and small (12,000) subunits, suggesting a hexameric (L6S6) structure (Andrews and Abel, 1981). However, the molecular weight of this enzyme was subsequently shown to be 530,000 by equilibrium sedimentation and electron microscopy revealed fourfold symmetry characteristic of an octameric (L8S8) structure (Andrews et al., 1981). The presence of two subunit types has been shown in all marine algal RUBISCOs investigated (Table II), with molecular weights in the same region as the better characterized higher plant enzymes. The molecular weights of the native enzymes are also given in Table I1 and these suggest an octameric structure. Amino acid analyses show an apparent homology between the large subunit of Halimeda cylindracea (Akazawa and Osmond, 1976) and Pilayella littoralis (Kerby and Evans, 1981) and large subunits from other sources, whereas small subunits appeared distinct. Apparent homology of amino acid analyses is not surprising due to the relatively large molecular weight of this polypeptide; studies on the primary structures are required if valid comparisons are to be made. Partial immunological identity has been shown between Halimeda and spinach RUBISCOs by double immunodiffusion tests (Akazawa and Osmond, 1976). Table I1 shows the limited kinetic data available for RUBISCOs of marine algae. Some of the data were obtained for unpurified enzyme (e.g., Giflordia rnitchellae) or are only an approximation due to sigmoidal Lineweaver-Burk plots (e.g., Bryopsis maxima and Spatoglossum pacificurn). The Kn,(co2)where reported is higher than that reported for fully activated higher plant enzyme (Jensen and Bahr, 1977). RUBISCOs from different higher plant species are not uniform with respect to their kinetic constants (Bird et a l . , 1982). It is difficult to compare Km(co2)values between laboratories because of the lack of standardization in the method of computing the CO, concentration from HC0,- concentrations. The reported Km(RuBP)values for marine algal carboxylases are variable and are considerably higher than those reported for higher plants. The Km(RuBP) for the oxygenase reaction was lower than for the carboxylase reaction in Synechococcus (Andrews and Abel, 1981) as is also found for spinach RUBISCO (Badger et al., 1980). Similar Km(RuBP) values have, however, been reported for both reactions in Halimeda (Akazawa and Osmond, 1976), and in this species the oxygenase activity was reported to be only 1% of the carboxylase activity,

TABLE I1 Properties of Marine Algal RUBISCOs ___

~

___

Mp Synechococcus sp. (Cyanobacterium) Halimeda cylindracea (Chlorophyceae) Bryopsis maxima (Chlorophyceae) Cylindrotheca sp. (Bacillariophyceae) Sparoglossum pacificum (Phaeophyceae) Giffordia mitchellae (Phaeophyceae) Pilayella littoralis (Phaeophyceae) Laminaria hyperboria (Phaeophyceae) a C

~~

___~

~~~

Mr

K, (carboxylation reaction) (kmol m-3)

4

with respect to:

(total)

(large subunit)

(small subunit)

530,000

57,000

12,000

45

X

10-6

NDb

56,000

13,500

200

X

10-6

ND

ND

ND

ND

650

X

10-6

30 x 10-6.

550,000

56,000

13,000

580,000

56,000

14,000

400

X

10-6

33

ND

ND

ND

260

X

10-6

60 X 10-6.

560,000

55,000

15,000

ND

ND

ND

RuBP

CO, 240

ND

ND 710 x

10-6

X

10-6

ND X

10-6~

ND 12 x 1 0 - 6 ~

HCO, 22.5

X

10-3

ND 6 X 10-3 (PH 8.3)

ND

6.7 X 10-3 (PH 8.3) 8.3 X 10-3 (PH 7.2)

ND

3.8 x 10-3 (PH 8 . 5 )

Reference Andrews and Abel (1981) Andrews et al. (1981) Akazawa and Osmond ( 1976) Yamada et al. (1978) Estep et al. (1978) Yamada et al. (1979) Weidner er al. (1975) Kerby and Evans (1978) Kuppers and Weidner ( 1980)

Mr, relative molecular mass. ND, Not determined. Where K,,co,, is not given, we have recalculated KmcHCo3,using a pK,, of 6.00 as for seawater at 25°C and 35%0 salinity (Skirrow, 1975).

CARBON METABOLISM IN MARINE ALGAE

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unlike higher plants and Synechococcus, where a ratio of approximately 9 between the maximal velocities of carboxylase and oxygenase reactions (VcIVo, see Section VI) have been determined. The low ratio report in Halimeda probably reflects deficiencies in the preparation and activation of the RUBISCO, since the assay buffer was sparged with C0,-free air and CO, is required for activation of the oxygenase reaction (Lorimer, 1981). It remains to be determined to what extent competition between 0, and CO, in the RUBISCO reactions accounts for the 0, inhibition of photosynthesis in marine plants (see Section VI). The K , values with respect to NaHCO, and the Km(RuRP) in Phaeodactylum tricornutum have been reported to vary depending on the method of enzyme extraction (Mukerji and Morris, 1976). Apparently lower K, values were obtained from freeze-thawed cells than from conventional extraction by passage through a French press; the significance of these findings is unclear. The RUBISCO from Cylindrotheca is stimulated by aspartate and malate and is inhibited by PEP (C4pathway products and substrate). This alga has an active PEPC which is thought to be involved in primary CO, fixation together with RUBISCO (Estep er al., 1978). RUBISCO has been shown to be associated with the pyrenoids of certain algae. Pyrenoids are found in the chloroplasts of many algae, and their structure is very diverse (for reviews see Griffiths, 1970; Dodge, 1973). The nature and function of pyrenoids in many algae remains unclear. The pyrenoid of the freshwater green alga Eremosphaera viridis has been characterized and was shown to be composed of about 90% RUBISCO (Holdsworth, 1971). The presence of two PCRC enzymes ribose-5-phosphate isomerase and ribulose-5-phosphate kinase, were also recorded in these pyrenoid extracts. Pyrenoid isolation from E . viridis was facilitated by the presence of a starch sheath which completely surrounds the pyrenoid core. Further support that algal pyrenoids contain RUBISCO comes from the characterization of the pyrenoid of Micromonas squamata (Salisbury and Floyd, 1978). Dissociation of the pyrenoid matrix with SDS followed by electrophoresis revealed three major polypeptides ( M , = 64,000, 54,000, and 12,000) and, additionally, isolated pyrenoids were shown to have RUBISCO activity. With the exception of the 64,000 polypeptide (which may be a dimer of the L + S subunits) the pattern closely resembles that obtained from pyrenoid extracts from the brown alga Pilayella littoralis (Kerby and Evans, 1978). Pyrenoids of P . littoralis occur in projecting portions of the chloroplast and are not bounded by reserves such as the starch shells of E . viridis and M . squamata which afford protection during isolation and subsequent purification. In order to isolate pyrenoids from P . littoralis the alga was pretreated with HgCl, and therefore RUBISCO activity could not be demonstrated in extracts even after attempts to reverse mercurial binding to proteins with thiol reagents. However, after purification of individual pyrenoid polypeptides, a comparison of amino acid analyses and peptide maps with purified (untreated) RUBISCO revealed that the major polypeptides were identical to those of RUBISCO (Kerby and Evans,

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N. W. KERBY AND J. A . RAVEN

1981). Indirect evidence for the presence of RUBISCO in pyrenoids comes from a consideration of the crystalline areas observed in some pyrenoids (Holdsworth, 1968; Kowallik, 1969; Leadbeater and Manton, 1971). Crystalline inclusions similar to those in algal pyrenoids also occur in higher plant chloroplasts (Cran and Possingham, 1972; Esau, 1975; Sprey, 1976) and isolated membrane crystals have been shown to have RUBISCO activity (Sprey, 1976). Baker et al. (1977) and Eisenberg et at. (1978) crystallized three forms of RUBISCO, one of these having lattice plane spacings consistent with those of crystalline areas in higher plant chloroplasts and algal pyrenoids. Since the majority of pyrenoid protein of one brown alga and two green algae has been shown to be RUBISCO it is likely that pyrenoids are of similar composition and function despite differences in structure. There is as yet no clear evidence to suggest pyrenoid RUBISCO is active in vivo as all pyrenoid preparations have been assayed in the presence of CO, and Mg2+, both known RUBISCO activators (see above). Due to the close proximity to pyrenoids of reserve materials such as starch, paramylon, and floridean starch, a role in reserve formation has been assigned to them. However, insufficient evidence is available to substantiate this role. Pyrenoids may act as a store of utilized enzyme(s) or as a protein reserve but this awaits confirmation. Brown algal pyrenoids are sometimes present in eggs or sporelings but are absent from the mature thallus (e.g.. in certain members of the Fucales and Ectocarpales) (Bourne and Cole, 1968; Evans, 1968; Chi, €971). As yet no characterization of red algal pyrenoids has been made. Clearly pyrenoids are not the sole localization of RUBISCO in chloroplasts since many algae do not possess them, but further studies are required to determine their function. VI. THE OCCURRENCE OF RuBPo ACTIVITY, AND OF THE PCOC, IN MARINE ALGAE All known RUBISCO enzymes, including those from marine algae, have oxygenase (RuBPo) activity competitive with RuBPc activity (Section V). The extent to which RuBPo and RuBPc activities are manifest depends on the steadystate O,([O,],) and CO,([CO,),] concentrations at the site of RUBISCO, the KI,, values for 0, in RuBPo (KO)and for CO, in RuBPc ( K J and the V,,, values for RuBPo (V,) and RuBPc (V,). The relevant equation is

where v, and v, are the respective rates of RuBPo and RuBPc activities achieved. In SI units rates are in mol substrate consumed (mol enzyme)- sec - I , and K,,, and concentrations are in mol m-3. The only marine algal or cyanobacterial enzyme for which sufficient data are available to use Eq. (8) is that from the cyanobacterium Synechococcus sp.

CARBON METABOLISM IN MARINE ALGAE

95

(Andrews and Abel, 1981), where the application of Eq. (8) shows that v,/v, = 0.56. This is at the high end of the range (Table 5.3 of Raven, 1984) for RUBISCO from freshwater cyanobacteria and chlorophycean and euglenoid algae, and for C, and C, terrestrial plants, where the range is from 0.25 (terrestrial C, and C, plants) to 0.59 (the freshwater cyanobacterium Anabaena variabilis). We shall take the A . variabifis value as a likely upper limit for the v,/v, for RUBISCO in air-equilibrated solution. Thus, if the inward diffusion of CO, and the outward diffusion of 0, in steady-state photosynthesis did not significantly alter the CO, and 0, concentrations at the site of RUBISCO activity, then for every 1.59 mol RuBP consumed, 1 CO, would be fixed yielding 2 PGA, and 0.59 0, would be fixed yielding 0.59 PGA and 0.59 phosphoglycolate. A very significant feature of this computation (cf. Raven and Beardall, 1981; Raven, 1984) is that, for 1 CO, fixed, (2 X 0.59) or 1.18 C are converted to glycolate. Thus, if there were no mechanism of metabolizing phosphoglycolate beyond the dephosphorylation to glycolate followed by glycolic acid excretion, there would be a net loss of organic carbon from the cell due to operation of RUBISCO! It is only when v,/v, is less than 0.5 that a glycolate-excreting, but not glycolate-metabolizing, organism could achieve net CO, fixation via RUBISCO. In practice all photolithotrophs growing at air-equilibrium concentration of carbon dioxide and oxygen have some capacity for glycolate metabolism. The classical pathway of glycolate metabolism via the photorespiratory carbon oxidation cycle (PCOC) (Fig. 2) as well as the tartronic semialdehyde pathway (see Raven and Beardall, 1981) yield 0.5 CO, produced per 1 glycolate (= 2 C)

Fig. 2 . The photorespiratory carbon oxidation cycle (PCOC) coupled to the PCRC (Fig. 1) with a ratio of RuBPc to RuBPo activity of 9 (a ratio only attainable with air-equilibrium C 0 2 and O2 concentrations in the medium surrounding an aquatic plant if a ‘ T O 2concentrating mechanism” is operative). Evidence for the occurrence of the enzymes of this pathway in marine algae may be found in Randall (1976) for phosphoglycollate phosphatase; Tolbert (1976) for glycollate oxidase and glycollate dehydrogenase. Fd - , reduced ferredoxin; PGA, 3-phosphoglycerate.

96

N. W. KERBY AND J. A. RAVEN

converted to 0.5 phosphoglycerate. For our example of RUBISCO from Anabaena variabilis with v,/v, = 0.59, we can see that the net carbon fixed after PCOC metabolism of glycolate relative to the gross carbon fixed by RuBPc is (vc - 0.5 v,)/vc or 0.705, so that there is a substantial carbon loss (as well as requirement for net energy requirement; see Raven and Beardall, 1981; Raven, 1984) involved in PCOC operation. For the RUBISCOs with lower vo/vc ratios in air-equilibrated solution (i.e., 0.25), the fraction of carbon fixed which is lost as glycolate in an organism which excretes, rather than metabolizes, glycolate is 0.25, while the fraction of carbon fixed which is lost as CO, during the operation of the PCOC (or tartronate pathway) is 0.125. Thus, with vo/v, = 0.25, glycolate excretion leaves the plant with 0.5 of the gross carbon fixed by RUBISCO, while glycolate metabolism leaves 0.875 of gross carbon fixation, albeit with reductant and ATP input over and above the 2 NADPH and 3 ATP used per CO, fixed in the PCRC (see Raven and Beardall, 1981; Raven, 1974). Since marine algae RUBISCOs probably have vJv, ir air-equilibrated solutions of 0.25-0.59, we might anticipate that, with diffusive entry of CO, and efflux of 0,, the in vivo vo/vc range is from 20.25 to 20.59. Accordingly, the minimal glycolate production as a fraction of carbon fixed (mol carbon in glycolate per mol gross carbon fixed) should be 0.5, while the minimal CO, production in the PCOC should be 0.125 mol CO, produced per mol gross carbon fixed. Even the most fervent supporters of glycolate excretion could scarcely support the occurrence of a glycolate efflux in steady-state photosynthesis which disposes of more than half of gross photosynthesis. Carbon dioxide production via the F i O C is not readily quantified. The techniques employed to estimate carbon dioxide efflux from illuminated marine algae include the measurement of net carbon dioxide efflux to a carbon dioxide-free gas stream (Lloyd et al., 1977), the loss of 14C02to a gas stream after a period of photosynthesis in 14C0, (Lloyd et al., 1977; Hough, 1976), and, best of all, short-term (<60 sec) measurements of I4CO, fixation and 'zCO, release (Lloyd et a!. , 1977). Reassimilation of carbon dioxide produced within the cells is a problem with all of these methods (Jackson and Volk, 1970; Raven, 1972). The loss of carbon dioxide to a gas stream free of this gas is open to the objection that the RuBPc:RuBPo ratio is not typical of growth conditions, while the pulse labeling with *4C0, may lead to different specific activities of decarboxylation substrates in light and in darkness, a point which can only readily be checked if the gas stream is free of carbon dioxide (Lloyd er al., 1977). Even the short-term measurements of 14C02uptake and 12C0, release must be really short term if recycling of I4C through the PCRC and PCOC is not to occur (Ludwig and Canvin, 1971). Having established the magnitude of carbon dioxide efflux from algae, the flux must then be apportioned into dark respiration continuing in the light, PCOC activity, and (if a C0,-concentrating mechanism is present) to leakage of carbon

CARBON METABOLISM IN MARINE ALGAE

97

dioxide from the intracellular pool (see Zenvirth and Kaplan, 1981; Badger and Andrews, 1982). The occurrence of a C0,-concentrating mechanism will, of course, increase the potential for reassimilation of carbon dioxide. Dark respiration and PCOC are commonly distinguished on the basis of oxygen sensitivity, the component of carbon dioxide efflux which is inhibited upon changing from air-equilibrium oxygen concentrations to low oxygen (- I kPa 0,) being attributed to “photorespiration,” in view of the much higher K+(02)for RuBPo than for cytochrome oxidase (Raven and Beardall, 1981; but see Raven, 1984, for a discussion of high in vivo KKoz, values for dark respiration as a result of diffusive limitation in some macroalgae). The data on CO, release show that it is uncommon for the CO, release in the light in air to exceed that in the dark in air; an exception is Caulerpa verticillata in a 14C0, release experiment in which specific activity differences between light and darkness could have been significant (Hough, 1976). In some cases the CO, release in the light in air is very substantially less than in the dark (Lloyd et al., 1977). This work is particularly important in view of the use of the “artificial leaf‘ ’ technique which minimizes problems of carbon dioxide retention in large volumes of aqueous media. Furthermore, the depression of CO, release at low 0, tensions is often small (Hough, 1976; cf. Lloyd et al., 1977). These data suggest that, even if dark respiration occurs at the same rate in the light as in the dark (an assumption which permits scaling of results for reassimilation), CO, release from the PCOC is usually less than one-tenth of net photosynthesis rather than the one-eighth or more predicted from RuBPc/RuBPo activity in air-equilibrated solutions with subsequent operation of the PCOC (Fig. 2). An alternative method of examining PCOC activity is the magnitude of any “postillumination burst” of carbon dioxide: in C, land plants this is taken to indicate the continued operation of the PCOC in the dark, consuming phosphoglycolate, glycolate, and glycine in the cycle at the time of cessation of illumination. It is thus considered to give a lower limit of steady-state PCOC activity when the component attributable to dark respiration is subtracted (Jackson and Volk, 1970). However, it is also found in some C, land plants where it is attributed to leakage from the bundle sheath CO, pool (Jackson and Volk, 1970), and this could be an alternative explanation of the CO, data of Burris (1977), which were interpreted in terms of PCOC activity (cf. Zenvirth and Kaplan, 1981; Badger and Andrews, 1982). However, the postillumination burst of oxygen uptake (Burris, 1977) is not susceptible to such a “CO, pool” explanation; where Bums (1977) reports parallel oxygen uptake and carbon dioxide efflux data (e.g., for Enteromorpha, Thalassiosira, and Glenodinium) the ratio of the postillumination increments of the two fluxes is 1.O-2.0: this could be accounted for by metabolism of a mixture of glycolate and glycine by the PCOC (Fig. 2). However, in the absence of additional data a role for a postillumination stimulation of dark respiration (see Raven, 1977) cannot be ruled out. We must conclude from these results that it is difficult to distinguish the

98

N. W.KERBY AND J. A. RAVEN

various components of carbon dioxide release from green cells. The data are frequently as consistent with any light-dependent carbon dioxide release in the presence of external carbon dioxide or postillumination burst being due to leakage from a free carbon dioxide pool as with its production from the PCOC. A further method for attempting to quantify the PCOC is the analysis of shortterm labeling products of 1802 or 14C0, (or I3CO,). Assimilation of IsO, into phosphoglycolate, glycolate, glycine, serine, glycerate, and phosphoglycerate have been very important in quantitatively establishing the occurrence of the PCOC in C, land plants (Lorimer et al., 1977; Lorimer and Andrews, 1981). The powerful technique (which suffers less than I4CO2 or I3CO, from internal recycling of isotopes) has rarely been applied to marine algae (cf. De Veau and Burris, 1981, 1982). Much data on I4C labeling of glycolate, glycine, and serine with 14C0, has been published for marine micro- and macroalgae (e.g., Burris, 1977, 1980; Burris et al., 1976; Bidwell et a l . , 1969, 1970). This generally shows that these three compounds are indeed short-term products of I4CO, fixation in the light, and that labeling is decreased as 0, is decreased below airequilibrium values. However, it is not yet possible to deduce quantitative estimates of PCOC from this data (see Raven and Glidewell, 1981); qualitatively it agrees with PCOC functioning. Two other lines of evidence as to the occurrence, and magnitude, of photorespiration are the Warburg effect (0, inhibition of photosynthesis) and the magnitude and 0, dependence of the CO, compensation concentration. Dealing with the CO, compensation concentration first, the photosynthesis - photorespiration (PCRC - PCOC) carbon balance is struck when v, (= gross CO, fixation) = 2 v, (2 v, = 2 glycolate synthesized = 1 CO, produced in the PCOC). For the lowest v,/v, (= 0.25) for RUBISCO in air-equilibrated solution, 2 v, = v, when [CO,] in a solution with air-equilibrium 0, concentrations is some 0.25/2 or 0.125 of the air-equilibrium level, i.e., some 1.25 mmol m-3, equivalent to some 4 X l o p 4 m3 CO, (m3 air)-I. At the other end of the range of VJV, values in air-equilibrated solution, i.e., 0.59, the computed CO, compensation concentration is 0.59/2 of the air-equilibrium concentration, i.e., some 2.95 mmol m-3, equivalent to some 9.5 X l o p 4 m3 CO, (m3 air)-l. We note that a continuation of “dark” respiration in the light would increase these CO, compensation concentration values by adding additional CO, efflux to the PCOC CO, efflux, and thus require more CO, fixation to achieve zero net CO, flux. If we compare the computed CO, compensation concentration for RUBISCO activity in marine algae [at least 1.25 mmol CO, m-3, or 4 X m3 CO, (m3 air)- ‘1 with measured values, we find that the great majority of published values are lower than our computed lower limit. Relatively few data are available for marine microphytes; Lloyd et al. (1977) found low CO, compensation concentrations (
CARBON METABOLISM IN MARINE ALGAE

99

Brown and Tregunna (1967) investigated a number of marine algae at external pH values which were as low as the plants would tolerate without damage (detected as inhibition of CO, uptake in light) over the experimental period. Of the algae tested, Ulva expansa, Enteromorpha linza, Polyneura lastissima, lridaea cordata, Gigartina latissima, and Fucus gardneii all had CO, compensation concentrations substantially below 1 mmol rn-, of free CO, in solution; only Sargassum muticurn had a CO, compensation concentration in excess of 1.25 mmol m--3. Coughlan and Tattersfield (1977) investigated the CO, compensation concentration of Enteromorpha intestinalis, Ulva lactuca, Porphyra umbicalis, Rhodymenia palmuta (now Palmaria palmata), Fucus serratus, and Pelvetia canaliculata over a 0-30°C temperature range; in all cases the CO, compensation concentrations were well below I mmol CO, n i r 3 . Raven et al. (1982) have demonstrated that the intertidal alga Ascophyllum nodosum has a low (well below I mmol CO, m-3) CO, compensation concentration under both submerged and emerged conditions. Glenn and Doty (1981) found that the CO, compensation concentration of the rhodophyte Eucheuma was about I mmol m-,. Generally low CO, compensation concentrations can also be deduced from “pH drift” experiments (see Raven, 1970), although complications arise through the occurrence of processes which increase the external pH independently of inorganic carbon uptake in photosynthesis (Thomas and Tregunna, 1968; Dromgoole, 1978a). Clearly measurements of total inorganic carbon concentration are required in “pH drift” experiments if valid conclusions are to be drawn, just as pH measurements are essential if valid conclusions are to arise from measurements of depletion of total inorganic carbon concentration in seawater containing algae in the light (cf. Tolbert and Garey, 1976; Kremer, 1981d). We may conclude that the few microalgae, and the great majority of the marine macroalgae (seaweeds) tested, show CO, compensation concentrations which are lower than would be expected for diffusive entry of CO, and RUBISCO activity. The final test for in vivo functioning of RuBPo and PCOC is oxygen inhibition of photosynthetic gas exchange. In C, terrestrial plants it is possible to account for the in vivo competitive effects of 0, and CO, on CO, fixation in terms of the in virro kinetics of RUBISCO (Laing et al., 1974). The extent of 0, inhibition of net photosynthesis depends on the taxon investigated, the experimental conditions (e.g., the inorganic carbon concentration and the pH), and whether the experiments involve 14C02 uptake, I2CO, uptake, or 0, evolution as the measure of net photosynthesis (see Turner and Britain, 1962; Beardall et d . , 1976; Burris, 1977, 1980; Black et a l . , 1976; Downton et a l . , 1976; Dromgoole, 1978b; Lloyd et a f . , 1977; Bidwell et al., 1969; Bjorkman 1966). In a number of cases (e.g., Ulva lactuca: Bjorkman, 1966; Chaetomorpha crassa: Downton et a l . , 1976; Enteromorpha sp.: Black et al., 1976; Chaetomorpha sp. and Symbiodinum sp. : Burris, 1977; Skeletonema costatum

100

N. W. KERBY AND J. A. RAVEN

and Gonyaulax tamarensis: Beardall et al., 1976; Dunaliella terliolecta, Thalassiosirafluviatilis,and Porphyridium sp.: Lloyd et al., 1977) there appears to be essentially no effect of 0, or CO, fixation when CO, is near the airequilibrium concentration, and 0, ranges from the air level (21 kPa) down to 12 kPa. This supports the view that many marine algae have a CO, fixation reaction in the light which is less 0, sensitive than one would expect of RUBISCO supplied with CO, from the medium by diffusion. In Acetabularia mediterranea chloroplasts, by contrast, the interactions of oxygen and carbon dioxide concentrations in determining the rate of net carbon dioxide fixation are in general accord with a C,-type physiology (Bidwell et al., 1970). Attempting to summarize these data, the following conclusions seem valid. 1. Algal RUBISCOs have, like all other such enzymes, substantial RuBPo activity which competes with RuBPc activity as a function of the O,/CO, ratio. 2. The RuBPo activity achieved in vivo at a given external CO,/O, concentration ratio is lower than that predicted from the properties of RUBISCO in vitro; the evidence for this comes largely from 0, inhibition and CO, compensation concentration measurements, with data consistent with this point of view coming from 14C0, labeling and CO, efflux experiments. ‘*O, uptake characteristics, and labeling of PCOC intermediates, should be studied in more detail. 3. The apparently widespread suppression of RuBPo activity in vivo in marine algae seems to be a result of a “CO, accumulation mechanism” (possibly based on active transport of HCO,- : see Section IV) rather than on an auxiliary “C, C1” carboxylation of the type found in C, terrestrial plants. We note that the occurrence of a CO, accumulation mechanism greatly complicates the “pulsechase” experiments which were so useful in showing that 14C0, fixed by a (C, + C,) carboxylation was on the pathway to phosphoglycerate and sugar phosphates in terrestrial C, plants (Raven and Beardall, 1981; Beardall and Raven, 1981). Even if I4CO, labeling of C, dicarboxylic acids is not on the direct route to sugar phosphates, pulse-chase experiments will suggest that they are if a CO, accumulation mechanism prevents escape of 14C0, released from these compounds as they are turned over.

+

Our conclusions on the occurrence of photorespiration in marine algae may seem to be at variance with recent critiques by Burris (1980) and by Kremer (1981d). The difference is essentially a quantitative one: we do not deny that RuBPo and PCOC are active in vivo in a number of marine algae at “normal” seawater inorganic carbon and oxygen concentrations. However, we do suggest that the assembled data only quantitatively support a fully “C,-type” biochemistry and physiology in a few marine algae (e.g, Sargassum muticum, Acetabularia mediterranea); in others there are varying degrees of suppression of RuBPo and PCOC .

CARBON METABOLISM IN MARINE ALGAE

101

VII. CARBON ISOTOPE DISCRIMINATION IN MARINE ALGAE

The major stable isotope of carbon in the biosphere is I2C (98.9%), with I3C making up the remaining 1 . 1%. Discrimination between these two isotopes occurs at equilibrium [e.g., between gaseous and dissolved CO,, and between dissolved CO, bicarbonate (thermodynamic discrimination)], and in the rate at which equilibrium is achieved (kinetic discrimination; again, w e may cite the CO,(,, - C02(aq)- HC0,- system (see O’Leary, 1981). Data on the carbon isotope composition of samples are commonly expressed as the 613C value, where [Eq. (9)]

where 613C is in parts per thousand, and PDB refers to the standard limestone of a Belemnite from the Pee-Dee formation. Mook et al. (1974) discuss the temperature dependence of the equilibrium between gaseous CO,, dissolved CO,, and dissolved bicarbonate; the 6 value for CO, is more negative than that for HCO, - , and the difference increases at low temperatures. It is vital to consider “source” 613C values in interpreting the 6I3C value measured for organic (plant) carbon (O’Leary, 1979; Raven et al., 1982). Limestone, and HC0,- in seawater, is usually at about O%O of PDB , while atmospheric CO, is some -7%0, as is dissolved CO,. Analysis of 613C values in terrestrial vascular plant sporophytes shows that C, plants have S values near -29%0 while C, and CAM plants have 6 values near - 15%0 (C,-C,, and C,-CAM intermediates, have intermediate values) (Troughton, 197 I ;Osmond, 1978). Analysis of these values has been undertaken by Farquhar (1980), Farquhar et al. (1982), O’Leary and Osmond (1980), and Holtum et al. (1982). An equation which describes the extent to which the diffusive and carboxylative reactions limit carbon assimilation is, for RUBISCO [Eq. (10113

c,Gj

=

-

6,,”,,

+ a ) / @ + b)

(10)

where Splant= S13C of plant material 6,,,rce = 6I3C of source (atmospheric) CO, a = 6I3C value associated with CO, diffusion from bulk air (source) to enzyme (sink) b = 6I3C value associated with CO, fixation by RUBISCO C , = CO, concentration (mol m-3) in the bulk medium C, = CO, concentration (mol m-,) at the site of RUBISCO activity

102

N. W . KERBY A N D J. A . RAVEN

A very negative value for €iplant (i.e., close to the value of fi, -30%0) means that CJC, is close to 1, i.e., diffusion plays a very small part in restricting the rate of

photosynthesis. Conversely, a relatively positive value for 6plant(i.e., close to the value of a, --5%0) means that C , is very small relative to C,, and diffusion plays a major role in restricting the rate of photosynthesis. In practice, the balance between stomata1 and biochemical conductances in C, vascular plants is obtained at CJC, of about 0.7 (Wong et al., 1979). For plants using PEPC as a preparatory (auxilliary) carboxylation (i.e., C, and CAM plants), the effective value for a (allowing for HCO,- as the “real” substrate for PEPC) is some -1%~. In practice, 6 is, in part, influenced by the incomplete isolation of ’RUBISCO from external CO,; the PEPC-based CO, pump is “leaky,” so that 6 values for whole plant carbon in C, and CAM plants are outwith the range of - I%c (complete limitation by PEPC activity) to - 11%0 (complete limitation by diffusion) given by the equation. The values for the carboxyl group of malate which originates from PEPC give the best direct estimate of 6I3C associated with CO, diffusion plus PEPC action (O’Leary and Osmond, 1980; Holtum et al., 1982). The situation is more complicated in aquatic plants. The only aquatic plant RUBISCO for which a 6I3C value has been measured in v i m is the diatom Chaetoceros sp. (Estep et al., 1978); the AI3C value [ b in Eq. (lo)] of -32%0 is within the range for terrestrial plant RUBISCO Ai3C values for which a mean of b = -30%~ is widely used (O’Leary, 1981). No estimates of b seem to be available for the multitude of other carboxylases in aquatic plants. The value of a is also less clear for CO, diffusion in solution; it is probably close to WOO (O’Leary, 198I), rather than the -4%0 for gas-phase diffusion (or the - 11%0 assumed by Raven, 1970; cf. Farquhar et a l . , . 1982). Finally, there is the problem of the discrimination value to be attributed to active HC0,- entry: this is often assumed (on rather flimsy evidence) to be negligible (see Beardall er al., 1982). Turning to the measured values for 6I3C, Table 111 shows the values for a variety of marine algae. The values are expressed as AI3C, i.e., as the difference between the 613C value of the organic material of the plant and the 6I3C value of the carbon dioxide in seawater. Where no specific data is given on the 6I3C value for this “source” carbon dioxide it is assumed to be - 7 % ~relative to the PDB standard. The AI3C values listed cover a very wide range, with some values less negative than the values commonly found for C, or CAM terrestrial plants (--7%0), and others as negative as the values most frequently found for C, terrestrial plants (--22%0) when values for plants grown in seawater in equilibrium with air are compared. For laboratory cultures of microalgae and cyanobacteria it is generally found that AI3C is more negative when the inorganic carbon supply to cells is improved by increasing the concentration of inorganic carbon in solution, by having a low cell density, or by more vigorously sparging the culture medi-

CARBON METABOLISM IN MARINE ALGAE

103

um. This is in agreement with the notion that such treatments reduce the extent of diffusive limitation, relative to biochemical limitation, of the rate of photosynthesis [see Eq. (lo)]. The finding that the (most negative) values for AI3C, i.e., (-20)-(-26)%0 for the rhodophyte Hafymenia duwillaei and a number of phytoplankton, suggests that the ratio CJC, in Eq. (10) can be as high as 0.8 (assuming AI3C plant = -26.4, a = 0, b = -32.6%0). This means that 0.2 of the “limitation” of photosynthesis is attributable to transport processes and 0.8 to biochemistry (carboxylation). Quantitatively, we can assume that inorganic carbon entry to the photosynthetic cells is by CO, diffusion, and use appropriate values in Eq. (8) to derive C,. For Macrocystis with A13C = - 10.5, it appears that C, = 4.6 mmol m - when C , = 13 mmol m - ,. To convert this concentration difference into a path length for CO, diffusion we may use Fick’s equation (Section 111). In situ rates of photosynthesis by Macrocystis are of the order of 20 pmol carbon fixed (m2 habitat)- I sec- (Jackson, 1977); with a frond area index (in terms of both sides of fronds) of 20 (Clendenning, 1971), the mean net rate of photosynthesis achieved is some 1 pmol (m2 both sides of fronds)-’, i.e., some 0.2 of the maximum (Raven, 1981), with light as possible limiting factor. Thus J in Fick’s m2 sec-I, and (C, mol m-* sec- l ; with Dcoz = 1.7 X equation is - C,) = 8.45 x lo-, mol mP3, the equivalent diffusion path length is 15 pm. This seems rather a small value (Wheeler, 1980; Smith and Walker, 1980; Raven et a / . , 1982). Even smaller equivalent diffusion path lengths may be computed for the likes of Halymenia; with A13C = -25.2%0, and other values as for Macrocystis, the equivalent diffusion path length is 4 pm. A great complication in the interpretation of these data is the possibility that many marine algae can utilize HC0,- as well as CO,. The 613C of marine HC0,- is about O%O, so that the A13C (relative to CO, at --7%0) for a plant which takes up HC0,- by diffusion through unstirred layers and mediated transport through membranes, and then converts all of the HC0,- to CO, with all of the CO, being fixed and none leaking, would have a AI3C of +7%0relative to CO, (6I3C = O%O relative to the PDB standard). The effect of leakage of CO, from the “concentrated” pool back to the medium would be to make 6L3Cplant more negative, as has been pointed out for the analogous “CO, pump” which terrestrial C, metabolism catalyzes (see Peisker, 1982; O’Leary and Osmond, 1980). The effect on 6I3C of the operation of the “C0,-concentrating mechanism” in freshwater microalgae is discussed by Beardall et a l ., (1982); algae with the concentrating mechanism (probably based on HCO,- transport) have a less negative A13C value. This switch could be at least part of the cause for the effects of CO, availability (concentration of inorganic carbon, pH, agitation, cell density) on the A13C value of marine phytoplankton (Table 111). This discussion of 613C and AI3C values of marine algae shows that the measurements are already useful in determining the relative limitations imposed by CO, diffusion and by RUBISCO in systems which use RUBISCO as their

TABLE 111 A13C of Marine Algae and Cyanobacteriaa

Organism Cyanobacteria Agmenellum quadruplicatum (strain PR6) (strain BG1) Coccochloris elebens

-g

Coccochloris elebens 17a Di Scytonema sp. Bacillariophyceae Chaetoceros didymus Chaetoceros lozenenzianur Coscinodiscus asteromphalus Cyclotella sp. Cyclotella nana

A'3C

(%o)

Comments

-15.9 -0 -8.0 -22.2 -23.9 - 12.3 -0 -8.0 -22.4 -19.6 -6.3

Grown in 1.5% CO, Grown in 0.2% CO, Grown in 0.03% CO, Grown in 1.5% CO,; 39°C Grown in 3.6% CO,; 39°C Grown in 1.5% CO, Grown in 0.2% CO, Grown in 0.03% CO, Grown in 3.6% CO, Grown in 2.7% CO, Wild; seawater

-20.0 -17.7 -19.2 - 16.4 - 10.7 -9.8 -6.0 -13.2 -13.8 -13.9 -18.6 -18.9

Seawater, 18°C Seawater, 18°C Seawater, 18°C Seawater, 18°C pH 8.6, seawater, 10°C pH 8.6, seawater, 20°C pH 8.6, seawater, 30°C pH 8.2, extra HC0,-, 10°C pH 8.2, extra HCO, - , 20°C pH 8.2, extra HCO, - , 30°C pH 5.8, 5% CO,, 10°C pH 5.8, 5% CO,, 20°C

Reference

Calder and Parker (1973) Pardue et al. (1976) Calder and Parker (1973) Pardue et al. (1976) Black and Bender (1976) Wong and Wong and Wong and Wong and

Sackett (1978) Sackett (1978) Sackett (1978) Sackett (1978)

Degens et al. (1968)

16.8 - 13.0 - 15.1 -16.9 -20.9 -8.4 -4.7 -5.6 - 12.7 -11.5 - 10.1 -10.0 -20.2 - 17.6

pH 5 . 8 , 5% CO,, 30°C Seawater Seawater, 18°C Seawater, 18°C Seawater, 18°C Seawater, 18°C pH 8.6. slow aeration, 8°C pH 8.6, slow aeration, 18°C pH 8.6, slow aeration, 28°C pH 8.0, slow aeration, 8°C pH 8.2, violent aeration, 18°C pH 8.3, violent aeration. 26°C pH 8.3, violent aeration, 26°C Seawater, 18°C Seawater, 18°C

Wong and Sackett (1978) Wong and Sackett (1978)

Chlorophyceae sensu stricto Chlorococcum sp. Dunaliella sp. Dunaliellu tertiolecta

-20.0 -22.9 -8.9

Seawater, 18°C Seawater, 18°C Seawater, 18°C

Wong and Sackett (1978) Wong and Sackett (1978) Degens el al. (1968)

Prasinophyceae Playmanas sp.

-19.8

Seawater, 18°C

Wong and Sackett (1978)

Seawater; Seawater; Seawater; Seawater; Seawater;

Craig (1954); Smith and Epstein (1971) Fry et al. (1982) Black and Bender (1976) Fry et al. (1982) Black and Bender ( 1 976)

- 18.4

Cylindrotheca sp. Nitzschiu closterium Nitzschia curvilineata Nitzschia frustulum Skeletonema costatum

3 VI

Thalassiosira pseudonanu Thalassiosira subtilis

Ulvophyceae Acetubularia sp. Avrainvillea (elliotii)? Avrainvillea erecta Avrainvillea nigricuns Boergesenia forbesii

-

-2.5, -5.3 -9.4 -11.2 -12.4 -8.9

wild wild wild wild wild

Estep et al. (1978) Wong and Sackett (1978) Wong and Sackett ( 1 978) Wong and Sackett (1978) Wong and Sackett (1978) Degens et ul. (1968)

(continued)

TABLE 111 (Continued) Organism

e

0 3

Caulerpa paspaloides Caulerpa prolifera Caulerpa racemosa Chaetomorpha linum Chlorodesmis fastigiata Enteromorpha jlexuosa Enteromorpha marginata Enteromorpha salina Halimeda sp. Halimeda sp. Halimeda cylindracea Halimeda macroloba Halimeda monile Halimeda opuntia Penicillus sp. Pencillus capitatus Penicillus dumetosus Phaeophyceae Calpomenia sinuosa Dictyota dentata Dictyota dichotoma Dictyota divaricata Macrocystis pyrifera Padina sanctae-crucis Padina tenuis Sargassum sp.

A'3C

(%o)

(- 19.8)-( -20.5)

-6.3 -6.9 (- 10.9)-( - 13.6) - 18.8 - 14.3 -9.6 -11.0 ( -8.0)- ( -8.3) -7.9 -8.3 -8.8 -5.5 - 14.3 -6.3 -11.6 - 10.2 -3.3 -7.1 -6.5 -5.7 - 10.5 -2.5 -4.4 -9.3

Comments

Reference

Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild

Fry et al. (1982) Fry et al. (1982) F r y et al. (1982) Fry et al. (1982) Black and Bender (1976) Black and Bender (1976) Smith and Epstein (1970) Parker (1964) Craig (1953) Fry et al. (1982) Black and Bender (1976) Black and Bender (1976) Fry et al. (1982) Black and Bender (1976) Craig (1954) Fry et al. (1982) Fry et af. (1982)

Seawater; Seawater; Seawater; Seawater; Seawater; Seawater; Seawater; Seawater;

Fry et al. (1982) Fry et al. (1982) Fry er al. (1982) Fry et al. (1982) Smith and Epstein (1976) Fry et al. (1982) Black and Bender (1976) Smith and Epstein (1976)

wild wild wild wild wild wild wild wild

Sargassum sp. Sargassum polyceriatum Turbinaria ornata

-

4 0

Rhodophyceae Acanthophora spicifera Amphinoa frugilissima Ceramium sp. Corallina sp. Corallina chitense Digenia simplex Galaxaura oblongata Galaxaura squalida Galoraing dermanema Gelidion acerosa Gigartina crustacea Gracilaria sp. Grateloupia setchelii Halymeniu durvillaei Hypnea sp. Laurencia sp. Laurencia obtusa Laurenciu obtusa Laurencia poitei Liagora ceranoides Nemulion sp. Plocamium sp. Spyridia aculeata (unidentified)

-7.1 -7.8 -4.3 -7.0 -11.5 - 12.4 - 10.0 -11.6 -11.0 -11.2 -7.5 - 17.2 -8.9 -17.2 -11.9 -15.7 -25.2 -12.9

-4.0)-(-8.3) -8.0

-12.7 -4.8 -11.5 -7.8 -9.7 - 10.6 (-27.7)

Seawater; wild Seawater; wild Seawater; wild

Black and Bender (1976) Fry et al. (1982) Black and Bender (1976)

Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild Seawater; wild (Seawater; wild)

Black and Bender (1976) Fry et al. (1982) Fry et al. (1982) Parker ( 1964) Smith and Epstein ( 1 970) Parker (1964) Black and Bender (1976) Fry et al. (1982) Black and Bender (1976) Fry et al. (1982) Smith and Epstein (1970) Fry et al. (1982) Smith and Epstein (1970) Black and Bender (1976) Fry et al. (1982) Black and Bender (1976); Craig (1954) Parker (1964) Fry et al. (1982) Black and Bender (1976) Fry et al. (1982) Craig (1954) Black and Bender (1976) Fry et al. (1982) Fry et al. (1982)

(continued)

TABLE IIl (Continued) Organism Dinophyceae Glenodinium foliaceum Symbiodinium microadriaticum (from Tridacna maxima) Prymnesiophyceae Coccolithus huxleyi

A'3C

(%o)

Comments

-18.2 -16.3

Seawater, 18°C Seawater; wild

-18.1

Seawater. 18°C: normal strain. coccoliths removed Seawater, 18°C; coccolith-less strain Seawater, 18°C Seawater, 18°C; normal strain, coccoliths removed Seawater, 18°C Seawater, 18°C

-12.5

Coccolithus pelagicus Hymenomonas carterae

-23.9 -10.7

Isochrysis galbana Pavlova (Monochrysis) lutheri

-26.4 -26.1

Reference Wong and Sackett (1978) Black and Bender (1976)

Sikes and Wilbur (1982) Wong and Sackett (1978) I

Sikes and Wilbur (1982) Wong and Sackett (1978) Wong and Sackett (1978)

CARBON METABOLISM IN MARINE ALGAE

I09

carboxylase, and in which CO, fluxes are purely diffusive (cf. Raven et al., 1982). In most marine algae there is independent evidence for HC0,- use and/or a “C0,-concentrating mechanism” (see Section 111) which introduces potentially rate-limiting processes of unknown carbon isotope discrimination into Eq. (8). A most important conclusion is that boundary layer effects, with or without active HC0,- influx, can account for the observed range of A13C and 8l3C values in marine algae without the need to invoke the operation of auxilliary carboxylations of low intrinsic discriminations before RUBISCO fixes the CO, in a “C,-like” mechanism. VIII. P-CARBOXYLASES All plant tissues contain enzymes which can interconvert three-carbon (C,) and four-carbon (C,) acids by carboxylation and decarboxylation (Coombs, 1979). As discussed earlier, certain algae have high capacities for light-independent CO, fixation (Section IV). Since aspartate, glutamate, and malate are strongly labeled during light-independent 14C0, fixation OAA was presumed to be the first labeled product and this has been confirmed by phenylhydrazine trapping experiments in certain marine algae (Akagawa et al., 1972~).Several enzymes can catalyze the addition of CO, to a C, unit to form a C, dicarboxylic acid utilizing either PEP or pyruvate as substrate and these are summarized below: Mn’+

Pyruvate + CO2 + NADPH2 F=== L-malate + NADP L-malate:NADP+ oxidoreductase (“malic enzyme”) (EC 1. I . I .40) Pyruvate

(1 1)

M$+

+ C 0 2 + ATP + H 2 0 F==+ OAA + ADP + Pi Pyruvate carboxylase (PC)(EC 6.4.1.1)

PEP + co2+ H~O%OAA + pi Phosphoenolpyruvate carhxylase (PEPC) (EC 4. I . 1.31) GDP

PEP

(13)

GTP

M“‘+

+ C02 + IDP

(12)

OAA

+ ITP

ADP

(14)

ATP

Phosphoenolpyruvate carboxykinase (PEPCK) (EC 4. I . 1.49) PEP

+ C02 + Pi .Mg’+

(Mn“)

’ OAA

+ PP,

Phosphoenolpyruvate carboxytransphosphorylase (PEPCTrP) (EC 4.1 . 1.38)

It should be noted that, in the above reactions, CO, is not intended to imply that CO, is the actual substrate for carboxylation. Akagawa er ul. (1972~)examined the enzymes responsible for light-independent CO, fixation in certain members of the Phaeophyceae. High activities of a Mn2+- and ADP-dependent CO, fixation were observed which utilized only

TABLE IV p-Carboxylases in Marine Algae K , (mol m-3) with respect to:

Alga Dictyota dictotoma (Phaeophyceae) Spatoglossum pacifcum (Phaeophyceae) Laminaria hyperborea (Phaeophyceae) Ascophyllum nodosum (Phaeophyceae) Phaeodactylum tricornuturn (Bacillariophyceae) Amphidinium carterae (finophy ceae)

Enzyme type

M,

PH (optimum)

PEPCK

-

7.3

PEPCK

-

PEPCK

HCO,

CO,

ADP

0.3

10

-

0.07~

Akagawa et al. (1972~)

-

0.2

10

-

0.080

Akagawa et al. (1972~)

-

7.6

0.23

0.46

1.1~

Kiippers and Weidner (1980)

PEPCK

60,000

7.9

0.036

50

0.87

0.012

Kerby and Evans (1983b)

PEPCK

62,000

6.2

0.048d

7.6

5.7 17.6 0.75

4.8 0.59

340,000

2.2 0.13 Pyruvate 0.13

PC

Kinetic constants determined at pH 7.0. Value given by Kremer (1981~). c Kinetic constants determined with a crude extract. Kinetic constants determined at pH 6.2. c Kinetic constants determined at pH 7.6. a

PEP

5.9

-

Reference

Holdsworth and Bruck 0.005~ (1977) ATP Appleby et al. (1980) 0.03

111

CARBON METABOLISM IN MARINE ALGAE

+

PEP as a substrate. Pyruvate Pi, ATP, and Mg2+ could not replace PEP, ADP, and Mn2 , respectively. It was concluded on this basis that PEPCK [Eq. (14)] was responsible for @-carboxylationsince PC and PEPC activities [Eq. (12) and (13)] could not be detected. However, a low level of “malic enzyme” [Eq. (1 I)] was detected. The PEPCK exchange reaction between the carboxyl group of oxaloacetate and 14C02 in the presence of ATP and Mn2 and the decarboxylation of oxaloacetate were also demonstrated. Kinetic constants for HCO, - , PEP, and ADP were 10, 0.3, and 0.07 mol m-3, respectively, for purified Dictyora dichofoma enzyme in the carboxylating direction (Table IV) and 0.05 and 0.4 mol m P 3 for OAA and ATP in the decarboxylating reaction. HC0,caused a competitive inhibition of the decarboxylating reaction. Other enzymes relating to light-independent CO, fixation were examined and activities of malate dehydrogenase, glutamate oxaloacetate transaminase, and fumarase were reported (Akagawa ef a l . , 1972~).Weidner and Kuppers (1973) reported the presence of PEPCK in another brown alga, Luminaria hyperborea, based on stimulation by ADP and M$+. PEPCK activity was greater than that of RUBISCO, probably reflecting either enzyme inactivation or improper assay conditions (see Section V). Despite these findings and those of Kremer and Willenbrink (1972), which imply the participation of PEPCK in light-independent CO, fixation, the occurrence of a C, pathway operating via PEPC in certain marine algae has been implied (Joshi and Karekar, 1973; Karekar and Joshi, 1973; Joshi er al., 1974). Kremer and Kuppers ( I 977) failed to detect significant activities of PEPC in marine macropbytes belonging to the Chlorophyta, Phaeophyceae, and Rhodophyceae. However, significant activities of PEPCK were found in those algae which had appreciable rates of light-independent CO, fixation and it was concluded that I4C labeling into C, compounds could be explained as a less important, light-independent carboxylation reaction via PEPCK parallel to the operation of RUBISCO (Kremer and Kuppers, 1977). The majority of evidence to suggest that PEPCK is responsible for @-carboxylation in marine algae is based on an ADP stimulation of a PEP-dependent carboxylation (Kremer and Willenbrink, 1972; Weidner and Kuppers, 1973; Kremer and Kuppers, 1977). Stimulation by ADP is insufficient to classify the enzyme as a PEPCK (Davies, 1979) due to reports of PEPC stimulation by nucleotides (Sanwal and Maeba, 1966; Taguchi e f al., 1977; Wong and Davies, 1973). Davies (1979) suggests that the rationale of Utter and Kolenbrander (1972) should be adopted to determine which enzyme is responsible for @carboxylation. This initially involves demonstrating whether the PEP-dependent CO, fixation is not merely stimulated by the presence of ADP or IDP. If the CO, fixation is not nucleotide dependent then it is probably catalyzed by PEPC or PEPCTrP [Eqs. (13) and (15)J. Having demonstrated a nucleotide dependence then PEPCK or PC [Eqs. (12) and (14)] are probably responsible. PC always contains biotin and should be inhibited by preincubation with avidin. This ra+

+

112

N. W. KERBY AND J. A. RAVEN

tionale was adopted to determine the enzyme responsible for P-carboxylation in the brown alga Ascophyllum nodosum (Table V) (Kerby and Evans, 1983a). The enzyme utilized only PEP as a substrate and was dependent on ADP and Mn2 ; enhanced activity was observed when Mg2+ was added to the complete reaction mixture. Preincubation with avidin did not inhibit the reaction and when PEP and ADP were replaced with pyruvate and ATP in the presence and absence of acetyl-CoA no fixation was observed (Table V), indicating the absence of PC. The enzyme was most active with ADP as phosphate acceptor but also responded to IDP at a reduced rate. In extracts of A . nodosum the formation of ATP corresponded to the amount of CO, fixed during PEP carboxylation (Table VI), providing further evidence that the enzyme is PEPCK. Studies by Kremer (1981b) have also shown that ATP is a product of P-carboxylation in other members of the Phaeophyceae, confirming the presence of PEPCK. The A. nodosum enzyme has a molecular weight of 60,000 as determined by gel filtration. After treatment with sodium dodecyl sulfate (SDS) and SDS polyacrylamide gel electrophoresis a single band was observed (M,-60,000), indicating that the enzyme may be monomeric (Kerby and Evans, 1983b) as has been shown for the bacterium Escherichiu coli (Goldie and Sanwal, 1980). Kinetic constants are given in Table IV and the enzyme from A . nodosum displays a high affinity for PEP and ADP but a poor affinity for HCO,- (Km(HC03-) 50 mol rn-,). Since PEPCK has been reported to utilize CO, rather than HC0,- the Km(coz) has been calculated to be 0.87 mol m-,. However, the reactive species +

TABLE V The Substrate and Cofactor Requirement of PEP-Dependent CO, Fixation in A . nodosum Extracts0 Reaction system Complete Complete + MgCI, -PEP -ADP -ADP, +ATP -MnCI, -MnCI,, +MgC12 Complete + acetyl-CoA -PEP, -ADP, +Q, fATP -PEP, -ADP, +Py, +ATP, +acetyl-CoA -PEP, -ADP, -MnC12, +Py, +ATP, fMgCI, -PEP, -ADP, -MnCI,, +Py, +ATP, +MgCI,, -ADP, +IDP -ADP, +CDP -ADP, +GDP

Activity

+ acetyl-CoA

0.251 0.348 0.013 0.015 0.026 0.015 0.036 0.277 0.015 0.015 0.015 0.017 0.052 0.02 0.017

f 0.004

* 0.012

f 0.001 f. 0.0002

f. 0.002

* 0.001 * 0.004 2 0.008 * 0.001 f 0.0002

* 0.0005

2 0.001 f. 0.002

f 0.0002

* 0.0003

a Activities in pmol min- 1 mg- I protein. Results mean of five replicates t standard error (after Kerby and Evans, 1983a).

113

CARBON METABOLISM IN MARINE ALGAE

TABLE VI Activities of Phosphoenolpyruvare Carboxykinase (PEPCK), Glutamate Oxaloucetnte Transurninuse (GOT), and Mnlute Dehydrogenase (MDH) in Ascophyllum nodosum Extractsu I4CO, fixation

ATP production

OAA consumption

GOT

MDH ~~

I 11

0.312 +- 0.012 0.385 0.001

*

*

0.32 0.03 ND

NDb 0.79 5 0.05

ND 0.61

k

0.01

~

ND 9.14 0.32

*

Activities are in pmol product formed or substrate consumed min- I mg-l protein as the mean of five replicates SE. I and 11 were separate extracts which had been passed through a Sephadex G-25 column, after concentration with 80% (NH&SO4 saturation. ND, Not determined (after Kerby and Evans, 1983a).

*

of inorganic carbon fixed via PEPCK in members of the Phaeophyceae is unknown and merits further study. PEPCK is not solely confined to members of the Phaeophyceae. The enzyme responsible for P-carboxylation and the early appearance of label in aspartate in the marine diatom Phaeodactylum tricornutum is also a PEPCK (Holdsworth and Bruck, 1977). Originally the enzyme was thought to be PEPC (Holdsworth and Colbeck, 1976; Beardall et al., 1976; Mukerji and Morris, 1976) but subsequent studies revealed that ATP was a product of P-carboxylation (Holdsworth and Bruck, 1977). The enzyme is similar to those reported above with respect to kinetic constants (Table IV) but has some unusual properties with respect to other PEPCKs from mammalian and bacterial sources. The enzyme reaction apparently lies strongly in the formation of oxaloacetate and favors HCO,- rather than CO, as substrate despite its poor affinity for HCO, - compared to CO, at pH 7.6 (Table IV). Other diatoms (Cylindrotheca closterium and Thalassiosiru pseudonuna) and a Prymnesiophyte (Puvlova lutheri) (Green, 1975) have also been shown to contain PEPCK (Appleby et al., 1980). Another diatom (Chaetoceros culcitrans) as well as the chlorophyte Dunaliella tertiolecta and the rhodophyte Porphyridium cruentum all possessed a PEPC which was stimulated by Mn2+ and inhibited by malate, aspartate, and ADP (Appleby et al., 1980). PEPC has so far not been purified or characterized from marine algae. Two dinoflagellates (Amphidinium carterae and a Gymnodinium sp.) have been shown to contain PC which had not previously been detected in plants (Appleby et al., 1980). The enzyme was characterized as containing biotin and was therefore inhibited by avidin. Unlike PEPC and PEPCK the enzyme was inhibited by 5 mol m-, MnCl,. The molecular weight was found to be over 300,000 and the kinetic constants are given in Table IV. Acetyl-CoA did not activate the enzyme in the range of 0.1-1 mol m-,. Since different enzymes have been shown to be responsible for P-carboxylation within marine algae more axenic cultures of phytoplankton are required for enzyme screening to determine whether the type of enzyme present is of taxonomic significance.

114

N. W. KERBY AND J. A . RAVEN

IX. C, METABOLISM IN THE PHAEOPHYCEAE Higher rates of light-independent CO, fixation are usually associated with young developing tissue as compared to older, fully developed tissue in members of the Phaeophyceae (Willenbrink et al., 1975, 1979; Kuppers and Kremer, 1978; Kremer, 1979, 1981~).Consistent with this, maximum PEPCK activity has been localized in young growing regions of members of the Laminariales and Fucales (Weidner and Kuppers, 1973; Kuppers and Kremer, 1978), whereas maximum RUBISCO activities are localized in fully differentiated regions of the thallus. Enzyme activities vary seasonally (Kuppers and Weidner, 1980) as do the rates of light-independent CO, fixation (Willenbrink et a f . , 1979). A maximum of enzymatic activities is achieved during April and May in Laminaria hyperborea. A consequence of this would be that young developing tissue can obtain a high metabolic activity from early spring onward when the water temperature has only risen slightly. Since the PCRC (Fig. 1) is the only pathway capable of regenerating substrate for carbon fixation it is interesting to consider the source and availability of substrate for P-carboxylation. Labeled aspartate formed after 10 sec light fixation contains 31% of its label in the P-carboxyl group in the diatom Phaeodactylum tricornutum (Holdsworth and Bruck, 1976). Therefore, since the substrate for P-carboxylation is PEP, one must assume that the remaining 69% of the label found in the a-carbon of aspartate has been derived from PGA, the primary product of photosynthesis. Similar findings have been made for members of the Phaeophyceae during I4C light labeling experiments (Kremer, 1981a). However, during dark CO, fixation low levels of label were detected in the a-carbon atom of aspartate which suggests that PEP must be provided by an alternative metabolic pathway, possibly provided by the degradation of reserve material (Kremer, 1981b). It has been shown that mannitol, a reserve material in the Phaeophyceae, is transported to young developing tissue in Laminaria species (Schmitz et al., 1972) and therefore the substrate of P-carboxylation in the dark can thus be provided if sufficient mannitol from either storage pools or from long distance translocation is available. Growth of certain brown algae, such as Laminaria hyperborea, begins during a season when the photon flux density in the sublittoral zone exceeds the compensation point for only a few hours daily (Liining, 1971). Growth measurements have shown that the new developing region is dependent on carbon compounds translocated from mature regions (Luning, 1969; Schmitz et al., 1972; Luning et al., 1973). Light-independent carbon fixation may be of importance in providing anaplerotic reactions in tissue the carbon requirements of which are supplied by translocation from older regions of the plant. However, translocation of reserve materials in other members of the Phaeophyceae, such as members of the Fucales, which have significant rates of light-independent CO, fixation, has yet to be established. Since, theoretically, 1 mol of mannitol yields 2 mol of PEP and could there-

115

CARBON METABOLISM IN MARINE ALGAE

fore result in the refixation of 2 mol of CO, it has been suggested that nonphotosynthetic CO, fixation be included into calculations of primary productivity (Kremer, 1981b). However, before this step is taken more detailed studies on the fate of the C, products of P-carboxylation is required. I4C labeling of ethanolinsoluble compounds has been reported after dark CO, fixation (Craigie, 1963; Willenbrink et al., 1979) but the amount appears to be low [2% of the label in insoluble material after 30 min dark fixation (Kremer, 1979)l. No attempts have been made to characterize these insoluble products. There is preliminary evidence that carbon fixed in the dark may be released during the light period (Johnston, 1984). Ascophyllum nodosum exhibits similar but less pronounced CAM characteristics (see Section 111). In the dark the acidity (free proton activity) of this alga increases and decreases with the onset of a light period (Fig. 3). These changes have been quantified by measuring the pH of ground material, and the titratable acidity (Figs. 3 and 4). At the end of a dark period the pH was 6.1 and the titratable acidity was 13 pmol H (g fresh wt)- I ; at the end of the following light period the pH rose to 6.58 and the titrable acidity fell to 6.5 pmol H+ (g fresh wt)-’. Measured changes in the malate concentration were great enough to account for these changes in titratable acidity. Certain members of the Phaeophyceae have been shown to contain “malic enzyme” (Akagawa et al., 1972c), which could account for the decarboxylation of malate directly. Bidwell (1967) showed that the labeled amino acid pool (the main product of light-independent CO, fixation) increased in the dark and decreased in the light. The possibility that there are changes in the concentration of amino acids over a light-dark cycle is currently under investigation. +

6,0[ 8 6.1

6.3 6.4 I a

6.6 6.5

28+6’

0

6.7I 8

I

I

10

12

1

I

14

16

18

I

I

20

22

Time o f Day (hr) ~////////~///////////~/

7 /

LIGHT

DARK

Fig. 3. The change in pH of aqueous extracts of Ascophyllum nodosum during light and dark regimes. (Johnston, 1984.)

N. W. KERBY AND J. A . RAVEN

116

* 0

1L

f 12

y f

10

L

- 8

Y P

55

6

I--

a

: - 2

8

L

1

I

I

I

1

I

10

12

14

16

18

20

22

Time of Day (hr)

LIGHT

DARK

Fig. 4. Titratable acidity of extracts of Ascophyllum nodosum in pmol H + (g fresh wt)light and dark regimes. (Johnston, 1984.)

I

during

X. CONCLUSIONS The analysis of the mechanism of inorganic carbon transport in marine algae is fraught with difficulties. In many cases the rate of inorganic carbon fixation at low inorganic concentration implies, if CO, diffusion is the mechanism of carbon entry, a very small unstirred layer thickness. More realistic aqueous unstirred layer thicknesses arise from the assumption that HC0,- is actively transported at the plasmalemma and/or the plastid envelope. The capacity to fix carbon at high pH values and the partial suppression of RuBPo activity are consistent with such a transport. When the characteristics consistent with partial suppression of RuBPo activity are found in algae exposed to air, including those algae which are not normally covered by only a thin layer of water, the normal recourse is to assume active HCO, - influx at the plastid envelope. However, it is not possible to use this explanation for cyanobacteria. It is almost certain that all RUBISCOs (including those from marine algae) have RuBPo as well as RuBPc activity. From the known in v i m range of affinities and substrate-saturated rates during CO, and 0, fixation by RUBISCOs from a variety of sources it is clear that many marine algae show lower in vivo RuBPo activity than expected on the basis of diffusive entry of CO, and loss of 0,, and the bulk phase concentrations of CO, and 0,. Evidence for the suppression of RuBPo activity in marine algae comes from measurements of the (low) CO, compensation concentration, the (low) 0, inhibition of net photosynthesis, and the (low) rates of CO, production in the light. However, some RuBPo and PCOC activity is

CARBON METABOLISM IN MARINE ALGAE

117

suggested by rapid labeling of glycolate, glycine, and serine and occurs in most marine algae. This evidence for suppression of RuBPo activity in vivo is consistent with the data, discussed above, for “C0,-concentrating mechanisms” in marine algae. Before the problem of the mechanism of inorganic carbon influx can be resolved we need to know much more about the properties and quantity of RUBISCO from marine algae, as well as better controlled experiments on the kinetics of inorganic carbon assimilation by whole cells and tissue and, it is hoped, by isolated chloroplasts. Central to these investigations will be further attempts to estimate intracellular concentrations of inorganic carbon species in marine algae. More quantitative data on in vitro RUBISCO kinetics from marine algae together with the occurrence of RuBPo and PCOC in vivo are also required to establish the magnitude, and taxonomic range, of “C0,-concentrating mechanism. Until more is known about the various reactions in vivo which contribute to carbon isotope discrimination in aquatic plants and in particular the isotope discrimination characteristics of HCO, - transmembrane transport, this potentially useful technique cannot be fully exploited. Clearly most St3Cvalues for marine algae are consistent with either limitation by CO, diffusion or by a lowdiscrimination HC0,- active transport process. In a few cases, however, no such low-discrimination process seems to be limiting. It should be noted that a high (more positive) St3Cin aquatic plants is not necessarily a good indicator of C, metabolism or CAM. Certain marine algae have high potentials for light-independent CO, fixation, with P-carboxylases being responsible. They do not, however, possess a C,-type photosynthesis. A range of P-carboxylases appears to be responsible for lightindependent CO, fixation in different species of marine algae and it would be of interest to know the taxonomic range of individual enzymes. The magnitude and role of C, metabolism in marine algae has yet to be clearly elucidated. The importance of PEPCK, particularly in members of the Phaeophyceae, may be clarified by the use of specific inhibitors in vivo such as mercaptopicolinic acid [a known inhibitor of Ascophyllum nodosum PEPCK activity in vitro (Kerby and Evans, 1983a)l. As suggested earlier a low 0, inhibition of photosynthesis is more probably equated with “C0,-concentrating mechanisms” rather than with the occurrence of a particular P-carboxylase as has been suggested by Krerner (1981~).More detailed studies are required on the kinetics of in vitro P-carboxylases and the species of inorganic carbon which serves as the substrate. PEPCK is the least favorable plant carboxylating enzyme when compared to RUBISCO or PEPC. Higher affinities of PEPCK for CO, may be revealed, as was found for RUBISCO (e.g., see Lorimer, 1981) with these further studies. Further studies are also required on the kinetics of in vivo light-independent CO, fixation together with the ultimate fate of products, before light-independent CO, fixation can be included in primary productivity measurements. ”

118

N. W. KERBY AND I. A. RAVEN

ACKNOWLEDGMENTS N.W.K. wishes to acknowledge financial support from the Agricultural and Food Research Council. We thank Mr. A. M. Johnston for permission to quote his unpublished data.

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Cell Wall Storage Carbohydrates in Seeds-Biochemistry of the Seed “Gums” and L6Hemicelluloses”

J. S. GRANT REID Department of Biological Science University of Stirling Stirling, Scotland

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Structures of Cell Wall Storage Carbohydrates in Seeds ........................ A. Background Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Structural Types and Their Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Formation and Postgerminative Catabolism ......... ..... ...... A. Galactomannan Metabolism in Leguminous Seeds .......................... B. Mannan Mobilization in the Date Endosperm ........................ C. Xyloglucan Metabolism in Tropaeolum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. “Galactan” Metabolism in Lupinus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1V. Considerations of Biological Function . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . V. Perspectives ....................... .................................. .................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION By the late nineteenth century it was clearly recognized by botanists that the massively thickened cell walls present in many seeds contained reserve substances. Reiss (1889) and others described “reserve celluloses” which were utilized following germination, while Tschirch (1 889) and Nadelmann (1 890) were able to demonstrate that the “mucilages” in the endosperm cell walls of some leguminous seeds had a storage function. Schleiden (quoted by Vogel and ADVANCES IN BOTANICAL RESEARCH. VOL. I I

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Schleiden, 1839) reported that the thickened cell walls of some seeds could be stained blue with iodine, and he named the substance responsible for the starchlike reaction “amyloid.” Amyloids were later shown to occur widely in seeds (Winterstein, 1893; Kooiman, 1960a) and to be mobilized following germination (Godfrin, 1884). The carbohydrate nature of the cell wall reserves of seeds was inferred from their microchemical staining reactions, and proven by the positive identification of sugars released from them on acid hydrolysis. For example, the reserve cellulose of the ivory “nut,” Phytelephas macrocarpa, released “seminose” or mannose (Reiss, 1889); the mucilage of the locust “bean,” Ceratonia siliqua, gave mannose and galactose (Bourquelot and Herissey, 1899), and the amyloid of the nasturtium seed, Tropaeofummajus, yielded glucose, xylose, and galactose (Winterstein, 1893). The combined ultrastructural, physiological, and “biochemical” approach which many of the early botanists adopted to study the cell wall storage carbohydrates of seeds was extraordinarily effective. It is unfortunate that it was not carried forward with vigor into the twentieth century. The mid-twentieth century (1930- 1970) saw the introduction of a series of new techniques for the determination of the structures of complex carbohydrates (Whistler and Wolfrom, 1965; Whistler and Bemiller, 1972) and most of our present knowledge of the structures of cell wall storage carbohydrates was obtained during that period. Seeds were generally treated with alkali to extract polysaccharides of the “hemicellulose” type or with water to extract “gum” polysaccharides. Consequently the molecules with which this article is concerned are still widely classified as seed gums and hemicelluloses. In recent years (from about 1970) there has been a reawakening of interest in the physiology and biochemistry of the cell wall storage carbohydrates of seeds, and a recent review article has treated them for the first time as a single, botanically coherent group of substances (Meier and Reid, 1982). It is the purpose of this article to outline the structures and occurrence of cell wall storage carbohydrates, to give an account of current research on their metabolism, and to explore their overall biological significance in the seeds which contain them. 11. STRUCTURES OF CELL WALL STORAGE CARBOHYDRATES IN SEEDS This section indicates the principal types of carbohydrate molecules stored in the cell walls of seeds, and their distribution. To allow the assessment of the status of published structural data of various kinds the section is prefaced by a brief resum6 of the methods which have been used to determine these structures, and their limitations. A. BACKGROUND METHODOLOGY

The determination of the primary structure of a cell wall polysaccharide normally involves the following procedures:

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1. Isolation of polysaccharide material from the plant tissue 2. Purification to a degree of homogeneity acceptable for structural determination 3. Total hydrolysis to release the constituent monosaccharides for qualitative and quantitative analysis 4. Determination of molecular weight, or degree of polymerization 5. Linkage analysis to determine linkage types ( 1 += 2, 1 + 3, etc.), linkage modes (a or p), ring sizes (pyranose or furanose), and to obtain some information concerning the distribution or ordering of monosaccharide residues within the molecule The isolation of cell wall polysaccharides from seeds has normally been carried out by treating the tissue with a solvent, usually hot water or dilute alkali. There may be a pretreatment to remove lipids and/or low-molecular-weight carbohydrates or to inactivate enzymes. Hot water is unlikely to cause extensive degradation of carbohydrate macromolecules, but it can bring about irreversible changes in their noncovalent interactions. (Once gelatinized and partially solubilized, a starch granule cannot be reconstituted.) Alkali is potentially degradative. In the presence of oxygen it can bring about the sequential oxidative cleavage of monosaccharide residues from the reducing end of the molecule (Whistler and Bemiller, 1958). Alkaline oxidation of this type can be avoided (Aspinall et al., 1961), but other types of alkaline modification are inevitable. Substituents bound by ester linkage can be cleaved or can migrate (Bouveng et al., 1960), p-elimination reactions can occur at uronic acid residues, and nonglycosidic linkages between wall components may be broken. It must also be borne in mind that polysaccharides are generally polydisperse (they span a range of molecular weights) and polymolecular (they encompass a limited range of molecular structures). Incomplete extraction from the tissue can, therefore, cause unwanted fractionation: the polysaccharide material passing into solution need not be identical in molecular weight and/or structure with that which is left behind in the tissue. Once extracted and isolated, polysaccharide preparations may be purified, usually by fractional or selective precipitation by organic solvents or metal ions (Whistler and Woifrom, 1965). These methods are simple to carry out, but they suffer from two disadvantages. Insufficient purification may lead to structural studies being carried out on a mixture of distinct molecular types while overzealous purification may subfractionate a polydisperse and polymolecular native polysaccharide. The complete hydrolysis of polysaccharides is effected by acids. Different workers routinely use different hydrolysis conditions-for example, 72% H,SO, at 30°C followed by 4% H,SO, at 120°C; 0.5 M H,SO, at 100°C; 1 M trifluoroacetic acid at 121°C (Whistler and Wolfrom, 1965; Albersheim er al., 1967). Different glycosidic linkages differ greatly in their susceptibility to acid hydrolysis, while the monosaccharides released differ greatly with regard to their

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stability in an acidic environment. Acidic sugar residues (uronic acids) present a particularly serious problem. When they are involved in a glycosidic linkage they often stabilize it, yet once released into an acid medium they are highly unstable. There is no fully satisfactory method of determining the uronic acid residues in a polysaccharide quantitatively. Ideally, published data on the composition of polysaccharides should be corrected for losses during the hydrolysis procedure; in practice this is seldom done. Molecular weight determinations may be carried out by physical techniques or by chemical (end-group) analyses. The former are often subject to errors arising from the self-association of polysaccharide molecules in solution, while the latter may be subject to disproportionate interference from low-molecular-weight, partially degraded material. At the heart of any linkage analysis of a polysaccharide there is a methylation analysis (Whistler and Wolfrom, 1965). The entire molecule is subjected to a procedure to convert all free hydroxyl groups to methyl ethers, and the permethylated product is then hydrolyzed with acid to give a mixture of partially methylated monosaccharides which can be derivatized, separated, and identified. This reveals which hydroxyl groups were involved in glycosidic linkage within the intact macromolecule provided that the original methylation was complete and that no extensive demethylation took place during the subsequent hydrolysis procedure. Methylation analysis gives no information on linkage modes (aor p), nor does it provide much information on the order of residues in a polysaccharide. Additional information can be obtained from selective cleavage of particular types of glycosidic linkages by acids or enzymes (if available). On the basis of methylation analyses and associated techniques, “structures” may be postulated for the molecule. These can be tested by periodate oxidation techniques (Whistler and Wolfrom, 1965) since periodate ion, IO,-, cleaves 1,2 diols in a stoichiometric reaction to give predictable products. The application of the above methods will give a “structure” for a polysaccharide preparation, but it must be understood that such structures are not necessarily definitive. They are, of course, only average structures, but they will also to some extent reflect the method and completeness of extraction, the degree of purification, the methods of hydrolysis and quantitative analysis of sugars, the method used to determine the molecular weight, and the procedures used in the methylation analysis. This should be borne in mind, particularly when comparing polysaccharide preparations. B . STRUCTURAL TYPES AND THEIR DISTRIBUTION

Full structural studies have been carried out on polysaccharides isolated from the seeds of relatively few species. Nevertheless, on the basis of these and of numerous more limited investigations, it is possible to discern several major types of cell wall storage carbohydrate molecules: the mannan group of polysaccharides,

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the xyloglucans, and a galactose- and arabinose-rich class which for convenience will be referred to here as the “galactuns.” 1. The Mannun Group The mannan group comprises three distinct structural types: the “pure” mannans, the glucomannans, and the galactomannans. They are structurally related in that all three are based on 1 -+4, P-linked D-mannopyranose residues. They are distributionally related also, in that they are found only in seed endosperms as opposed to storage cotyledons or axes. “Pure” mannans, that is polysaccharides yielding over 90% mannose on hydrolysis, have been obtained from the seed endosperms of two palms, Phoenix dactylifera, the date palm, and Phytelephas macrocarpa, the ivory nut tree, and their structures have been subjected to thorough investigation over a long period (Ludtke, 1927; Klages, 1934; Aspinall et al., 1953, 1958; Meier, 1958). Both seeds have yielded two mannans (A and B) differing in their solubilities in alkali and cuprammonium solutions. All four polysaccharides have similar structures: a linear 1 -+ 4, @-linkedD-mannan backbone carries a small proportion (less than 2%) of single-unit a-D-galactopyranosyl substituents linked 1 + 6 to mannose. The mannans A and B differ in molecular weight (Meier, 1958) but it is not clear whether they are two functionally distinct molecular species or subfractions of a polydisperse native mannan. Meier’s (1958) observation that the mannans A give an X-ray diffraction pattern in the native state and that mannans B do not suggests that they are distinct. Mannan preparations from the endosperms of three other palm seeds have been investigated in less detail, but they are clearly similar in structure to the mannans of ivory nuts and dates (Mukherjee and Rao, 1962; El Khadem and Sallarn, 1967; Robic and Percheron, 1973). Yet other palm seeds are known to have endosperms which release D-mannose on hydrolysis (Lienard, 1902, cited by Herissey, 1903). It seems reasonable to assume that the cell wall storage carbohydrates in the hard endosperms of all palm seeds are mannans. Mannans with structures similar to those of the date and the ivory nut have been obtained by alkali extraction of coffee beans (Co#eu arabica) (Wolfrom et al., 1961) and of the endosperm of the seed of the umbellifer Carum r a n i (Hopf and Kandler, 1977). The endosperms of other umbelliferous seeds are known to contain reserve celluloses (Hegnauer, 1973) which are probably also mannans. Glucomannans have been obtained by alkali extraction of the seeds of Asparagus oficinalis (Goldberg, 1969; Jakimow-Barras, 1973), Endymion nutans (Goldberg, 1969), Scilla nonscripta (Thomson and Jones, 1964), Iris ochroleucu, and I . sibirica (Andrews et al., 1953). All have been subjected to structural investigation, including methylation analysis, and all are similarly constituted. A linear 1 -+ 4, P-linked backbone contains almost equal numbers of Dglucopyranosyl and D-mannopyranosyl residues. Their distribution is uncertain, and could be random. To the backbone is attached a small percentage ( 3 to 6%)

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of single-unit o-galactopyranosyl branches attached 1 + 6, probably by a linkages (Goldberg, 1969). All of the above species are from the Iridaceae and Liliaceae, other species of which are known to have seeds which are rich in mannose- and glucose-containing polysaccharides (Jakimow-Barras, 1973) or which have thick-walled endosperms (Elfert, 1894). Glucomannans may be characteristic of the hard endosperm of seeds from these families. Galactomannuns are the best characterized of all the cell wall storage carbohydrates of seeds (Whistler and Smart, 1953; Smith and Montgomery, 1959; Stepanenko, 1960; Dea and Morrison, 1975), a fact which reflects the industrial importance of some seed galactomannans (see Glicksman, 1953; Carlson et al., 1962; Saxena, 1965; Chudzikowski, 1971; Kovacs, 1973; Nurnberg and Rettig, 1974, for examples of their uses). The galactomannans are typical of the leguminous seed endosperm (Anderson, 1949) and they can be completely extracted from isolated endosperms or seed tissue with hot water. Numerous leguminous seed galactomannans have been subjected to full structural analysis, and they conform to a common structural type. A 1 44, P-linked D-mannan backbone is heavily substituted by single-unit a-D-galactopyranosyl side chains linked 1 46 to mannose. The degree of galactose substitution in galactomannans varies from about 20 to nearly 100% and is apparently genetically controlled and chemotaxonomically useful (Reid and Meier, 1970; Kooiman, 1971; Campbell, 1978). The lower degrees of substitution are characteristic of the LeguminosaeCaesalpinioideae, which are generally held to be more primitive than the Leguminosae-Faboideae. Only one endospermic leguminous seed has so far been found to contain a storage polysaccharide other than galactomannan. The seed of the Judas tree (Cercis siliquastrum: Leguminosae-Caesalpinioideae)contains a galactoglucomannan; its structure is similar to those of the galactomannans but the backbone contains D-glucopyranose residues (McCleary el al., 1976). The only nonleguminous species whose mature seeds contain galactomannans are from the Convolvulaceae (Khanna and Gupta, 1977; Kooiman, 1971). Interestingly, however, galactomannans have been isolated from the immature seeds of palm species which contain “pure” mannans at maturity (Mukherjee et al., 1961; Kooiman, 1971; Balasubramaniam, 1976). A developmental relationship between galactomannan and mannan in the palm seed endosperm has been suggested (Balasubramaniam, 1976), but has not been investigated experimentally. The mannans, the glucomannans, and the galactomannans are clearly related structurally, but in their physical properties the mannans and glucomannans differ considerably from the galactomannans. The former are almost completely insoluble in water by virtue of their cellulose-like structures, and endosperms that contain them are hard. In the cell wall they may be crystalline (Kooiman, 1960b). The highly substituted galactomannans are water soluble: presumably the galactosyl substituents effectively prevent the self-association of the main chain to give crystalline aggregates. In their properties, the mannans and

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glucomannans are ‘‘reserve celluloses” whereas the galactomannans are ‘‘seed mucilages.” 2 . The Xyloglucans The xyloglucans are amyloids, that is they can be stained blue with iodine both in situ and when extracted from the cell wall. The physical basis of the iodine coloration is not known, but it has been exploited both to survey seeds for the presence of amyloid (Kooiman, I960a) and to analyze xyloglucans quantitatively (Kooiman, 1960b). Kooiman (1960a) carried out iodine staining on endosperm and/or embryo tissue from seeds of over 2600 species and found 230 of them to be amyloid positive. In the Leguminosae-Caesalpinioideae, amyloid was restricted to the nonendospermic tribes Cynometreae, Amherstieae, and Sclerolobieae. All investigated species of the Primulales, Annonaceae, Limnanthaceae, Melianthaceae, Pedaliaceae, Thunbergiaceae, and Tropaeolaceae contained amyloid as did a number of species of Balsaminaceae, Acanthaceae, Leguminosae-Faboideae (all amyloid-positive species were nonendosperrnic), Linaceae, Ranunculaceae, Sapindaceae, and Sapotaceae. Detailed structural analyses have been carried out on “amyloids” extracted with alkali from seeds of only 4 of the 230 species, namely: Tamarindus indica (Leguminosae-Caesalpinioideae) (Kooiman, 1961), Tropueolum majus (Tropaeolaceae) (Le Dizet, 1972), Impatiens balsamina (Balsaminaceae) (Courtois and Le Dizet, 1974), and Annona muricuta (Annonaceae) (Kooiman, 1967). All have similar structures: a linear 1 + 4, P-linked glucan (cellulosic) backbone carrying substituents of two types, a-D-xylopyranosyl and P-D-galactopyranosyl(1- 2) a-D-xylopyranosyl. Both types of side chain are attached to C-6 of D-glucose. Methylation analysis has indicated that there may be some branching of the backbone and that (1 + 3) linkages may be present (Courtois et al., 1976). These results could arise from contaminating D-glucans. The ratio ga1actose:xylose:glucose is 1:2:3 in the xyloglucans isolated from Tamarindus and Tropaeolum seeds, 1 :2:4-5 in that from Impatiens, and 1:1:4 in the polysaccharide from Annona. It is not clear to what extent these differences reflect differences in methods of isolation, purification, and structure determination; nor is it clear how wide a variation in seed xyloglucan structures may yet be encountered. Even the assumption that all amyloids are xyloglucans need not necessarily be valid. 3. The Galactans The cotyledon cell walls of several lupins, notably the agriculturally important species Lupinus angustifolius, L. albus. and L. luteus, are massively thickened. Schulze and Steiger ( I 889) demonstrated that the cell wall material of L . luteus released D-galactose and a pentose on hydrolysis and named it “Paragalaktan. ” The total cell wall polysaccharides of L. angustifolius cv. Unicrop release galactose (76%) arabinose (13%), xylose (4%) and a uronic acid (7%) on hydrolysis (Crawshaw and Reid, 1984) while isolated walls have a similar composition

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(Hutcheon and Reid, unpublished). Linkage analysis of the isolated walls is underway. A water-soluble 1 + 4, P-linked D-galactan has been isolated from the cotyledons of L . albus in very low yield and using isolation techniques which would certainly lead to extensive degradation of polysaccharides (Hirst et a l . , 1947). This “lupin galactan” is clearly not representative of the water-insoluble native wall, but it indicates the presence within it of a 1 + 4, P-linked galactan component. Methylation analysis of the native walls confirms that the main galactosidic linkage is 1 + 4 (Wilkie, Reid, and Hutcheon, unpublished). 4 . Other Cell Wall Storage Carbohydrates The cell wall storage carbohydrates of so few species have been subjected to any chemical investigation that the list of major structural types given here can scarcely be considered complete. Further structural studies are necessary. Valuable ultrastructural and physiological studies have even been carried out on the postgerminative mobilization of cell wall storage carbohydrates of unknown or partially known structure. The perisperm of Yucca, for example, contains deposits of cell wall carbohydrate, the mobilization of which has been carefully documented (Homer and Arnott, 1966); yet their structure has not been determined. Similarly the mobilization of the cell wall storage carbohydrates in the endosperm of lettuce (Lactuca sativa) has been observed (Jones, 1974), one of the enzymes responsible has been identified (Halmer et a l . , 1978), and the control of the mobilization process has been investigated (Halmer and Bewley, 1979). Yet it is not clear whether the storage carbohydrate in the lettuce endosperm is a “pure” mannan, a glucomannan, a galactomannan, or some intermediate type. The control of cell wall storage carbohydrate metabolism following germination has also been studied in the coffee bean (Takaki and Dietrich, 1979) although so far only a small proportion of the cell wall material has been positively identified as “pure” mannan (Wolfrom et al., 1961; Wolfrom and Patin, 1965). The structures of quantitatively minor deposits of cell wall storage carbohydrates have received little attention, with the notable exception of the cell wall carbohydrates present in the endosperms of the commercially important cereal grains, alongside massive deposits of starch. These “cereal gums” will not be considered here (but see Meier and Reid, 1982). 111. FORMATION AND POSTGERMINATIVE

CATABOLISM This section deals in detail with a limited number of systems: galactomannan metabolism in leguminous seeds, mannan mobilization in dates, xyloglucan breakdown in nasturtium seeds (Tropaeolum majus), and “galactan” catabolism in Lupinus. All of them were first studied in the late nineteenth century and are being reinvestigated now.

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A. GALACTOMANNAN METABOLISM IN LEGIJMINOUS SEEDS

The occurrence, the formation, and the mobilization of leguminous seed galactomannans were first investigated by Nadelmann (1890). In his scholarly dissertation “Ueber die Schleimendosperme der Leguminosen” he documents the morphology of “mucilage” storage in the endosperms of several seeds chosen to represent most of the endospermic tribes in the Leguminosae. He also describes mucilage deposition during seed development in the endosperm cell walls of Trigonella foenurngraecurn, Colutea brevialata, and Tetragonolobus purpureus, and delineates the process of mucilage mobilization following germination in Trigonella foenumgraecum and Tetragonolobus purpureus. Although Nadelmann was not aware of the chemical nature of his seed “mucilages,” it was he who established a storage role for the galactomannans. In recent years there has been renewed interest in the biochemistry and physiology of galactomannan mobilization. The overall morphology and physiology of the process has been studied in two seed systems, fenugreek (Trigonella foenurngraecurn) and carob (Ceratonia siliqua), which exemplify the two extremes of galactomannan structure. The fenugreek seed contains a highly galactose-substituted galactomannan, while the galactomannan of carob is representative of the comparatively low-galactose galactomannans of the LeguminosaeCaesalpinioideae (see Section II,B, I ) . The enzymology of galactomannan degra-

Fig. 1. Cryostat sections of mature, imbibed fenugreek seeds stained with periodic acid-Schiff‘s reagent to reveal periodate-reactive polysaccharides. A, Axis; Al, aleurone layer; C, cotyledons; E, endosperm; T, part of testa. The intense staining in the endosperm is due to galactomannan. (After Reid and Bewley, 1979.)

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dation has been most fully studied with respect to the guar seed (Cyamopsis tetragonoloba), which contains a galactomannan of intermediate galactose substitution. Galactomannan formation has so far been studied only in developing fenugreek seeds. 1. Galactomannan Formation and Mobilization in the

Fenugreek Seed The mature fenugreek seed contains about 30% by weight of a high-galactose galactomannan (mannose : galactose = 53 : 47) which is localized in the endosperm. Most of the endosperm cells appear to be completely filled with the polysaccharide (Fig. 1) and must be considered to be nonliving; the only living cells in the endosperm are those of the one-cell-thick aleurone layer which surrounds the storage tissue (Reid and Meier, 1972). Studies of endosperm development have confirmed that the galactomannan which “fills” the storage cells is a cell wall polysaccharide, and germination studies have established a key role for the aleurone layer during galactomannan degradation. a. Galactomannan Formation during Endosperm Development. The time course of galactomannan deposition in the developing endosperm is shown in Fig. 2, while Figs. 3 and 4 illustrate the morphological changes which occur in the endosperm during that period. Galactomannan is deposited in the form of secondary thickenings on the cell walls of the endosperm, and the deposition continues until the galactomannan occupies the whole volume of the cell. Cytoplasm and vacuole disappear and are replaced by a mass of galactomannan. Galactomannan deposition is a cell-by-cell process; the endosperm cells next to

Fig. 2. Time course of galactomannan (0) and stachyose fenugreek seeds. (After Campbell and Reid, 1982.)

(a) accumulation

in developing

Figs. 3 and 4. Cryostat sections of a fenugreek seed nearing completion of galactomannan deposition in the endosperm: sections examined by Nomarski interference contrast. Al, Aleurone layer; C, cotyledon; G ,galactornannan; PW, primary wall; T, part of testa. The endosperm cells nearest the cotyledons are already “filled” with galactomannan (Fig. 3) while the outermost cells, nearest the aleurone layer, are still depositing galactomannan (Fig. 4).

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the embryo are the first to be “filled” (Fig. 3) while those adjacent to the aleurone layer are last (Fig. 4).The mechanism which protects the aleurone cells from being “filled” with galactomannan is not understood; some galactomannan-like material is laid down on these cells but this deposition soon ceases (Meier and Reid, 1977). The galactomannan seems to be formed initially in the intracisternal space or “enchylema” of the rough endoplasmic reticulum (RER). which swells greatly and stains for periodate-reactive carbohydrate. The enchylema swells to such an extent that the cytoplasmic lamellae between the ER cisternae become pinched off to give “inside out” RER vesicles, with the ribosomes on the “inside” (poculiform ER). Where the enchylema makes contact with the plasmalemma its contents appear to be discharged into the growing wall space (Meier and Reid, 1977).

Fig. 5 . Electron micrograph of a cell almost “filled” with galactomannan ( G ) . Irregularly distributed residues of protoplasm (RP) enclose small pockets of galactomannan. Section contrasted with periodic acid, thiocarbohydrazide, and silver proteinate. (After Meier and Reid, 1977.)

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Days after onthesis

Fig. 6 . Incorporation of label from GDP-D-[U- ‘‘C]mannose into galactomannan by whole homogenates prepared from developing fenugreek seed endosperms. (Campbell and Reid, 1982.)

The degradative processes leading to the disappearance of the cytoplasm and its organelles have not yet been investigated. Some cytoplasmic material may even be sloughed off and lost into the growing cell wall (Fig. 5). It is interesting to observe that the final remnants of cytoplasm in a cell nearing the completion of galactomannan deposition remain in contact with neighboring cells, via plasmodesmata (Meier and Reid, 1977). This may be the route of cell to cell transport of cytoplasmic degradation products, which may themselves serve as substrates for galactomannan formation. During the period of galactomannan deposition (Fig. 2), the endosperm contains high levels of an enzymatic activity which catalyzes the transfer of Dmannosyl residues from GDP-D-[U-14C]mannoseto a soluble product which has been shown to be galactomannan (Campbell and Reid, 1982). Enzyme activities transferring o-galactosyl units are also present but they have not yet been thoroughly investigated. The GDPmannose : galactomannan mannosyltransferase activity peaks twice during galactomannan deposition; once at the beginning of the deposition and once at the height of galactomannan accumulation (Fig. 6 ) . The early peak corresponds largely to light particulate material ( 100,000g pellet) while the latter peak consists mainly of grossly particulate material (Fig. 7). It is possible that the early peak largely represents transferase activity still associated with endoplasmic reticulum, while the later peak is almost certainly enzyme associated with, or occluded within, large particles of galactomannan. The light particulate enzyme of the early peak sediments with ER markers and has a density of 1.06 g ml-’ (Campbell, 1978). It requires divalent metal ions for activity (Table I). The enomlous stimulation of the enzyme activity by Co2+ and Ni2+ may be due to their interaction with the galactomannan product rather than the enzyme. Both

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&&

Fig. 7. Incorporation of label from GDP-~-[U-"+C]mannose into galactomannan by enzyme preparations, obtained by differential centrifugation of endosperm homogenates. 0-0, Gross particulate enzyme (1000 g pellet); @-a,particulate enzyme (100,000 g pellet); 0.0, soluble enzyme (100,000 g supernatant). (Campbell and Reid, 1982.)

ions are known to form insoluble complexes with galactomannans (Campbell and Reid, 1982). The natural cofactor of the enzyme is probably Mg2+. b. Galactomannan Mobilization following Germination. The breakdown of galactomannan in the endosperm of the fenugreek seed begins 16 hr after germination, and is complete in a further period of 24 hr (Reid, 1971; Reid and TABLE I Effect of Cations on the Particulate GDPmannose:palactomannan Mannosyltransfera& of the Fenugreek Endosperm0 Cation None

Mg2 + Mn2 +

cu2+ Ca2 + co2+

K+

Relative incorporation level

1 23 348 9 24 524 2

" All cations were 10 mM in the assay. For details see Campbell and Reid (1982). (After Campbell, 1978.)

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Bewley, 1979). During this time mannose, galactose, and traces of mannooligosaccharides can be detected in the endosperm, but they do not accumulate to any extent. The galactornannan breakdown products are rapidly absorbed by the embryo and converted to sucrose and starch (Reid, 1971). If endosperm halves are isolated from dry fenugreek seeds and incubated under “germination” conditions, galactomannan breakdown occurs, but the end products of galactomannan degradation accumulate quantitatively and have been identified as galactose and mannose. The galactomannan is therefore depolymerized by hydrolytic as opposed to phosphorolytic cleavage, and the enzymes involved are produced within the endosperm itself (Reid and Meier, 1972). Galactomannan breakdown is paralleled in the endosperm (but nor in the embryo) by a-galactosidase and Pmannanase activities (Reid and Meier, 1973b). In isolated endosperm halves galactomannan breakdown is partially or totally inhibited by metabolic inhibitors, the site of action of which can only be the living cells of the aleurone layer. These inhibitors also suppress a-galactosidase and P-mannanase activities, suggesting a key role for the aleurone layer in the production or activation of these enzymes (Reid and Meier, 1973b). When fenugreek seeds are allowed to germinate in the presence of 80% D,O the endosperm a-galactosidase becomes density labeled (Reid and Davies, unpublished). It is probably synthesized de novo in the aleurone layer. Inhibitor studies suggest that the P-mannanase is probably also synthesized de novo (Reid et al., 1977). According to Reese and Shibata (1965) the complete hydrolytic breakdown of a leguminous galactomannan would require at least three enzymes-an a-galactosidase, a P-mannanase, and a pmannoside mannohydrolase (P-mannosidase). The fenugreek seed endosperm does contain P-mannosidase activity which increased fourfold during galactomannan breakdown (Reid and Meier, 1973b). Recent studies on galactomannan breakdown in guar (Cyamopsis tetragonoloba) seeds have shown conclusively that the P-mannoside mannohydrolase of the guar endosperm is not synthesized de novo and that it is present in association with galactomannan even in the resting endosperm (McCleary, 1983). McCleary (1982) has demonstrated that complete extraction of the P-mannoside mannohydrolase from the endosperm is possible only if the galactomannan is first depolymerized. Consequently it is probable that the P-mannoside mannohydrolase of the fenugreek endosperm is also present in an active form in the resting seed. The mode and timing of its synthesis during seed development await investigation. A role for the aleurone layer in the production of enzymes for galactomannan breakdown is indicated by ultrastructural observations. The breakdown of galactomannan is first visible next to it, in a “dissolution zone” which increases in size inward toward the cotyledons (Fig. 8). Electron microscopic examination of the aleurone layer provides evidence of intensive synthesis of secretory proteins just prior to and during galactomannan breakdown (Reid and Meier, 1972). The galactomannan of fenugreek constitutes about 30% of the total reserve material in the seed, the remainder being mainly protein and oil localized within

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hours

Fig. 9. Dry weight changes accompanying germination of and early seedling development from fenugreek seeds. (Reid and Bewley, 1979.)

the cotyledons (Reid and Bewley, 1979). The overall pattern of reserve mobilization, shown in Fig. 9, illustrates the extraordinary rapidity of the breakdown of the endosperm reserves relative to the cotyledon reserves, The influx of material from the endosperm into the embryo during galactomannan breakdown is so rapid that it causes a transitory increase in the dry weight of the cotyledons. This strongly suggests that the mobilization of the endosperm reserves is not subject to the same regulatory constraints as are the breakdown processes in the embryo. Certainly there is no positive hormonal control by the embryo over the activity of the aleurone layer (Reid and Meier, 1972), as has been established for certain cereal grains (Yomo and Varner, 1971).

2 . Galactomannan Mobilization in the Carob Seed Structurally the galactomannan of carob or locust “bean,” Ceratonia siliqua (Leguminosae-Caesalpinioideae)typifies the low-galactose galactomannans of the Caesalpinioideae. Its degree of galactose substitution is only 20%, whereas that of fenugreek galactomannan is almost 100%.The carob seed is very much larger than that of fenugreek, but its gross anatomy is similar, the galactomannan-rich endosperm completely surrounding the embryo. In the carob seed, however, the endosperm is particularly massive, accounting for about 60%of the dry weight of the seed. The endosperm of the carob seed does not exhibit the same degree of anatomical specialization as that of fenugreek: there is no clear division of it into aleurone layer and storage tissue, and all the cells have living Fig. 8. Top: Cryostat section of part of a fenugreek seed after imbibition but prior to galactomannan mobilization: stained with periodic acid-Schiff‘s reagent to reveal galactomannan. C, Part of a cotyledon; G ,galactomannan; A, Aleurone layer. Note that all the storage cells of the endosperm are “filled” with galactomannan. (After Reid, 1971 .) Midd/e: Section comparable with that above, but during galactomannan mobilization. Note that the dissolution zone (D) in the endosperm is adjacent to the aleurone layer. (After Reid, 1971.) Bottom; Section comparable with those above but after galactomannan mobilization. Only a remnant of the endosperm (E) remains. (After Reid, 1971 .)

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protoplasts. The galactomannan is present in the thickened cell walls. Galactomannan formation in the carob endosperm has not been studied, but its mobilization has been investigated by Seiler (1977). In many respects the physiology of galactomannan utilization in carob resembles that in fenugreek. Again, the enzymes involved are a-galactosidase, pmannanase, and p-mannosidase, and they are present within the endosperm itself. The a-galactosidase at least is synthesized de novo following germination. The pattern of galactornannan mobilization in carob shows an interesting variation from that in fenugreek. Initially some wall dissolution occurs around all the endosperm cells, presumably because they all secrete hydrolytic enzymes. But

Fig. 10. Electron micrograph of the outer part of the carob seed endosperm in the early stages of galactomannan breakdown. AZ, Outer cell layer of endosperm; FS, fibrillar wall layer next to cell lumen-probably not composed of galactomannan; GM, galactomannan. Note the limited digestion of galactomannan (arrows). (After Seiler, 1977.)

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this soon ceases (Fig. LO); bulk galactomannan mobilization then takes place from the cotyledons outward toward the testa. It seems probable that enzyme production in the endosperm cells is inhibited by the accumulation of breakdown products, and the inhibition is relieved only as the breakdown products are transported into the cotyledons. 3 . Enzymatic Interactions in Galactomannan Hydrolysis Although it is clear that an a-galactosidase, a (3-mannanase, and a P-mannoside mannohydrolase contribute to galactomannan hydrolysis in fenugreek and carob seeds, none of the fenugreek or carob seed enzymes has been purified to homogeneity. The corresponding enzymes from the guar seed (Cyamopsis fefragonoloba)have, however, been purified and their molecular and catalytic properties have been investigated (McCleary, 1982, 1983). Furthermore, the cooperative interaction of the three enzymes in the hydrolysis of galactomannan has been elegantly demonstrated in vizro using the purified enzymes (McCleary , 1983). To determine the relative importance of these three enzymes in galactomannan hydrolysis and sugar uptake by guar cotyledons, McCleary (1983) incubated washed endosperm-free embryos with galactomannan alone or in the presence of the enzymes singly and in combination. Five embryos were incubated in the presence of a quantity of galactomannan equivalent to that normally present in the endosperms of five guar seeds. If enzymes were added, the amounts were equivalent to the amounts present in’five seeds at the time of the maximum rate of galactomannan breakdown. The galactomannan substrate was guar galactomannan which had been pretreated with a pure fungal 9-mannanase to reduce its viscosity without altering its mannose : galactose ratio. When embryos were incubated in the presence only of galactomannan, and of galactomannan plus the (3-mannoside mannohydrolase, there was no uptake of carbohydrate. Similarly, galactomannan plus P-mannanase gave no uptake of carbohydrate, since the limit galactomannan was resistant to P-mannanase attack. The a-galactosidase gave quantitative removal of the galactose from the galactomannan, and the galactose was rapidly taken up by the cotyledons. When a-galactosidase plus (3mannanase were used, the galactomannan was hydrolyzed to galactose and a series of manno-oligosaccharides, ranging from mannobiose to mannopentaose; the galactose was rapidly absorbed by the embryo, but the uptake of the oligosaccharides occurred more slowly and was incomplete after 24 hr. (In a separate series of experiments it was shown that the cotyledons took up mannobiose and mannotetraose intact, but that higher oligosaccharides were probably hydrolyzed at the surface of the cotyledons by a cotyledonary (3-mannosidemannohydrolase with properties similar to that of the endosperm.) Only when all three enzymes were present was the galactomannan completely hydrolyzed and carbohydrate uptake complete within 48 hr-the time taken in vivo (McClendon et a l . , 1976; McCleary, 1983).

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The only mannan-containing seed in which storage carbohydrate mobilization has been studied in detail is that of Phoenix dactylifera, the date palm. In his classic paper of 1862 Sachs describes the anatomy of the date seed, in its resting state and at different times after germination, noting the enormously thick walls of the endosperm cells and their content of aleurone and oil. He describes the germination of the tiny cone-shaped embryo and the mode of its absorption of the endosperm’s reserves. The cotyledon of the embryo acts as a haustorium and grows inward into the endosperm, absorbing the reserves in a narrow zone in front of it. Sachs recognized that the cell walls of the date endosperm constitute a major substrate reserve, and noted that they are completely broken down, with the exception of the thin primary walls which accumulate in the dissolution zone. Sachs’ highly accurate drawings of the date seed and its germination are reproduced unmodified in Fig. 1 1 . The pioneering work of Sachs was confirmed and extended by Keusch (1968) who demonstrated unequivocally that the mannan reserves of the date endosperm are broken down in the dissolution zone surrounding the advancing haustorium, and showed that the end products of the breakdown process are mannose, manno-oligosaccharides and traces of galactose. Using 14C-labeled D-mannose, Keusch was able to demonstrate the uptake of mannose by the haustorium and its rapid conversion to sucrose. The enzymes responsible for mannan hydrolysis were not directly investigated by Keusch (1968); he did however demonstrate that the breakdown of the polysaccharide was hydrolytic rather than phosphorolytic and reasoned that a pmannanase and a (3-mannosidase mannohydrolase had to be involved. We have detected these enzymes in endosperm homogenates (De Mason, Reid, and Sexton, unpublished). The p-mannanase activity is present exclusively in the “dissolution zone” while the p-mannoside mannohydrolase activity is present throughout the endosperm, even prior to germination. The site of production of the P-mannanase is not yet clear. Keusch (1968) incubated isolated, washed haustoria for periods of 15 to 40 hr in solutions containing ivory nut mannan A, 0.01 M Na/K phosphate buffer, toluene, and ammonium molybdate, and observed extensive breakdown of the mannan. The conclusion was drawn that it is the haustorium which secretes the enzymes necessary for mannan breakdown. The endosperm cells of the date are living (DeMason, Reid, and Sexton; unpublished), and there is no reason why they should not be capable of synthesizing and/or secreting p-mannanase. The question of the origin of the hydrolytic enzymes is receiving further attention. C. XYLOGLUCAN METABOLISM IN TROPAEOLUM

Apart from the early reports of Heinricher (1888), Reiss (1889), and others that ‘‘amyloids” in a variety of seeds including Impatiens balsamina, Tropaeolum

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Fig. 11. Reproduction of Sachs’ (1862) line drawings of date seed germination. Drawing No. 4 shows the anatomy of the endosperm (E) during reserve mobilization. p, Primary cell wall; WS, weakened zone; PZ, residual compressed primary walls; Ep, epithelium of haustorium.

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majus, and Cyclamen europaeum are mobilized following germination, there is little information in the literature relevant to the biochemistry of the breakdown of seed xyloglucans. Gould et al. (1971) observed that a “pectic” xyloglucan is broken down following germination in mustard seed cotyledons; xyloglucan, however, is not a major storage material in mustard. We are investigating xyloglucan mobilization in the seeds of Tropaeolum majus, and have established that the cell walls of the cotyledons lose their ability to undergo “amyloid” staining following germination (Ward, Reid, and Sexton, unpublished). Furthermore, the cotyledons of the germinated seeds develop xyloglucan-degrading enzymatic activities which vary par1 passu with the breakdown of the polysaccharide. These enzymes include endoxyloglucanase, f3-galactosidase, a-xylosidase, and P-glucosidase. The endoxyloglucanase has been purified to homogeneity (Edwards et at., 1984). D. “GALACTAN” METABOLISM IN LUPINUS

The physiological role of the cotyledon cell walls of lupin seeds was a subject of scientific controversy in the late nineteenth century! Nadelmann (1890), on the basis of microscopic observations, concluded that the thickened cell walls in the cotyledons of L. angustifolius, L. albus, and L. luteus were mobilized following germination and were substrate reserves. Elfert (1894) vigorously refuted this claim, asserting that Nadelmann’s observations were erroneous; he concluded that the changes in wall morphology which followed germination in Lupinus were simply a “metamorphosis” of the wall brought about by cotyledon expansion. Schulze (1895-1896) pointed out that neither Nadelmann nor Elfert appeared to be aware of an earlier paper (Schulze and Steiger, 1889) in which it had been shown that the seed of L . luteus contained a water- and alkali-insoluble carbohydrate material (“Paragalaktan”) which was probably present in the cotyledon cell walls and which was mobilized following germination. The observations were later extended to L . angustifolius (Schulze, 1895-1896). “Paragalaktan,” which released galactose and arabinose on acid hydrolysis, was, according to Schulze, clearly a storage material. In more recent times the ideas of both Elfert and of Nadelmann and Schulze have received support. Matheson and Saini (1977) conducted an investigation of the polysaccharides in the cotyledons of L. luteus following germination, with particular attention to the “pectic” fractions which were soluble in hot water and oxalate/EDTA solutions. They noted a net depletion of galactose- and arabinosecontaining polysaccharides, and concluded that the later stages of cotyledon expansion were accompanied by the selective hydrolysis of certain wall polymers. Although Matheson and Saini (1977) noted that depletion of wall polysaccharide was accompanied by the transitory accumulation of a glucan which they later indicated was starchlike (Saini and Matheson, 1981), they do not seem to have considered that the galactose- and arabinose-containingpolysaccharides of

-i 15

P

*;=EL.

15

-

10-

5-

\\

Nonstarch polysaccharides

\\

o Galactose residues A

Arabinose residues

o Xylose residues

Y\

'--4=-f:,y\ -7

- e- _ - _m-4%: -~

Fig. 12. Mobilization of major stored reserves in cotyledons of Lupinus angusrifolius cv. Unicrop. Open symbols, day/night conditions; filled symbols, continuous darkness. (After Crawshaw and Reid, 1984.)

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TABLE I1 Monosaccharide Residues in Storage Mesophyll Cell Walls Isolated from Cotyledons of Lupinus angustifolius cv Unicrop before and after Reserve Mobilization“ Neutral monosaccharide residues Arabinose

Xylose

Galactose

Glucose

Rhamnose

Uronic acid

Walls from 16hr-imbibed seeds6

12

15

66

4

2

8

Walls from 14day-germinated seedsc

16

20

30

23

11

35

a

Hutcheon and Reid (unpublished). Before mobilization of cell wall storage polysaccharides. After mobilization of cell wall storage polysaccharides.

L. luteus might be reserves. Evidence in support of a reserve function for the cell wall polysaccharides of Lupinus has come from Parker’s (1 976) observation that the cell wall thickenings in L . albus and L. angustifolius cotyledons disappear following germination and from cognate biochemical studies being carried out in the author’s laboratory. Fig. 12 shows the changes in the major stored reserves in the cotyledons of L. angustifofius following germination, while Table I1 shows the composition of storage mesophyll cell walls isolated from the cotyledons before and after reserve mobilization. Clearly nonstarch carbohydrates localized in the cell wall constitute a major reserve in L. angustifolius. The linkages present in the cell walls and the enzymes responsible for their hydrolysis are currently under investigation. IV. CONSIDERATIONS OF BIOLOGICAL FUNCTION Although the cell wall storage carbohydrates of seeds are utilized as substrate reserves following germination it is nevertheless pertinent to raise the question of their overall biological function in the seeds which contain them. Are they exclusively storage macromolecules, or do they have other functions? This question is by no means original. It was effectively posed by Nadelmann (1890) who set out to investigate whether or not the mucilages present in leguminous seed endosperms were reserve substances in addition to being involved in water imbibition. Nadelmann went on to demonstrate that the mucilages have a reserve function and concluded that they are first and foremost (“in erster Linie”) reserves. Marloth (1883) commented on the possibility that a “hard endosperm” with thick cell walls serves to protect seeds from mechanical damage. Gould et al. (197 1) have also suggested a protective function for storage polysaccharides

CELL WALL STORAGE CARBOHYDRATES IN SEEDS

149

of the celI wall type. Unlike starch, which is stored intracellularly, the cell wall storage carbohydrates are interposed between living cells and the external environment. It is perhaps not unreasonable to expect that they might play a direct role in the seeds’ interaction with that environment. It is the author’s personal opinion (a physical scientist’s view, perhaps) that the most telling argument in favor of a nonstorage role for the cell wall storage carbohydrates of seeds can be based on their bulk properties as materials. Mannans and glucomannans are crystalline, insoluble materials which confer extraordinary hardness on seeds which contain them in their endosperms. On the other hand the galactomannans and xyloglucans are hard only in the unimbibed state. They are essentially hydrophilic molecules. As materials these polysaccharides, particularly the galactomannans, are commercially important because of their complex interactions with water (Dea and Momson, 1975). It was the naive(?) expectation that these same interactions might be important to the germinative strategy of seeds which prompted us to investigate the role of galactomannan in the water relations of fenugreek seed during germination (Reid and Bewley , 1979). Figure 13 shows the movement of water which accompanies the imbibition of the fenugreek seed. Clearly the seed as a whole exhibits a normal water uptake curve-initial hydration of the tissue, followed by a lag phase culminating in the completion of germination and a further uptake of water. Analysis of the individual contributions of the endosperm, cotyledons, and radicle, however, shows

hours

Fig. 13. Pattern of water uptake by fenugreek seeds. Dry seeds were placed on wet cotton at cotyledons; W-W, axis. time = 0. 0-0,Whole seed; 0-0, endosperm and testa; 0-0, Quadruplicate batches of six seeds were analyzed: error bars represent 2 X SEM; G , completion of germination (radicle breakthrough). (After Reid and Bewley, 1979.)

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J. S . GRANT REID

I

I

I

L

U

I

1

f

I

--

.\a' -o-n

I

drying time (hours)

and naked Fig. 14. Drying curves for whole fenugreek seeds (O-O), testa-free seeds (0-O), embryos (m-m) at 52% relative humidity. Triplicate batches of 20 seeds were processed. (After Reid and Bewley, 1979.)

that the uptake of water in the initial hydration phase is predominantly into the endosperm: it represents 30% of the dry weight of the seed, but takes up over 60% of the water. Nadelmann's (1890) premise that the leguminous seed endosperm imbibes water is correct! Figures 14 and 15 show the analogous changes in water content which occur when a fully imbibed seed is allowed to dehydrate in a drying atmosphere: the seed as a whole loses water more or less linearly with time (Fig. 14) but the same is not true of the individual tissues (Fig. 15). Water is initially lost only from the

2LO -

0

drying time (hard

cotyledons

d r y ' q time (hars)

Fig. 15. Patterns of water loss from imbibed, whole fenugreek seeds (A) testa-free seeds (B),and naked embryos (C) at 52% relative humidity. Data derived from single batches of 10 seeds, testa-free seeds, or naked embryos, dissected and processed at each time point. (After Reid and Bewley, 1979.)

CELL WALL STORAGE CARBOHYDRATES IN SEEDS

151

endosperm: the cotyledons and axis lose no water until several hours after the initiation of the drying process. In the absence of the endosperm the cotyledons and axis lose water immediately (Fig. 15). The physical basis of the ability of the endosperm of the fenugreek seed to protect the embryo against desiccation can be deduced from Fig. 16, in which the water potential ($,) of the endosperm is shown in relation to water loss from the endosperm and the embryo. Clearly the endosperm is capable of losing water with little change in its water potential, until its water content falls to 100% of its dry weight. Thereafter further water loss is accompanied by a rapid change in water potential. The embryo does not lose water initially because it is not directly subjected to the very low water potentials of the surrounding atmosphere. It is challenged only by the water potential of the endosperm which for a period of some hours remains above - 1.5 MPa, a value which living, turgid plant tissues can resist (Wiebe, 1966). The endosperm’s hydrodynamic properties can be directly attributed to its high content of galactomannan [cf. Fig. 8 (top) and compare Figs. 16 and 17). The biological ‘role of the galactomannan of the fenugreek seed is therefore complex. During imbibition it is responsible for the uptake of relatively large amounts of water, and its distribution around the embryo. During germination it effectively buffers the embryo against water loss. Following germination it acts as a substrate reserve for the developing seedling. To assess the relative importance of the storage and nonstorage functions of the fenugreek and galactomannan, it would be necessary to study the seed’s overall germinative strategy in its natural environment. Nevertheless, it is interesting to note that there is no purely nutritional reason for a proportion of the substrate reserves of the fenugreek seed to take the form of galactomannan (Reid and Bewley, 1979). In contrast, a clear structure-function relationship exists for the role of galactomannan in the water relations of germination.

d r y q time Iholas) Fig. 16. Water loss at 52% relative humidity from the endosperm testa (W-M) and from the embryo (0-0) of the hydrated fenugreek seed in relation to the water potential of the endospermltesta (0-0). At each time point six seeds were used for dry weight determination and four seeds for the determination of JI, of the endosperm. (After Reid and Bewley, 1979.)

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drying time (hours) Fig. 17. Water content (0-0) and water potential (0-0) of a purified sample of fenugreek seed galactomannan (Campbell, 1978) hydrated and then allowed to dry at 25% relative humidity. (After Reid and Bewley, 1979.)

V. PERSPECTIVES There is now enough interest in the physiology and biochemistry of the cell wall storage carbohydrates of seeds to allow the confident prediction that the enzymatic mechanisms of their breakdown will soon be understood with respect to the major seed systems. It is to be hoped that the cell wall-degrading enzymes themselves will be purified to homogeneity and their specificities studied in detail, as is already happening in the galactomannan field. Germinated seeds could provide a convenient source of moderate quantities of highly purified enzymes capable of specifically degrading complex plant cell wall polysaccharides. Such enzymes would be of incalculable value in probing the structures of native cell walls, in determining the fine structures of cell wall polysaccharide preparations and in positively identifying products of polysaccharide biosynthesis in vitro. There is, for example, a striking structural resemblance between the xyloglucan storage carbohydrates of seeds and the xyloglucans which are now assumed to comprise the main noncellulose component of primary cell walls in dicotyledonous plants (Albersheim, 1976). Similarly there are indications (Wilkie, Reid, and Hutcheon, unpublished) that the “galactan” complex of lupin cell walls is structurally related to the “pectic” galactan-rhamnogalacturonan complex of primary cell walls (Albersheim, 1976). Xyloglucan- and galactan-containing seeds could provide enzymes to investigate primary cell wall structure, and will certainly provide insight into the types of enzymes which bring about the modification or turnover of primary cell walls which accompanies plant cell growth.

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The enzymology of cell wall storage polysaccharide biosynthesis has received relatively little attention. With the exception of the GDPmannose : galactomannan mannosyltransferase of the developing fenugreek seed endosperm (Campbell and Reid, 1982) there is no information concerning the properties of the enzymes catalyzing the formation of the major cell wall storage carbohydrates. To acquire such information should be relatively straightforward. Over a defined period of time virtually all the cells of developing endosperms of cotyledons are dedicated to the production of a single type of cell wall polysaccharide; levels of the relevant biosynthetic enzymes should therefore be relatively high. Furthermore, the structural features of synthetic products formed in virro should be easily compared with those of the carbohydrates known to be present in vivo. Once acquired such information would be relevant not only to seed systems but also to our understanding of the biosynthesis of analogous noncellulose polysaccharides in vegetative tissues. Perhaps the most interesting question surrounding the cell wall storage carbohydrates is that of their overall biological function. Do they all, like the galactomannan of fenugreek (Reid and Bewley, 1979), have a nonstorage function which is dependent upon their physicochemical properties? The answer will be provided only by the more widespread adoption of a combined biochemical and ecological approach to the study of seed “storage’ ’ carbohydrates. ACKNOWLEDGMENTS The financial support of the Agricultural Research Council and of Unilever Ltd. is gratefully acknowledged.

REFERENCES Albersheim, P. (1976). In “Plant Biochemistry” (J. Bonner and J. E. Vamer, eds.), 3rd ed., pp. 225-274. Academic Press, New York. Albersheim, P., Nevins. D. J.. English, P. D., and Karr, A. (1967). Curbohydr. Res. 5, 340-345. Anderson, E. (1949) Ind. Eng. Chem. 41, 2887-2890. Andrews, P., Hough, L., and Jones, J. K. N. (1953). J . Chem. Soc. 1186-1192. Aspinall, G. 0.. Hirst, E. L., Percival, E. G. V., and Williamson, I. R. (1953). J . Chem. Soc. 3 184-3 188. Aspinall, G. 0 . . Rashbrook, R. B., and Kessler, G . (1958). J . Chem. Soc. 215-221. Aspinall, G. 0.. Greenwood, C. T., and Sturgeon, R. J. (1961). J . Chem. SOC.3667-3674. Balasubramaniam, K. (1976). J. Food Sci. 4, 1370-1373. Bourquelot, E., and Herissey, H. (1899). C. R. Hebd. Seances Acad. Fr. 129, 228. Bouveng, H. O., Garegg, P. J . , and Lindberg, B. (1960). Acta Chem. Scand. 14, 742-748. Campbell, J. (1978). Ph.D. thesis, University of Stirling, Scotland. Campbell, J., and Reid, J. S. G . (1982). Planta 155, 105-1 1 I . Carlson. W. S., Ziegenfuss, E. M . , and Overton, J. D. (1962). Food Techno/. 16, 50-54. Chudzikowski, R. J. (1971). J. Soc. Cosrner. Chem. 22, 43-60. Courtois, J.-E., and Le Dizet, P. (1974). C. R . Hebd. Seances Acud. Fr. Ser. C 278, 81-83. Courtois, J.-E., Le Dizet, P., and Robic, D. (1976). Curbohydr. Res. 49, 439-449.

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Crawshaw, L., and Reid, J. S . G. (1984). Plunta 160, 449-454. Dea, 1. C. M., and Morrison, A. (1975). Adv. Curbohydr. Chern. Biochem. 31, 241-312. Edwards, M., Dea, I. C. M., Bulpin, P. V., and Reid, J. S. G. (1984). Plunra (in press). Elfert. T. (1894). Bibl. Bor. 30, 1-25. El Khadem, H., and Sallam, M. A. E. (1967). Curbohydr. Res. 4, 387-391. Glicksman, M. (1953). “Gum Technology in the Food Industry.” Academic Press, New York. Godfrin, M. J . (1884). Ann. Sci. Nut. 19, 5-158. Goldberg, R. (1969). Phyrochemisrry 8, 1783-1792. Could, S. E. B, Rees, D. A,, and Wight, N. J. (1971). Biochem. J. 124, 47-53. Halmer, P., and Bewley, J. D. (1979). Plunta 144, 333-340. Halmer, P., Bewley, J. D., and Thorpe, T. (1978). Plunta 139, 1-8. Hegnauer, R. (1973). “Chemotaxonomie der Pflanzen,” Vol. 6. Birkhauser, Basel. Heinricher, E. (1888). Flora (Jenu) 71, 163-185. Herissey, H. (1903). Rev. Cen. Bor. 15, 345-392, 406-417, 444-464. Hirst, E. L., Jones, J. K. N., and Walder, W. 0. (1947). J . Chem. Soc. 1225-1229. Hopf, H., and Kandler, 0. (1977). Phytochemistry 16, 1715-1717. Homer, H. T., and Amott, H. J. (1966). Bor. Gar. 127, 48-64. Jakimow-Bmas, N. (1973). Phytochemistry 12, 1331-1339. Jones, R. L. (1974). Planfa 121, 131-146. Keusch, L. (1968). Plunru 78, 321-350. Khanna, S. N., and Gupta, P. C. (1967). Phytochemistry 6, 605-609. Klages, F. (1934). Ann. Chem. 509, 159-181; 512, 185-194. Kooiman, P. (1960a). Acra Bor. Neerl. 9, 208-219. Kooiman, P. (1960b). K. Ned. Akud. Wet. C 63, 634-645. Kooiman, P. (1960~).Rec. Trav. Chim. Pays-Bas 79, 675-678. Kooiman, P. (1961). Rec. Trav. Chim. Pays-Bus 80, 849-865. Kooiman, P. (1967). Phytochemistry 6, 1665-1673. Kooiman. P. (1971). Curbohydr. Res. 20, 329-337. Kovacs, P. (1973). Food Technol. 27, 26-30. Le Dizet. P. (1972). Curbohydr. Res. 24, 505-509. Ludtke, M. (1927). Ann. Chem. 456, 201-224. McCleary, B. V. (1982). Curbohydr. Res. 101, 75-92. McCleary, B. V. (1983). Phytochernistry 22, 649-658. McClendon, J. H., Nolan, W. G., and Wenzler, H. F. (1976). Am. J. Bor. 63, 790-797. Marloth, R. (1883). Bor. Juhrb. Sysr. Pflanzengesch. 4, 225-265. Matheson, N. K., and Saini, H. S. (1977). Phytochemistry 16, 59-66. Meier, H. (1958). Biochim. Biophys. Acru 28, 229-240. Meier, H., and Reid, J . S. G. (1977). Plunta 133, 234-248. Meier, H., and Reid, J. S. G. (1982). Encycl. Planr Physiof. New Ser. 13A, 418-471. Mukhejee, A. K., and Rao, C. V. N. (1962). J . fndiun Chem. Soc. 10, 687-692. Mukherjee, A. K., Choudhury, D., and Bagchi, P. (1961). Can. J. Chem. 39, 1408-1418. Nadelmann, H. (1890). Juhrb. Wiss. Bor. 21, 1-83. Numberg, E., and Rettig, E. (1974). Drugs Made Ger. 17, 26-31. Parker, M. L. (1976). Ph.D. thesis, University of Wales, Bangor. Reese, E. T., and Shibata, Y. (1965). Can. J . Microbiol. 11, 167-183. Reid, J. S. G. (1971). Planta 100, 131-142. Reid, 1. S. G., and Bewley, J. D. (1979). Plunra 147, 145-150. Reid, J. S . G., and Meier, H. (1970). 2. Pji’anzenphysiol. 62, 89-92. Reid, J . S. G . , and Meier, H. (1972). Pluntu 106, 44-60. Reid, J. S. G., and Meier, H. (1973a). CuryotogiuSuppl. 25, 219-222. Reid, J. S. G., and Meier, H. (1973b). Planfa 112, 301-308.

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Reid, J. S. G., Davies, C., and Meier, H. (1977). Plunra 133, 219-222. Reiss, R. (1889). Landwirrsch. Jahrb. 18, 71 1-765. Robic, D.. and Percheron, F. (1973). Phytochemisfry 12, 1369-1372. Sachs, J. (1862). Bor. Zrg. 20, 241-246, 250-252. Saini, H. S., and Matheson, N. K. (1981). Phytochemistry 20, 64-646. Saxena, V. K. (1965). Res. I d , 10, 101-106. Schulze, E. (1895-96). Hoppe-Seyler’s 2. Physiol. Chem. 21, 392-41 1 . Schulze, E., and Steiger, E. (1889). Landwirtsch. Versuchs-Stn. 36, 391-476. Seiler, A. (1977). Planra 134, 209-221. Smith, F., and Montgomery, R. (1959). “Chemistry of Plant Gums and Mucilages.” Van NostrandReinhold, Princeton, New Jersey. Stepanenko, B. N. (1960). Bull. SOC. Chim. Biol. 42, 1519-1536. Takaki, M., and Dietrich, S. M. C. (1979). Rev. Bras. Bot. 2, 125-127. Thompson, J. L., and Jones, J. K. N. (1964). Can. J . Chem. 42, 1088-1091. Tschirch, A. (1889). “Angewandte Pflanzenanatomie.” Urban & Schwarzenberg, Vienna. Vogel, T., and Schleiden, M. J. (1839). Poggendofs Ann. Phys. Chem. 327-330. Whistler, R. L., and Bemiller, I. N. (1958). Adv. Carbohydr. Chem. 13, 289-329. Whistler, R. L., and Bemiller, J. N. (1972). “Methods Carbohydr. Chem. Vol. 8.” Academic Press, New York, London. Whistler, R. L., and Smart, C. L. (1953). “Polysaccharide Chemistry”, Academic Press, New York. Whistler, R. L., and Wolfrom, M. L. (1965). Methods Carbohydr. Chem. 5. Wiebe, H. H. (1966). Plant Physiol. 41, 1439-1442. Winterstein, E. (1893). Hoppe-Seyler’s 2. Physiol. Chem. 17, 353-380. Wolfrom, M. L., and Patin, D. L. (1965). J. Org. Chem. 30, 4060-4063. Wolfrom, M. L., Laver, M. L., and Patin, D. L. (1961). J. Org. Chem. 26, 4533-4535. Yomo, H . , and Varner, J. E. (1971). Curr. Top. Dev. Biol. 6, 111-144.

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Welwitschia mirabilis-New Aspects in the Biology of an Old Plant

D. J. VON WILLERT Institut fur Angewandte Botunik Universitat Miinster Miinster, Federal Republic of Germany

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . Life Cycle ........................ 11. Osrnoregulation and Chemical Cornpositi 111. Water Economy and Water Uptake . . . . 1V. Photosynthesis and Carbon Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................... V . Energy Balance .................... VI. Concluding Remarks . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . ....................

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189

I. INTRODUCTION On the third of September, 1859, the Austrian physician Dr. Friedrich Welwitsch discovered Welwitschia mirabilis in southern Angola and introduced it to science. It is not surprising that such a strange plant quickly attracted the attention of botanists. Four years later a detailed scientific description of the morphology and anatomy of Welwitschia appeared (Hooker, 1863). With the report of the British painter Thomas Baines-again in 1859-that he had seen a “bulbous plant with 4 leaves” east of Swakopmund (Kers, 1967) the whole range of distribution of Welwitschia was marked. In fact, the distribution of Welwitschia is restricted to a strip about 1200 km long along the west coast of southern Africa ADVANCES IN BOTANICAL RtSEARCH, VOL II

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Copyright 0 1985 by Academic Pres5, Inc (London) Ltd All rights of reproduction in any form reserved ISBN 0-12-005911-8

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D.J. VON WILLERT

from the Nicolau River north of Mosslmedes (Angola) to the Kuiseb River (South-West Africa, Namibia). Welwitschiu never reaches the coast. Its westemmost stand is about 10 km inland and its easternmost stand about 150 km inland. In its south-north extension, Welwitschiu occupies only the northern and central part of the Namib Desert while in its west-east extension Welwitschia leaves the Namib Desert, stretches over subtropical grassland, and penetrates into the Mopane Savanna. Hence, Welwitschiu occupies an area with a wide

a

40

.-

-

_____....-

:i 111

Relative humidity

100 80

'1,

60

30 40

20 20

10

0

.-

.

0 100 80

z

c

9 m

60 40

s.

; c

3. $ '

-ae -

i

20

0

Local time (hr)

Fig. I . Air temperature and relative air humidity in a daily course at four different habitats of Welwirschiu rnirubilis in March, 1977. (a) Fifteen kilometers east of Torrabay; (b) 90 km far inland in subtropical grassland; (c) west of the Brandberg in grassland about 60 km far inland; (d) Mopane Savanna about 150 km from the coast; (e) air temperature and relative humidity in the Welwitschia Flats about 45 km east of Swakopmund on a "normal winter day"; (f)on a winter day with fohnwind conditions.

Welwitschia mirabilis

159

ecological amplitude. Welwirschia mainly grows in an area with annual rainfall between 0 and 100 mm. Except for dew and fog, precipitation occurs only in the summer months from January to March. At its easternmost border-the Mopane Savanna-annual rainfall can exceed 200 mm, but fog characteristic for the Namib is missing here. Except for the desert research station at Gobabeb, the southernmost location of Welwirschia, no continuous recordings of the climate of Welwitschia habitats are available. In order to characterize the differences between the near coastal, grassland, and savanna habitats of Welwitschia, the daily courses of air temperature and relative humidity are presented in Fig. 1. Although these examples must not be considered totally representative, they show that with increasing distance from the coast temperatures, especially night temperatures, increase and relative humidities decrease. Characteristic for the Namib Desert is that during winter, a heavy east wind (fohn or “berg wind”) can blow which, as a typical berg wind, carries hot and dry air down the escarpment. This leads to the curiosity that, at the coastal habitats, the maximum day and night temperatures in the course of a year occur in winter. For the Welwitschia Flats (about 45 km east of Swakopmund) where 5000-6000 specimens grow, Fig. 1 compares the air temperature and relative humidity of a normal winter day with a fohn day. LIFE CYCLE

With respect to hereditary relationships, Welwitschiu is completely isolated. Systematically, Welwitschia is placed with the gymnosperms. It is the only species in the family of the Welwitschiaceae and is together with the Gnetaceae and Ephedraceae combined in the Gnetales. No fossils exist. Phylogenetically, Welwitschiu must be very old as there are no recent forms known that relate to other taxonomic groups (Markgraf, 1926). Seeds of Welwitschia germinate within a fortnight and display rather quickly two cotyledons which are up to 5 cm long. Carbon is allocated to the root which, for a 10-week-old seedling, is 35 cm long (Fig. 2). The apical meristem of the shoot degenerates early and besides the cotyledons, which live more than 2 years, only one further pair of leaves develops. A seedling 18 months old is shown in Fig. 3. Development from the stage shown in Fig. 3 to that shown in Fig. 4,which shows the oldest specimen in the Welwitschia Flats, takes about 2000 years. With such a big old plant, the existence of only one pair of leaves is difficult to understand. The hypocotyl has folded up to a bizarre form and can be up to 2 m high, similar to the diameter of the stem or trunk. The leaves are generated from a basal meristem which is inserted into a groove 2-3 cm deep in the hypocotyl. In the natural habitat, the leaves grow very slowly (10-15 cm per year). The growth occurs at the base and the tip dies back continuously. Leaves can be up to 3 m long and thus have a continuous developmental gradient spanning many

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D.I. VON WILLERT

Fig. 2. A 10-week-old seedling of Welwitschia mirabilis.

years. This allows one to examine the behavior of every tissue age in a way not feasible with any other plant. Environmental factors affecting growth and drying back determine the length of the leaf. It has been reported that a leaf can dry completely and start growing at the base when conditions improve. Antelopes and zebras will feed on Welwitschiu leaves during prolonged drought periods and sometimes tear leaf ribbons out of the groove. As long as the meristem is not damaged, the leaf will grow further. Welwitschiu has a tap root 1 to 1.5 m long. Depending on the soil, Welwitschia forms lateral roots from the tap root at various depths. Frequently, anastomoses in the roots have been observed (Giess, 1969; see Fig. 2). Welwitschiu is dioecious. The inflorescences emerge from buds in the leaf axils that are hidden in the groove and show a dichotomously branched cyme carrying cones at the end of the stalks. There is still a controversy whether Welwifschiu is insect- or wind-pollinated, but detailed studies are in progress (Marsh, 1982). Plants grown from seeds in the greenhouse flowered for the first time at an age

Welwitschia mirabilis

161

Fig. 3. Eighteen-month-old seedling of Welwitschia mirubifis with the two still-living cotyledons and the only two further leaves this plant will ever generate.

Fig. 4. About 2000-year-old specimen of Welwitschia mirabilis, the so-called “Groot Welwitschia” from the Welwitschia Flats. (Photos by E. Brinckrnann.)

162

D. I. VON WILLERT

of 10-12 years and gave ripe seeds after an artificial pollination. Nothing is known about the age at which plants enter the reproductive stage in the natural habitat. Although the anatomy and morphology of the Welwitschia leaf has been described in detail (Hooker, 1863; Rodin, 1958a,b; Bornman et al., 1972) its leaf character is still equivocal. Welwirschia is found in many books of succulent plants but its leaf is in no way succulent. It is tough and leathery, contains about 50% water and is 1-1.5 mm thick. The leaf is amphistomatic with sunken stomata. The outer walls of the epidermal cells are heavily thickened. Bundles of fiber cells stretch longitudinally just underneath the epidermis, and the parenchyma contains numerous large ramifying crystalliferous sclereids. The leaf obviously has a scleromorphous character. The assaults and challenges of the natural habitat open a wide field for physiological and ecophysiologicalinvestigation to elucidate the adaptational properties of Welwitschia. As it is not easy to reach the places where Welwitschia grows, comparatively little is known about the physiological reactions of this plant in its natural habitat. We shall focus here mainly on four aspects: 1. The osmoregulation and chemical composition 2. The water economy with special regard to water uptake from the atmosphere 3. Photosynthesis and carbon balance 4. Energy balance 11. OSMOREGULATION AND CHEMICAL COMPOSITION Generally, xeromorphic plants growing in deserts have low water potentials (Richter, 1976). Welwitschia is no exception in this respect. For a near coastal and hence very dry habitat leaf water potentials as low as -7.0 MPa have been reported (Eller et ul., 1983). But the method used in this investigation could give only a rough estimate (Shackel, 1984). Unfortunately, no simultaneous measurements of the water potential in the rooting horizon or determinations of the osmotic potential of the roots were made but the low water potentials of the leaf must reflect an osmotic adaptation to the dry environment. The osmotic potential (Jln) of the leaf is also low. Walter (1936) gives -3.96 MPa and -3.48 MPa for the years 1932 and 1939, respectively. During a prolonged drought period in September, 1981, much lower Jln values were observed. The mean qnof 58 leaf extracts of plants growing in the Welwitschia Flats was -6.2 MPa (Eller et al., 1983). Osmotic potentials from four different growing sites on a transect from the coast to the Mopane Savanna are compiled in Table I. There is no clear correlation between the Jll, and the growing site, a fact

163

Welwitschia mirabilis

TABLE I Range of Osmotic Potentials of Welwitschia mirabilisa Distance from coast Site

(kmf

Torrabay (a) Wereldsend (b) Brandberg (c) Mopane Savanna (d)

90 65 150

Osmotic potential (-MPa) 2.5-4.5 4.0-4.5 4.0-5.5 3.3-6.5

15

a Taken from plants at four different growing sites, same localities as in Fig. la-d, along a transect from west to east as determined in March, 1977.

which is in agreement with the results of the chemical composition of plants from different habitats (see below). Little is known about the chemical constituents of Welwitschiu mirabilis. The only available data concerning the concentration of inorganic ions are those of Walter (1936). He found chloride in a concentration of 159 to 204 mmol/dm3 cell water, which accounts for 20 to 25%of the osmotic potential. The amount of sulfate is about 1/20 of the chloride content and the concentration of monosaccharides is higher than that of disaccharides. The sugars together account for 10 to 15% of the overall +n. He concluded that Welwitschiu is not a typical halophilic species, although it can-while growing in a soil with only traces of salt-accumulate considerable amounts of chloride. Recently, we performed detailed studies of the distribution and composition of inorganic and organic ions in Welwitschia leaves. Fig. 5a-c shows the content of sodium, potassium, calcium, and chloride in four different Welwitschia plants from the Welwitschia Flats. Plants 14 and 23 grew in a shallow dry water course while plants 25 and 26 grew about 800 m apart and uphill. The gradients of the inorganic ions along a Welwitschiu leaf are consistent with the distribution of these ions in other plants (Kinzel, 1982). However, the absolute amounts were extraordinarily high, especially for K and Na . K and C1- contents declined with increasing leaf age while Na and Ca2 are accumulated. This is in full agreement with the opinion that potassium and chloride are freely mobile ions that can be easily retranslocated from old to young parts while sodium and calcium are only transported with the transpiration stream and consequently accumulate in older parts (Kinzel, 1982). Fig. 5a-c makes clear that the distribution of ions in the various leaves followed the same principle but with marked differences in absolute amounts. Plants from the same growing site (plants 25 and 26) and from sites some 800 m apart (plants 14 and 23) exhibited pronounced differences in their ion contents, which is consistent with the ranges of osmotic potentials (Table I) and implies that the physiological behavior might also differ significantly. All attempts to characterize Welwitschia specimens from different +

+

+

+

+

164

D.J. VON WILLERT

Fig. 5 . The sodium (a), potassium (b), and calcium and chloride (c) contents of leaves of four different plants of Welwitschiu rnirubilis and their distribution with leaf age. Plants 23 and 14 grew in a shallow dry watercourse; plants 25 and 26 grew 800 m westward and uphill. Samples were taken in a grid pattern with 5 cm distance between the samples. For all samples with the same leaf age ( i x . , across the leaf blade) the mean value of the ion content and the standard deviation were calculated and plotted against leaf age. The samples below zero were taken from the leaf part hidden in the groove of the trunk.

165

Welwilschia mirabilis

800 700 600

-

500 400

b

c 300

2

t

200 100

-0 " o

-E,

300

200 100

0 0

20

40

60

0

20

40

60

80

Distance from leaf base (cm)

Fig. 5c. See legend on page opposite.

habitats by their content of inorganic ions failed because the variance at one habitat exceeded that between two different habitats. This is illustrated in Fig. 6, which shows widely different ion contents of three specimens growing in the grassland near the Brandberg. A more detailed analysis of the ion distribution in a single leaf presented in Fig. 7 shows that potassium was distributed in a 45-cm-broad and 55-cm-long leaf blade with marked variations across the leaf (i.e., for the same leaf age). However, in different longitudinal rows, there were either low (i.e., in a row 10 cm from the leaf margin) or high (i.e., in a row 20 cm from the leaf margin) contents of potassium. This might indicate that the transport of potassium is mainly along the leaf rather than across. There can be no doubt that potassium and chloride are translocated from the older parts of the leaf to the younger parts. Due to the very slow growth, these transport processes need not be rapid. One would expect that the die-back process at the tip of the leaf is the result of senescence and that the Welwitschia leaf with its fantastic leaf age gradient would allow one to define senescence precisely, e.g., in terms of a threshold in the concentration of particular ions. The gradients together with the markediy differing absolute ion contents just in front of the dry tip do not favor this opinion, but indicate that senescence is a continuous and slow change rather than a rapid transition and that the die-back process is under the control of other parameters. There is a visibly distinct border between the living green tissue and the dry brown tissue at the tip of the leaf. However, samples taken from the brown and dead parts revealed that none of the organic and inorganic compounds

D.J. VON WILLERT

166 f

a

1000

1

b

800

600 400 0

t 200

D

-z

-

600400 . 200 .

c

d

are remobilized. The gradients continue into the dead dry parts without any discontinuity. Despite the pronounced gradients, the sum of all inorganic cation equivalents was constant for all leaf ages, indicating that the cations are not arbitrarily distributed. A rapid decline in potassium is associated with a steep increase in sodium and vice versa, and can be seen in Figs. 5a,b and 6. In contrast to sclerophyllous species, malacophyllous plants in arid environments show pronounced diurnal fluctuations in their leaf water content and consequently in their osmotic potential. Due to an overnight accumulation of malate, CAM species have much lower osmotic potentials in the morning than in the evening (Luttge et al., 1982; von Willert et al., 1984a). A comparison of the predawn osmotic potential with that in the early afternoon reveals that in three out of four plants no significant differences exist (Fig. 8). The same is true for the water content (Fig. 9). In these investigations, one leaf was taken in the morning and the other in the evening, and so the observed differences might reflect preexisting differences with no relation to a prevailing water deficit. On

Welwitschia rnirabilis

167

Fig. 7. Distribution of potassium in a single leaf of Welwirschia rnirabilis shown in a threedimensional diagram.

the other hand, plant 14 had by far the smallest leaf area and this can be taken as an indication that its behavior could be different from the other three. It is surprising to find the expected gradients in the water content of the leaf but not a gradient in the osmotic potential in a way generally exhibited by other plants where the youngest part has the lowest osmotic potential. Based on the data given for the metabolically most active part of the leaf (20 cm distal to the base) of plant 26, an evaluation was made of the contribution of all solutes to the overall osmotic potential. For this calculation we used the van’t Hoff equation

where C, is the molal concentration of the solutes, R is the gas constant, and T the temperature (“K). For a temperature of 20°C one obtains -4.6 MPa, which is

D.3. VON WLLERT

168

0

10 20 30 40 50

0 10 20 30 40 50 60 70

Distance from leaf base (cm) Fig. 8. Comparison of the predawn osmotic potential with that in the afternoon of four Welwirschia plants. Error bars represent the standard deviation of the combined sample with the same leaf age (i.e., across the leaf blade).

150 130 k

:: 110

2

290

'0 Y-

O

0 150

i

8 130

c

110

90 0

10 20 30 40 50 60

0 10 20 30 40 50 60

Distance from leaf base (cm) Fig. 9. Comparison of the predawn water content in percent of dry matter with that in the afternoon of four Welwitschia plants. Error bars as for Fig. 8.

Welwitschia mirabilis

169

75% of the osmotic potential of -6.1 MPa determined directly on the expressed sap by a cryoscopic method. If one takes into account the nonideal behavior of the solutes, i.e., activity coefficients different from 1 , one gets a deviation of less than 1% from this calculation. Thus, the use of the above equation for a rough estimation of the osmotic potential of the measured solutes is justified. In algae, low-molecular-weight carbohydrates play an important part in osmotic adaptation (Hellebust, 1976; Kirst and Bisson, 1979; Munns et al., 1983). Their participation as compatible solutes in halophilic angiosperms has been demonstrated for sorbitol with Planrago species (Ahmad et al., 1979; Briens and Larher, 1983), for pinitol with Honkenya peploides (Gorham et a l . , 1981) and Sperguluria media (Albert and Popp, 1978). Since no information about the lowmolecular-weight carbohydrates in Welwitschia leaves is available except for the above-mentioned data (Walter, 1936), the results of a detailed analysis of this fraction will be presented (Fig. 10). Sucrose is the most abundant sugar, and in young leaf parts chiro-inositol is also an important constituent. All the other compounds occur in low amounts and show no pronounced gradients. Considering the high concentrations of inorganic ions and of organic acids the fraction of low-molecular-weight carbohydrates hardly contributes significantly to the osmotic potential. However, nothing is known about the compartmentation of these compounds in the cell and therefore their exact osmotic role is unknown.

Fig. 10. Distribution of low-molecular-weight carbohydrates in a 4.5-cm-long leaf of Welwifschia mirabilis.

170

D. J. VON WILLERT

O J ,

0

,

,

20

,

I

,

40

,

,

60

0

20

40

60

80

Distance from leaf base (cm)

Fig. 11. Distribution of proline in leaves of four Welwitschiu mirubilis plants in relation to leaf age and daytime either predawn (solid symbols) or in the afternoon (open symbols). Error bars as for Fig. 8.

Irrespective of whether proline is an indicator of, or a protective agent against, drought and heat stress, one might expect high proline levels in Welwitschia as drought and heat are the prevailing conditions in these habitats. In fact, proline predominates among the organic compounds in Welwitschiu. In parts close to the meristem, contents between 480 and 590 pmol/g dry matter were frequently observed. This corresponds to a proline concentration between 299 and 339 mmol/dm3. Besides Triglochin maritimum (Stewart and Lee, 1974), these are the highest proline contents so far reported for higher plants. The proline content depends markedly on leaf age (Fig. 11). In several cases proline seems to accumulate slightly during the day and is seen at significantly higher amounts in the evening, at least in the photosyntheticallymost active leaf part around 20 cm

171

Welwitschia mirabilis

from the base. This can be taken as a sign of a diurnal fluctuation of proline, as was described for other plants in the Namib Desert (Treichel et al., 1984). There is no relationship between the proline content and other parameters (TIr, water, sodium, or chloride content). Hence the significance of the proline accumulation is difficult to interpret. The high concentrations in the meristem area suggest a protective function for proline, as this is the most important part for the development of the leaf, and they also may indicate that the proline is localized in the cytoplasm, since vacuolar volume should be smallest in the immature cells. The other amino acids are present in minor amounts. Only alanine, y-aminobutyric acid (GABA), glutamic acid, and arginine exceed 1 ~ m ogl dry matter. In order to complete the picture of the chemical composition of Welwitschia,

'

200

180

160

-

b ;

140

120

t 7J Frn 100 0

-

80

0

-E,

60 40

20 0 0

10

20

30

40

50

Distance from leaf base (cm)

Fig. 12. Distribution of organic acids and inorganic phosphate in a 45-cm-long leaf of Welwitschia mirabilis.

D. J . VON WILLERT

172

TABLE I1 Chemical Composition of Arthraerua leubnitziae, Zygophyllum stapfii, and Welwitschia mirabilisa

Content (pmol/g dry matter)

Arthraerua Zygophyllum Welwitschia mirabilis

Malate

Citrate

Proline

Na

K

Ca

11 61

21 54 183

0 92 139

1843 7129 580

528 48 705

896 143

141

10

CI

Water content (% fresh wt)

398 1144 233

61.2 83.2 52.8

a For Welwirschia the given data are the mean values of 58 leaves. (For each leaf a complete gradient as given in Fig. 5a and b was established and subsequently the mean leaf content estimated.) For Arthraerua and Zygophyllum the mean of six specimens is given. Data for Arthraerua and Zygophyllum were from 1980, that for Welwitschia from 1981, all obtained in the Welwitschia Flats.

the fraction of the organic acids shall be mentioned briefly. A detailed description will be given in Section IV. The dominating acids are malate and citrate but isocitrate and quinate are significant. The distribution of these acids together with that of inorganic phosphate depends on leaf age and is given in Fig. 12. Phosphate exhibits a remarkable peak 15 cm from the base. This part of the leaf is considered to be the most active metabolically. Arthraerua leubnitziae and Zygophyllum stapfii frequently grow together with Welwitschia. Zygophyllum is a succulent halophytic species while Arthraerua is a xeromorphic plant. A comparison of the chemical composition of these three species (Table 11) shows that besides their different growth forms they have highly contrasted amounts of the same compound, although they root in the same soil and exist in the same climate. 111. WATER ECONOMY AND WATER UPTAKE

The water balance is determined by relative rates of water uptake and loss. For many plants of arid zones where temporary water loss exceeds water gain, some sort of store (succulence in its widest sense) will balance diurnal fluctuations of the leaf water content. Consequently, succulence appears to be an adaptation to withstand a greater water loss than gain for a considerable time and must be emphasized as a drought avoidance mechanism as described recently for CAM succulents of the southern Namib Desert (von Willert et al., 1984b). For Welwitschia nothing is known about the water uptake from the soil. The conspicuous carrot-like tap root penetrates between 1.O and 1.5 m deep before it splits into numerous thin roots (Giess, 1969). How far downward the roots extend is still uncertain. According to the classification introduced by Walter (1960), Welwitschia belongs to the hydrostable species because even a prolonged drought does not alter

Welwitschia mirabilis

173

its water content significantly (Eller et ul., 1983). As shown in Fig. 9, there is little or no diurnal change in the water content of a Welwitschiu leaf. This means that the water conduit functions well and that the water loss by transpiration is balanced without much time delay. In the case of plant 14 (Fig. 9), where the water conduit might be disturbed, water loss seems to be regulated by reduction of leaf area. Little is known about the capacity for water storage in Welwitschia. The spongelike roots may act as a water store balancing temporary water deficits. The leaf with its low water content is hardly able to provide the necessary water to keep the observed transpiration going. At noon between 25 and 30% of the apparent leaf water content is consumed by transpiration in 1 hr. This should result in a decreasing leaf water content if the water cannot be delivered by or via the roots. The only available data of the transpiration of Welwitschia in the course of a day concern measurements during a prolonged drought period in September, 1981 (vun Willert e t a f . , 1982). Figure 13 illustrates that the transpiration rates of different specimens correspond well in the morning and afternoon but scatter considerably around noon. Taking all parameters that influence transpiration into account, this is not surprising and underlines the conclusion drawn from the chemical composition that each Welwitschiu specimen has to be treated as an individual. While the transpiration is not markedly reduced during midday, the leaf conductance shows that the stomata are widely open at dawn, close substantially when the deficit of water vapor partial pressure is highest, but open again in the late afternoon. This is in full agreement with the observation of Gaff (1972) that in the natural habitat the stomata are open during the day rather than at night. The high water loss of a Wefwirschiu leaf during noon reported by Walter (1936) is also consistent with our results. From the daily course of transpiration a water loss of about 1 dm3/m2 total leaf area and 12 hr was calculated. For comparison, Zygophyllum stapfii loses only 0.09 dm3/m2during the same time. The waste of water exhibited by Welwitschia becomes evident if the transpiration of Welwitschia is compared with the transpiration of different plants in the southern Namib Desert 4 days after an abundant rainfall (Table 111). Although there is plenty of water available, these plants, except for one species, handle water much more economically than Welwitschia during a severe drought. The data on transpiration of Welwirschiu require three critical comments. (1) Transpiration was measured in leaves that, after being grazed by animals, remained at a length of about 10 cm. The transpiration at the end of a 1-m-long leaf blade could be different from that of a 10-cm leaf. (2) Leaf damage caused by animals will change the ratio of transpiring leaf surface to water-harvesting root surface significantly in favor of water harvesting. Consequently, water supply of a short regrown leaf will be much better than of the former large leaf blade and this might facilitate transpiration. (3) Transpiration was measured by a weighing

174

D.J. VON WILLERT

Fig. 13. Daily course of transpiration (a) and leaf conductance (b) of four different plants of welwitschia mirubilis. The changes in other environmental parameters are shown (c and d). (After von Wilbrt ef al., 1982.)

175

Wetwitschiu mirubitis

1000

800 Global radiation

600

7 E

400

-B

Dew point temperature

200 0

7

8

9

10

11

12

13 14 Local time (hrl

15

16

17

18

19

0

Fig. 13c and d. See legend on page opposite.

method. Due to the washboard-like leaf earlier attempts to measure transpiration with the use of a clamp-on porometer failed as it was impossible to seal the cuvette tightly. Experience with a recently developed portable steady state porometer for measuring CO, and water vapor exchanges of leaves (Schulze et al., 1982) under the conditions of the Namib Desert was no improvement. Since the measuring cuvette has no cooling device, overtemperatures of up to 8°C were obtained. Also, in rapidly transpiring plants, the relative humidity inside the cuvette increased considerably during the measurement. consequently, the driving force for transpiration, i.e., the deficit of water vapor pressure inside and outside the cuvette, was changed and the results did not reflect true transpiration. As shown

176

D. J . VON WILLERT

TABLE 111 Transpiration of I1 Plants Growing in the Southern Namib (Richtersve1d)a ~~

~

~

Water loss (drn3/rn2/ 12 hr)

Leaf consistency

Rhus populifolia Ozoroa dispar

0.20 0.16

Sclerophy llous Sclerophyllous

c 3

Solanum incanum Acacia erioloba Monechma mollissimum

0.77 1.16 0.79

Malacophyllous Malacophyllous Malacoph yllous

c3

Didelta carnosa Zygophyllum longicapsulare Psilocaulon subnodosum Othonna opima Cotyledon orbiculata

0.58 0.32

Succulent Succulent

c3

0.23 0.98 0.28

Succulent Succulent Succulent

c3

Welwitschia mirabilis

0.96

Sclerophyllous

c3

Species

Mode of photosynthesis

c 3

c3

c3

c 3

CAM CAM

Measured on March 27, 1981.4 days after an abundant rainfall and of Welwitschia mirubilk on October 10, 1981, in the Welwitschia Flats after 3 years of drought. The calculation of the transpiration is based on total leaf area.

in Table 111, the transpiration of Acacia erioloba is fairly comparable to that of Welwitschia. The transpiration of Acacia erioloba was determined by two different methods, the porometer of Schulze et al. (1982) and the weighing method fist applied by Stocker (1929). Table IV shows the comparability between the two methods. Measurements were done on a clear day at noon with a vapor pressure deficit (VPD) of the air at 2.15 kPa. Due to the overtemperature in the cuvette the starting VPD was 3.12 kPa. Only for leaf I did both methods give similar results. In this case, a gusty wind blew during the weighing experiment, which might have reduced the boundary layer of the detached leaf. Thus, as far as the boundary layer is concerned, the conditions probably resembled those in the ventilated cuvette. For all other leaves, the weighing method resulted in about 20% lower transpiration values. This example demonstrates how difficult the precise determination of a seemingly simple reaction like the transpiration can be, but exact transpiration data are necessary to understand the water balance. One should therefore repeat the transpiration measurements if possible with different methods in different seasons. A detailed investigation of the influence of leaf age could answer the question whether the water supply of a leaf is uniform in all parts or whether there is an increasing water deficit with leaf age. The available data must be looked upon only as a first approach and will need further confirmation.

177

Welwitschia mirabilis

TABLE IV

Comparison of the Transpiration of Four Different Leaves of Acacia eriolobau

mg H,O/min

Porometer technique

Weighing method

I 2 3

1.7 3.0

4

1.8 3.7 3.7 2.4

Starting VPD

3.12 kPa

2.15 kPa

Leaf

3.0 1.95

Obtained with either a porometer technique or with a weighing method. Experiments were performed around noon at Numees in the southern Narnib Desert on October 20, 1983.

Welwitschia is obviously able to balance the daily loss of water, but it seems reasonable to ask from where the water comes, especially during a severe drought. There should be no doubt that the demand is mainly supplied by water from the rooting horizon. As the accompanying flora suffers much more from prolonged droughts, Welwitschia must be connected to water sources that cannot be exploited by other plants. It is still a matter of controversy and argument whether Welwitschia is able to make use of dew and fog to balance the daily water losses. I will contribute to this discussion with two considerations and will start with a quantitative analysis of the problem. The maximum possible precipitation of dew and fog for the coastal area of the Namib is 50 mm/year (Walter, 1936). If we assume the daily water loss of WeEwitschia to be 1 dm3/m2 total leaf surface, then this means a water loss of 2 dm3/m2 projected leaf area as dew and fog will only precipitate upon the upper leaf surface. Hence, daily water loss by transpiration is equivalent to 2 mm precipitation per day. If dew and fog are quantitatively harvested, the annual gain would compensate the water loss of 25 days, which is about 7% of the annual demand. One must, however, consider that a quantitative uptake of water is most unlikely. The Welwitschia leaf has no special absorption tissue, and a direct uptake of liquid water via the stomata or the cuticle cannot be assumed. During the night dew and fog are ineffective because the stomata are closed and water harvesting can only start when the stomata open at dawn. Hence, the period of possible water uptake is shortened and consequently its efficiency reduced. If 25% of dew and fog precipitation can be exploited then the transpirational water loss of 1 week can be compensated, which is about 2% of the annual water demand. Taking into account that the frequency of fog decreases with increasing

178

D. I. VON WILLERT

distance from the coast, the possible water gain is further diminished. A calculation based on an annual dew and fog precipitation of 25 mm and a harvesting efficiency of 25% results in less than 1% of the annual water demand that can be met by this mechanism, and presumably even this value is much too high. The second consideration is a physical one concerning water vapor. An osmotic potential of -6.2 MPa means that the water vapor pressure of a leaf is at 95.5% of saturation. Due to reradiation on clear and calm nights, the Welwitschia leaf also will be cooler than the ambient air. Schulze et al. (1980) reported up to 4°C undertemperature of the leaf after sunset. Detailed investigations in 198 1 with thermocouples and infrared thermometers gave evidence for leaf temperatures up to 2°C cooler than the ambient air. The low osmotic potential together with the undertemperature of the leaf means that, when the air is saturated, a gradient in water vapor partial pressure exists from the atmosphere into the air spaces in the leaf. Because this gradient depends on the degree of water vapor saturation in the leaf, the prevailing relative humidity, the absolute air temperature, and the gap between air and leaf

.

0.4

0.3

-

2 Y

0

C

f

w-

%

0.2-

2 3 8 ?!

n

bP

J

& 0.1-

c

3 0'

I

1

I

1

I

I

I

I

Fig. 14. Water vapor partial pressure difference between the interior of the leaf and the atmosphere around the leaf at various air temperatures. The air is at 100% relative humidity and the osmotic potential of the leaf is -6.2 MPa. The temperature of the leaf and the air are equal or the leaf is 1 or 2°C cooler than the air.

Welwitschia mirabilis

179

temperature, the gradient can be significant. Figure 14 shows this relationship for a relative humidity of 100% in the air. For a given temperature, the gradient declines with decreasing relative humidity. The advantage of water vapor uptake is that it does not depend on dew and fog, which increases the number of days it can occur. The efficiency of this water harvesting mechanism would be substantially increased if, as in CAM plants, the stomata stayed open through the night. This would allow a water vapor uptake of at least 10 hr on a night like that given in Fig. 1E. Unfortunately, we have no information whether the stomata of Welwitschia are open at night or not. This should be proved before one judges the possibility of water vapor uptake from the atmosphere, a mechanism which would be facilitated by dew, fog, undertemperature, and the osmotic potential of the Welwitschia leaf. As we have no idea of the amount of water that can be gained by this mechanism, we must leave the question open whether it contributes to the water economy of Welwitschia or not. But it seems rather unlikely.

IV. PHOTOSYNTHESIS AND CARBON BALANCE The atmosphere CO, contains 98.9% I2CO2 and 1.1% I3CO2. One of the C0,-fixing enzymes, ribulose 1,5-bisphosphate carboxylase/oxygenase (“RUBISCO,” Lorimer, 198I), discriminates against 13C while another, phosphoenolpyruvate carboxylase (PEPC) does not. Consequently, C, plants have less of the heavy C isotope in their dry mass than C, plants. As CAM plants take up CO, via both enzymes, their content of the heavy isotope 13C is intermediate and values range between those of C, and C, plants. For several years it was assumed that the determination of the portion of l3C of the total carbon which is expressed as the value Si3Callows a classification of the plant in question as C,, C,, or CAM. However, this simple conclusion is no longer completely valid. As shown in Fig. 15, Welwitschia exhibits Si3C values that place it in the group of CAM plants. In the whole range of habitats, the 8I3C values of Welwitschia differ significantly from those of the accompanying C, plants (Schulze et al., 1976). The findings of Dittrich and Huber (1974) that greenhouse-grown Welwitschia incorporates 14C02 in the dark much better than in the light and that there is a diurnal rhythm of acidification and deacidification with an amplitude of 12 peqig fresh weight (total acidity), support this conclusion. On the other hand, preliminary experiments in the Namib Desert (Gaff, 1972) gave no sign of CAM. The stomata were found to be open during the day and not at night, which is consistent with the high daytime transpiration (Walter, 1936; von Willert et al., 1982). In the meantime, several new findings also convincingly demonstrate that, in the natural habitat, Welwitschia does not have CAM features. For the first time diurnal measurements of the gas exchange of Welwitschia in the Namib Desert are available (von Willert et al., 1982). They make clear that

180

-11

1

-I

-13-15-

- -17. ” -190

0, I*

‘a

-21

-

-23

..

-25

0

r

20

Coastal desert

1

I

I

1

1

1

40

60

80

100

120

140

Grassland

I

(km)

Savanna

Fig. 15. 613C values of C4 and C3 species and of Welwitschia mirabilis as related to the distance from the coast and the according vegetation zones in the northern Namib. (Data taken from Schulze et ai., 1976.)

Welwitschia does not have a net CO, uptake in any part of the night. These measurements took into account different climatic night conditions (i.e., water vapor partial pressure deficit), leaf age, and water supply. All these factors are known to affect nocturnal CO, uptake of CAM plants (Jones, 1975; von Willert et al., 1976, 1980; von Willert, 1979; Osmond, 1978; Hanscom and Ting, 1978). Figures 16 and 17 give two examples. The gas exchange of Welwitschia is that of a C, plant under water stress, which is best documented in Fig. 17. As gas exchange measurements only answer the question of the CAM criterion “net CO, uptake in the night,” a concerted effort was made to test the possibility of a partial refixation of respiratory CO, (Szarek and Ting, 1974). This refixation should become visible in an accumulation of acids overnight. Figures 18 and 19 compare the contents of malate and citrate in four Welwitschia plants in the evening and morning. There is no indication of acidification overnight. Again the specific distribution of the acids with leaf age can be seen and is further illustrated three-dimensionally in Fig. 20. The large variations in acid distribution with leaf age emphasize the difficulty of taking representative samples in physiological experiments. In contrast to Dittrich and Huber (1974) but consistent with a recent report (Ting and Burk, 1983) only a small uptake of I4CO, in the dark was found with

181

Wclwitschia mirabilis

l i . . . . . . . . . . . . . . . . . . . . . . . . I

351

Air temperature

30 1

25

i

t

:1

, Relative humidity ~

40

,

-

8

- 30-

lo

i

61 - I 0

1

40 1

Cuvette temperature

30

\g/

w lo00 800

Global radiation 200

19 20 21 22 23 24

1

2

3

4

5

6

7

8

9

10 1 1

12 13 14 15 16 17 18 19 20

0

Local time lhr)

Fig. 16. CO2 gas exchange of a horizontally growing leaf of Welwirschiu mirubilis; 9-17 cm: about I-year-old leaf part; 65-73 cm: about 6-year-old leaf part, plus related experimental data. (After von Willert er a l., 1982.)

D.J. VON WILLERT

182 3

-

1 OM)

H 2 Global radiation

'E s"l

E 0

CO release

I

.O

0 J

Relative humidity

30 -

.

- 20. 0

'

$ 50

.

e

10. 0 - . .

,.

. . . . . . . . . . . . . . . . . . . . . . . .0

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

4

b

-- 3

Global radiation

'#

1000

:2

8 -

500

3

-$ 1 0

0

100

50

01.. 6 7

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

8

9

10 1 1

12 13 14 15 16 17 18 19 20 21 22 23 24 Local time (hrl

1

2

3

4

5

0

Fig. 17. C02 gas exchange of Welwitschia mirabilis during a long drought period (a) and after irrigation (b). (After Eller er al., 1983.)

E

I83

Welwitschia mirabilis

0

20

40

60

0

20

40

60

80

Distance from leaf base (crn) Fig. 18. Distribution of malic acid in leaves of four Welwitschia mirahilis plants in relation to leaf age and day time. Error bars as for Fig. 8.

240

200 160

-

120

W

2>

L

U

80 40

7

0

.-g

200

-

160

'F 246 +

Plant 23

0

f -3

120 80

40 I

O J , ,

0

I

20

,

1

40

,

1

,

60

0

20

40

60

80

Distance from leaf base (crn) Fig. 19. Distribution of citric acid in leaves of four Welwirschia mirabilis plants in relation to leaf age and day time. Error bars as for Fig. 8.

184

D. J. VON WILLERT

.200 .180

.160 & 1140 ,120

,100

.80 .60

f 6

-

-5

.40 '

20

.O

Leaf base

Fig. 20. Distribution of citric acid in a single leaf of Welwitschia rnirabilis shown in a threedimensional diagram.

Welwitschia in the natural habitat. The incorporation in the light was 100-fold higher (von Willert et al., 1982). Of the small amount that was exchanged during the night 84% was incorporated into the organic acid fraction (only malate and citrate were labeled). The remainder was recovered in amino acids (not fractionated). Irrigation did not increase the nocturnal uptake of I4CO, but altered the amount of label found in the various fractions. Label in organic acids dropped to 68%, that in amino acids to 11%, and 20% appeared in the fraction of lowmolecular-weight carbohydrates, suggesting gluconeogenesis. When an intact, attached leaf of Welwitschia was allowed to fix I4CO, for 60 sec in the morning during the time of highest CO, uptake, the following distribution of label was found: organic acids 42.1%, amino acids 27.0%, sugars 21.4%, and organophosphorous compounds 9.5%. This distribution corresponds with earlier data from excised leaf tissue from greenhouse-grown material exposed to 14C0, at the end of the light period (Dittrich and Huber, 1974): organic acids labeled to 40.9%, amino acids to 12.0%, sugars to 2.3%, and 3-PGA to 16.3%. Irrespective of when during the light period I4CO, was incorporated, it seems

185

Welwitschia mirabilis

TABLE V Percentage Distribution of Label in Organic Acids, Sugars, Amino Acids, and Organic Phosphates in Relation to Leaf Age"

Organic acids

Distance from base (cm)

irri-

Irri-

gated

1 12 26

30.9 53.1 -

Non-

Sugars

gated

Nonirrigated

43.0 27.7 15.3

54.7 31.7 -

Amino acids

Organic phosphates

gated

Nonirrgated

Irrigated

Nonirrigated

Irrigated

37.0 56.7 65.6

12.5 13.9 -

16.6 14.0 16.5

4.5 4.1 -

3.4 1.6 2.5

Im-

a After a 14C02 feeding experiment of 15 min. The irrigated plant was watered with an equivalent of 80 mrn precipitation.

that organic acids were the most highly labeled fraction. In contrast to night fixation, this label was entirely recovered in malic acid, which might suggest the possible operation of the Krebs cycle in the light. Distribution of label in various fractions varies with leaf age (Table V). Surprisingly, the gradients in organic acids and sugars were reversed in irrigated plants. An important question concerns the malate pool. Is this pool stationary or has it a considerable turnover? The available information is limited to a single pulsechase experiment with a pulse of 45 min and a chase of the same length (Table VI) . The shift of label from organic acids to sugars might be taken as an indication of a prevalent turnover of malate, but further detailed pulse-chase experiments are necessary to work this out properly. Nevertheless, there are additional hints for a turnover in the malate pool. Its remarkable labeling in all experiments performed in the light is consistent with the high activity of PEPC reported for Welwitschiu (Dittrich and Huber, 1974). Furthermore, the considerable activity TABLE VI Percentage Distribution of Label in Organic Acids, Sugars, Amino Acids, and Organic Phosphatesa

Pulse of 45 min Pulse of 45 min plus chase of 45 min

Organic acids

Sugars

Amino acids

Organic phosphates

57.0 46.3

31.5 49.3

7.4 4.1

5.0 0.3

Determined immediately after a I4CO2 feeding of 45 min and again after a chase of a further 45 min.

186

D. J. VON WILLERT

of the malate decarboxylating malic enzyme in Welwitschia (Dittrich and Huber, 1974), together with the shift of label from malate to sugars, might indicate that malate acts as a form of a temporary CO, store. For several C, plants of the southern Namib, a malate formation at dawn together with stomatal opening was observed (von Willert et al., 1984a). With stomatal closure at midday or in the afternoon, the malate disappeared again. Nothing is so far known about similar reactions in Welwitschia. There are principally two functions for organic acids in a plant cell. The first is their role as counter ions in balancing an excess of cations. We observed that the chemical constituents of Welwitschia growing on two sites only 800 m apart had varied significantly only with respect to the contents of sodium and malate, which suggests that malate is indeed the counter ion for sodium. As the amount of inorganic ions should not fluctuate diurnally, the counter ion should not either. This was also found with Welwitschia (Figs. 18 and 19). The second function for organic acids, particularly malate, is to act as a CO, store as in CAM plants, or as a CO, transport form as in C, plants. Since there is no CAM-like acidification/deacidification rhythm in Welwitschiu, malate, which is synthesized in the light, should act preferentially as a temporary CO, store. One would therefore expect I4CO, to enter malate during light fixation but not stay there for a long time. Although neither CO, gas exchange nor acid contents indicate a CAM plant the 6I3C values of the plants we worked with in the Welwitschia Flats were characteristic of CAM ranging from - 17.77 to - 19.64 %o. This corresponds well with the data of Schulze et al. (1976) from the northern Namib. Is this contradictory or can it be explained? One must raise the question: has Welwitschia any features of a C, plant? Whatley (1975) found in chloroplasts of the incomplete bundle sheath of Welwitschia cotyledons a peripheral reticulum which in some respects resembled that of C, plants. The distribution of label following exposure of glasshousegrown plants of Welwitschia to I4CO, for 5 sec resulted in 28.1% label in 3-PGA and 35.3% in malate (Dittrich and Huber, 1974). This distribution is not that expected of a C, plant, nor is it that of a typical C, plant. Short-term pulse chase experiments were not performed, but a turnover in both pools was detected (Dittrich and Huber, 1974). Label in both compounds declined with increasing time. The possibility that photosynthesis of Welwitschia could be intermediate between C, and C,, in a fashion similar to that in several other plants from dry habitats (Kennedy and Laetsch, 1974; Sayre and Kennedy, 1977; Ape1 et al., 1978), has been discussed by Eller et al. (1983). Without a detailed determination of the 0, dependence of CO, uptake, the CO, compensation point, and other important criteria that have been established for C, and C, intermediates (Goldstein et al., 1976), this question cannot be answered conclusively. The following facts are relevant:

187

Welwitschia mirabilis

1. In vitro experiments with RUBISCO revealed that increasing temperature decreases the 6°C value of the products from -33.7%0 at 24°C to - 18.3%0at 37°C (Goodwin and Mercer, 1983). 2. According to Dittrich and Huber (1974) the RUBISCO to PEPC ratio is 1.5 for Welwitschiu,while it is 15.8 for the C, plant wheat, and Latzko e t a l . (1979) have shown that this ratio determines the SL3Cvalue in wheat and oat. 3. Organic acids account for about 6% of the total dry mass of a Welwitschiu leaf and have less negative S13C values than other cell constituents (Ziegler, 1979).

b

100-

50

CO, gain

fi:a

*.: : :a

-

'L

=

d N

-0

-5

#;:

o

: co,

loss

-50i

.

1

.

1

.

,

.

1

.

1

.

1

.

1

.

r

+

188

D. J. VON WILLERT

4. Respiration involves preferential loss of 13C, with the remaining organic carbon becoming heavier (Smith, 197I). 5. A leaf cell originated by the meristem needs at least 3 months before it comes into sunlight in Wefwitschiu. During this period, CO, fixation via the nondiscriminating PEPC is the only possible way of CO, fixation. 6. Increasing drought and other environmental factors influence the 613C value of plants (Schmidt and Winkler, 1979; Farquhar el a f . , 1982). These may well explain the high content of the heavy isotope 13C in the dry matter of Welwitschia. Consequently, taking all these arguments into account, together with the reported CO, gas exchange, absence of day-night oscillation of acids, transpiration, and leaf conductance, the measured 613C values cannot be taken as evidence of CAM. If we look at the photosynthetic capacity of a Welwitschia leaf and the carbon balance over a period of 24 hr, it becomes obvious that all leaf ages are able to take up CO, during parts of the day, but with increasing leaf age the CO, uptake decreases (Fig. 21). The CO, balance over a period of 24 hr shows clearly that only the first half of the leaf has a carbon gain while the older parts show a carbon loss. These parts may live at the expense of the younger parts. Nevertheless, the balance for the entire leaf is still positive. This is presumably how the die-back process at the tip is regulated. The overall balance must be positive or if it is not the leaf length has to be reduced. At first glance, this behavior seems wasteful. However, within a short time irrigation will improve CO, uptake (Fig. 17). This was obtained for all leaf ages and means that when water supply is good all-even the old parts-will contribute to a carbon gain. A plant with such a slow growth rate as Welwitschia must keep as much of the leaf blade alive as possible. Only this guarantees an immediate response to improved conditions. It is impossible for Wefwitschiu to rely or depend on growth to meet fluctuating environmental conditions. Welwitschia behaves very opportunistically but successfully. V. ENERGY BALANCE

It is generally accepted that plants in desert environments are characterized by small leaves. The lack of sufficient transpiration, together with the high solar radiation, would increase leaf temperatures to a lethal level unless the efficiency of convective energy transfer is kept high. Consequently, small leaves are considered to be an important adaptation. Welwitschia is an exception. A first calculation of the energy balance of Welwitschiu in its natural habitat has been made by Schulze et ul. (1980). The most important feature is without doubt the high reflectivity of the Welwitschia leaf. Only 55% of the incident global radiation is absorbed and as much as 40% is reflected, in contrast to most mesophytic leaves that reflect only 20-25%. This

Welwitschia mirubilis

I89

is in fact the best strategy a desert plant with large leaves can have. And it might be the high reflectivity that allows Welwitschiu to stretch the leaves horizontally. A second point is that the temperature of the soil shaded by the leaf is cooler than the temperature of the lower leaf surface, which results in a net loss of long wave radiation. A more detailed investigation (Eller et ul., 1983) revealed that, contrary to earlier assumptions, transpiration is not negligible and plays an important role in energy dissipation. As much as 14%of the total energy input is balanced by transpiration. The measured high transpiration of Welwitschiu is without doubt a waste in an arid environment, but necessary to prevent lethal temperatures of the leaf. Where (due to water shortage) this transpiration cannot be maintained the tissue dies, probably due to lethal temperatures; perhaps this is the reason the tip dies. Besides the negative carbon balance, lethal temperatures at the leaf tip may regulate leaf length.

VI . CONCLUDING REMARKS If botanists should construct a plant best adapted to a desert environment, they would never come up with a monster like Welwitschiu mirubilis. In a desert it seems suicidal to have such large evergreen leaves. The water storage capacity is limited. Despite earlier assumptions, Welwitschia is not a CAM plant. This means CO, is taken up during the day via open stomata. A tremendous loss of water is the consequence. The existence of Welwitschia is a challenge for a botanist because no survival strategy is visible and yet the plant was able to survive two rainless years during which all the other plants died off completely. The key to understanding the biology of this peculiar plant must lie in the fact that Welwitschia normally does not suffer from water shortage but has connection to water sources in the soil which cannot be exploited by other plants. Once a sufficient water supply is guaranteed a large leaf can be maintained, a high transpiration is possible, and this prevents lzthal leaf temperatures. If the stomata can be opened during the day, it is not necessary to perform a CAM photosynthesis. If the anatomy, morphology, and physiology of Welwitschia mirabilis are taken into account, the species name mirubilis seems to be the only justified one. REFERENCES Ahmad, J . , Larher, F., and Stewart, G. R . (1979). New Phytol. 82, 671-678. Albert, R . , and Popp, M . (1978). Oecol. Plant 13, 27-42. Apel, P., Ticha, J . , and Peisker, M. (1978). Biochem. Physiol. Pji’unzen 172, 547-552. Bornman, C. H., Elsworthy, J . A., Butler. V . , and Botha, C. E. J . (1972). Mudoqua 1, 53-66. Bornman, C. H., Botha, C. E. J . , and Nash, L. J. (1973). Madoqua 2 , 63-68. Briens, M . , and Larher, F. (1982). Plant Cell Environ. 5 , 287-292.

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Dittrich, P., and Huber, W. (1974). Proc. Inr. Congr. Photosynth. (M. Arron, ed.). Elsevier, Amsterdam. Eller, B. M., von Willert, D. J., Brinckmann, E., and Baasch, R. (1983). S. Afr. J . Bot. 2, 209223. Farquhar, G. D., Ball, M. C., von Caemmerer, S., and Roksandie, Z. (1982). Oecologia 52, 121124. Gaff, D. J. (1972). Dinteria 7 , 3-7. Giess, W. (1969). Dinteriu 3, 3-55. Goldstein, L. D., Ray, T. B., Kestler, D. P., Mayne, B. C., Brown, R. H., and Black, C. C. (1976). Plant Sci. Lert. 6, 85-90. Goodwin, T. W., and Mercer, E. I. (1983). “Introduction to Plant Biochemistry.” Pergamon, Oxford. Gorham, J., Hughes, L. L., and Wyn Jones, R. G. (1981). Physiol. Plant. 53, 27-33. Hanscom, Z., and Ting, 1. P. (1978). Oecologiu 33, 1-15. Hellebust, Y. A. (1976). Annu. Rev. Plant Physiol. 27, 485-505. Hooker, J. D. (1863). Trans. Linn. Soc. N . S . W. 14, 1-48. Jones, M. B. (1975). Planta 123, 91-96. Kennedy, R. A., and Laetsch, W. M. (1974). Science 184, 1087-1088. Ken, L. E. (1967). Svensk. Bot. Tidskr. 61, 97-125. Kinzel, H. (1982). “Ptlanzenokologie und Mineralstoffwechsel.” Ulmer, Stuttgart. Kirst, G. O., and Bisson, M. A. (1979). Aust. J. Plant Physiol. 6 , 539-556. Schmidt, H. L., Winkler, F J., and Fischbeck, Latzko, E., Kelly, G. J . , Wirth, J . , Meyer, A. 0.. G. (1979). Ber. Dtsch. Bot. Ges. 92, 153-156. Lorimer, G. H. (1981). Annu. Rev. PLnt Physiol. 32, 349-383. Luttge, U., Smith, J. A. C., and Marigo, G. (1982). In “Crassulacean Acid Metabolism” (I. P. Ting and M. Gibbs, eds.), pp. 69-91. Am. SOC.Plant Physiol. Markgraf, F. (1926). In “Naturliche Pflanzenfamilien” (Engler und Prantl, eds.), Vol. 13, pp. 419429. Marsh, B. (1982). Namib Bull. Suppl. 4, 3-4. Munns, R., Greenway, H., andKirst, G. 0. (1983). Encycl. Plant Physiol. New Ser. 12C, 59-135. Springer-Verlag. Heidelberg, New York. Osmond, C. B. (1978). Annu. Rev. Plant Physiol. 29, 379-414. Richter, H. (1976). Ecol. Stud. 19, 42-58. Rodin, R. J. (1958a). Am. J . Bor. 45, 90-95. Rodin, R. J. (1958b). Am. J . Bot. 45, 96-103. Sayre, R. T., and Kennedy, R. A. (1977). Plunra 134, 257-262. Schmidt, H. L., and Winkler, F. J. (1979). Ber. Dtsch. Bot. Ges. 92, 185-191. Schulze, E.-D., Ziegler, H., and Stichler, W. (1976). Oecologia 24, 323-334. Schulze, E.-D., Eller, B. M., Thomas, D. A., von Willert, D. J., and Brinckmann, E. (1980). becologia 44, 258-262. Schulze, E.-D., Hall, A. E., Lange, 0. L., and Walz, H. (1982). Oecologia 53, 141-145. Shackel, K. A. (1984). Plant Physiol. 75, 766-772. Smith, B. N. (1971). Plant Cell Physiol. 12, 451-455. Stewart, G. R., and Lee, J. A. (1974). Planta 120, 279-289. Stocker, 0. (1929). Ber. Dtsch. Bot. Ges. 47, 126-136. Szarek, S. R., and Ting, I. P. (1974). Planr Physiol. 54, 76-81. ring, I. P., and Burk, J. H. (1983). Plant Sci. Lett. 32, 279-285. Treichel, S., Brinckmann, E., Scheitler, B., and von Willert, D. J . (1984). Plunru 162, 236-242. von Willert, D. J. (1979). Ber. Dtsch. Bot. Ces. 92, 133-144. von Willert, D. J., Kirst, G. O., Treichel, S., and von Willert, K. (1976). Plant Sci. Lert. 7, 341346.

Welwitschia mirabilis

191

von Willert, D. J . , Brinckrnann, E., Scheitler, B., Schulze, E.-D., Thomas, D. A., and Treichel, S. (1980). Natunvissenschaften 67, 21-28. von Willert, D. J., Eller, B . M.. Brinckmann, E., and Baasch, R . (1982). Oecologia 55, 21-29. von Willert, D. J . , Brinckrnann, E., Scheitler, B . , and Eller. B. M. (1984a). Oecologia, in press. von Willert, D. J., Brinckmann, E., Eller, B . M . , and Scheitler, B. (1984b). Planta 61, 393-397. Walter, H. (1936). Jb. Wiss. Bot. 84, 58-221. Walter, H. (1960). “Einfuhrung in die Phytologie,” Vol. 3 . Ulrner, Stuttgart. Whatley, J. M. (1975). New Phytol. 74, 215-220. Ziegler, H. (1979). Ber. Drsch. Bot. Ges. 92, 169-184.

This Page Intentionally Left Blank

AUTHOR INDEX The numbers in italics indicate the pages on which names are mentioned in the reference lists.

A

Barker, C. A. V., 50, 66 Bassharn, J . A,, 85, I f 8 Abel, K. M., 91, 92, 95, 118 Bayley, P. M., 44, 69 Acton, J. D., 33, 67 Bean, R. C., 85, 118 Ahmad, J., 169, 189 Beardall, J . , 72, 77, 81, 82, 86, 87, 95, 96, Akagawa, H., 86, 87, 88, 109, 110, 11 I, 115, 97, 99, 100, 102, 103, 109, 113, 118. 119. 118, 121 122 Akazawa, T., 79, 90, 91, 92, 118 Bemiller, J . N., 126, 127, 155 Albersheim, P., 127, 152, 153 Benbasat, J. A , , 38, 65 Albert, R., 169, 189 Bender, M. M., 104, 105, 106, 107, 108, I f 8 Aldrovandi, S., 4, 65, 69 Benedek, G . B., 61, 69 Al-Faour, 0. M.. 27, 66 Benson, A. A,, 85, 118 Allen, N. S., 6, 7, 65 BergC, P., 15, 50, 65 Allen, R. D., 4, 6, 65, 69 Berman, H. J . , 61, 67 Anderson, E., 130, 153 Berne, B. J., 15, 69 Andrews, P., 129, 153 Berry, J., 74, 83, 101, 102, ff9 Andrews, T. J . . 80, 83, 84, 90, 91, 92, 95, Berry, J. A., 84, 120, 121 97, 98, 118, 121 Bewley, J . D., 132, 133, 138, 139, 149, 150, Apel, P., 186, I89 151, 152, 153, 154 Appleby, G., 110, 113, 118 Bhosale, L., 86, I20 Arefiev, I. M., 38, 68 Bidwell, R. 0. S., 79, 80, 86, 96, 97, 98, 99, Arnott, H. J., 132, 154 100, 115, 118, 121 Asakura, T., 33, 61, 68 Billard, R., 15, 50, 65 Ascoli, C . , 53, 54, 55, 65 Bird, I. F., 91, 118 Aspinall, G . 0.. 127, 129, 153 Bishop, D. G., 79, 99, 119 Bisson, M. A., 74, 80, 81, 84, 119, 169, 190 Bjorkman, O., 99, 118 B Black, C. C., 86, 87, 122, 186, 190 Black, C. C., Jr., 98, 99, 104, 105, 106, 107, Baasch, R., 162, 172, 173, 174, 179, 181, 108, 118 182, 184, 186, 189, 190, 191 Blackman, F. F., 79, 118 Badger, M. R . , 80, 83, 84, 90, 91, 92, 97, Bloomfield, V. A., 3, 37, 38, 65 98, 118, 120. 121 Bommenson, J . C . , 101, 121 Bagchi, P., 130. 154 Bonner, R., 61, 65 Bahr, J. T., 90, 91, 120 Boon, J . - P . , 51, 52, 67 Baker, T. S . , 94, 118, I19 Borghi, L., 4, 65, 69 Balasubramaniam, K., 130, 153 Born, G. V. R., 61, 65 Ball, F., 90, 122 Bornmann, C. H., 162, 189 Ball, M. C., 188, 190 Borowitzka, M. A., 80, 118 Barbi, M., 53, 54, 65 Bosq-Rolland, J . , 50, 69 Barclay, G. F., 5, 65 Botha, C . E. J., 162, 189 I93

194

AUTHOR INDEX

Borne, V. L., 94, 118 Bourquelot, E., 126, 153 Bouveng, H. O., 127, 153 Bovee, E. C., 6, 67 Briens, M., 169, 189 Brinckmann, E., 162, 166, 171, 172, 173, 174, 178, 179, 180, 181, 182, 184, 186, 188, 189, 190, 191 Brittain, E. G.. 99, 122 Brokaw, C. J., 6, 65 Brown, D. C., 79, 81, 99, 118 Brown, D. H., 90, 122 Brown, R. H., 186, 190 Bruck, K., 110, 113, 114, 120 Buchan, P. B., 56, 57, 69 Burk, J . H., 180, 190 Burr, G . O., 86, 120 Bums, J. E., 97, 98, 99, 100, 118, 119 Butler, V . , 162, 189 Byme, D., 46, 65

Cohen, A. L., 90, 122 Colbeck, J., 86, 87, 110, 113, 118, 120 Cole, K., 94, 118 Colflesh, D., 45, 67 Colman, B., 81, 121 Colombo, P. M., 80, 119 Cooke, D., 50, 66 Coombs, J., 86, 109, 119 Cornelius, M. 1.. 91, 118 Coughlan, S . , 99, 119 Courtois, J.-E., 131, 153 Cowan, I . R., 102, 123 Craig, H., 105, 106, 107, 119 Craig, T . , 5 1 , 5 5 , 66 Craigie, J. S . , 72, 87, 88, 115, 119 Cran, D. G., 94. 119 Crawford, G. E., 46, 48, 66 Crilly, J. F., 46, 47, 66 C u m i n s , H. Z., 3, 8, 20, 37, 44, 66, 67

D C Caemmerer, S., 79, 118 Calder, J. A., 104, 118 Callow, J. A., 89, 121 Calvin, M., 85, 118 Campbell, J., 130, 134, 137, 138, 152, 153, 153 Canvin, D. T., 91, 96, 118. 121 Careri, G., 4, 69 Carlson, F. D., 3, 45, 65, 66 Carlson, W . S . , 130, 153 Carpenter, E . J., 72, 118 Cathers, I. R., 81, 123 Chartier, P., 79, 118 Chen, S.-H., 3, 16, 49, 51, 66, 68 Chi, E. Y.,94, 118 Choudhury, D., 130, 154 Chu, B., 3, 27, 34, 35, 36, 45, 66, 67 Chudzikowski, R. J., 130, 153 Clark, D. C., 44, 69 Clauwaert, J., 38, 68 Clegg, J., 4, 66 Clegg, J. S . , 4, 66 Clendinning, K. A,, 103, 118 Cobb, A. H., 79, 119 Cochrane, T., 27, 29, 61, 66 Codd, G . A,, 90, 119, 121

Dalgleish, D. G., 42, 68 Dalling, M. J., 89, 121 David, G . , 50, 66, 67 Davies, A. R., 27, 66 Davies, C., 139, 154 Davies, D. D., 111, 119, 123 Dea, I . C . M., 130, 149, 154 De Ambrosis, A , , 4, 65, 69 Degens, E . T., 104, 105, 119 Deguent, P., 50, 67 Denman, H. H . , 11, 66 DeVeau, E. J., 98, 119 Dewey, M. M., 45, 67 Dietler, G., 44, 69 Dietrich, S. M . C., 132, 155 DiGiovanni, P. R., 61, 67 Dinapoli, A,. 34, 66 Dittrich, P., 179, 180, 184, 185, 186, 187, 190 Dodge, J. D., 93, 119 Dolan, T . , 87, 120 Doty, M. S., 99, 119 Downton, W . J. S., 79, 99, 119 Drorngoole, F . I . , 99, 119 Drost-Hansen, W . , 3 , 66 Dubois, M., 50, 66 Dwyer, J. D., 38, 65

195

AUTHOR INDEX

E Earnshaw, J. C . , 3, 14, 16, 29, 30, 36, 39, 40, 45, 46, 47, 48, 52, 60, 61, 63, 64, 65, 66, 67, 69 Edsall, J. T., 73, 119 Edwards, A , , 146, 154 Egle, K., 81, 122 Einav, S., 61, 67 Eisenberg, D., 94, 118, 119 Elfert, T., 130, 146, 154 El Khadem, H., 129, 154 Eller, B. M . , 162, 166, 172, 173, 174, 178, 179, 181, 182, 184, 186, 188, 189, 190, 191 Elsworthy, J. A., 162, I89 English, P. D., 127, 153 En-Shinn, W., 5 , 69 Epstein, S . , 105, 106, 107, 122 Esau, K., 94, 119 Eskov, A. P., 38, 68 Estep, M . F., 91, 92. 93, 102, 105, 119 Evans, L. V., 88, 89, 90, 91, 92, 93, 94, 110, 112, 113, 117, 119, 120, 12J Everson, R. G., 99, 118

F Fagerburg, W. R., 81, 119 Fan. S.-F., 45, 67 Farquhar, G. D., 74, 79, 101, 102, 118, 119, 123, 188, 190 Farrell, K., 90, 121 Feke, G. T., 61. 67 Ferguson, J. F., 79, 119 Finch, E. D., 5 , 67 Fine, S . , 61, 67 Finsy, R., 51, 67 Fischbeck, G., 190 Floyd, G. L., 90, 93, 122 Foissner, I., 6, 67 Ford, N. C . , 58. 68 Forrester, A . T., 20, 67 Frediani, C., 5 3 , 54. 5 5 , 65 Fridman, J. D., 61, 67 Frith, G. J. T., 89, I21 Fry, J., 107, 119 Fry, B., 105, 106. 107, 119 Fuhro, R. L., 61, 67

Fujime, S . , 25, 67 Fulton, A. B., 4

G Gaff, D. J., 173, 179, 190 Garegg, P. J., 127, 153 Gavey, W., 99, 122 Gavis, J., 79, I19 Gee, R . , 87, 120 Gelman, R. A,, 43, 67 Gibbons, I. R., 6, 65 Giess, W . , 160, 172, 190 Glenn, E. P . , 99, 119 Glicksman, M . , 130, 154 Glidewell, S. M . , 75, 77, 80, 81, 98, 122 Glover, H. E., 86, 87, 99, 100, 113, 118, 119 Godfrin, M. J . , 126, 154 Goldberg, R., 129, 130, 154 Goldie, A . H., 112, 119 Goldsmith, H. L., 61, 67 Goldstein, L. D., 186, 190 Goldstein, R. J . , 61, 68 Goodwin, T. W., 187, 190 Gorham, J . , 169, 190 Gould, S . E. B., 146, 148, 154 Gowda, C. A,, 86, 120 Graham, G., 75, 119 Gratton, E., 4, 69 Gray, I. C . , 89, 119 Green, D. J., 37, 5 5 , 56, 57, 69 Green, J. C . , 113, 119 Greenway, H., 169, 190 Greenwood, C . T., 127, 153 Griesmar, C., 46, 68 Griffiths, D. J., 93, 119 Griffiths, H., 102, 103, 109, 118. 122 Grigoriev, V. B., 38, 68 Guillard, R. R. L., 104, 105, 119 Gulari, E., 35, 66 Gulari, E. S., 34, 35, 66 Gupta, P. C . , 130, 154 Gutknecht, J., 74, 80, 81, 84, 119

H Haberli, A , , 44, 69 Hageman, R. H., 99, 121

196

AUTHOR INDEX

Hall, A. E., 175, 176, 190 Hallett, F. R . , 16, 49, 50, 51, 55, 66, 69 Halmer, P., 132, 154 Hamelin, A., 15, 50, 65 Hamilton, D. V., 61, 67 Hanscom, Z . , 180, 190 HBrd, S., 46, 67 Harris, A. Z., 85, 118 Hartmann, R., 5 1, 69 Hartt, C . D., 86, 120 Hartt, C . E., 86, 120 Harvey, J. D., 51, 52, 67, 69 Hashitani, T., 73, 120 Hassid, W. Z . , 85, 118 Hatch, M. D., 86, 87, 119, 120 Haug, A., 89, 119 Hayama, T., 7, 68 Hazelwood, C. F., 5, 67 Hegnauer, R., 129, 154 Heinricher, E., 144, 154 Heldt, H. W., 90, 121 Hellebust, J. A., 86, 89, 104, 105, 119 Hellebust, Y . A., 169, 190 Heller, W . , 1I , 66 Herbert, T . J., 33, 67 Herissey, H., 126, 129, 153, 154 Herpigny, B., 51, 52, 67 Hill, R., 79, 120 Hirst, E. L., 129, 132, 153, 154 Hofmann, N., 51, 69 Hofmann, V., 44, 69 Holdsworth, E. S . , 86, 87, 110, 113, 114, 118, 120 Holdsworth, R. H., 90, 93, 94, 120 Holligan, M. S . , 89, 119 Holm-Hansen, 0.. 98, 118 Holtum, J . A. M., 86, 101, 102, 120, 121 Holz, M., 51, 66 Hooker, J. D., 157, 162, 190 Hopf, H., 129, 154 Homer, H. T., 132, 154 Horowitz, S . B.. 5, 68 Hough, L., 129, 153 Hough, R. A., 96, 97, 120 Huber, W., 179, 180, 184, 185, 186, 187, 190

Hughes, A. I . , 22, 67 Hughes, L. L., 169, 190 Hwang, J. S . , 37, 67

I Ikawa, T . , 86, 87, 88, 91, 92, 109, 110, I I I , 115, 118, 121, 123 Ishiwata, S., 25, 67 Izui, K., 111, 122

J Jackson, G . A , , 103, 120 Jackson, W. A., 96, 97, 120 Jacobi, G., 86, 89, 120 Jacobson, K. A., 5, 69 Jahn, T. L., 6, 67 Jakeman, E., 7, 22, 67 Jakimow-Barras, N., 129, 130, 154 Jarosch, R., 6, 67 Jensen, R. G . , 90, 91, 120 Johnson, C. S., Jr., 10, 67 Johnson, H. S . , 87, 120 Johnson, K. S., 120 Johnson, R. P. C., 5, 29, 31, 32, 37, 38, 65, 67 Johnston, A. M., 81, 82, 11.5, 116, 120, 122 Jones, C . R., 10, 67 Jones, H. G., 79, 120 Jones, J. K. N., 129, 132, 153, 154, 155 Jones, M. B., 180, 190 Jones, R. L., 132, 154 Joshi, G . V., 86, 87, 120 Jouannet, P., 50, 66

K Kageyama, A,, 86, 120 Kamitsubo, E., 7, 67 Kamiya, N., 59, 67, 68 Kandler, O., 129, 154 Kang, S.-M., 89, 120 Kanzig, W., 44, 69 Kaplan, A., 83, 84, 97, 120, 123 Karekar, M. D., 86, 120 Karr, A., 127, 153 Katsuki, H., 1 1 1 , 122 Kaufmann, R., 5 1, 69 Kay, L. D., 85, 118 Kekwick, R . G. 0.. 89, 119

AUTHOR INDEX Kelly, G. J., 190 Kennedy, R. A , , 186, 190 Kerhy, N. W., 88, 89, 90, 91, 92, 93, 110, 112, 113, 117, 120

Kerker, M. L., 11, 68 Kers, L. E., 157, /90 Kessler, G., 129, 153 Kestler, D. P., 186, 190 Keusch, L., 144, 154 Keys, A . J., 91, 118 Khanna, S. N., 130, 154 Kharitonenkov, 1. G . , 38, 68 Kinzel, H . , 163, 190 Kirst, G. 0.. 169, 180, 190 Kirst, J. D., 72, 120 Klages, F., 129, 154 Klimontovich, A . V., 38, 68 Kluge, M., 86, 120 Kogelnik, H . , 8, 68 Kogoshi, K., 73, I20 Kolenbrander, H. M., 1 1 I , 122 Kooiman, P., 126, 130, 131, 154 Kortschak, H. P., 86, 120 Kovacs, P., 130, 154 Kowallik, K., 94, 120 Koyama, T., 33, 61, 68 Kreid, D. K., 61, 68 Kremer, B. P., 72, 81, 86, 87, 88, 89, 90, 92, 99, 100, 110, 1 1 1 , 112, 114, 115, 117,

197

Laver, M. L., 129, 132, 155 Lavery, A . N., 36, 66 Leadheater, B. S. C . , 94, 121 Le Dizet, P., 131, 153, 154 Lee, J. A , , 170, 190 Lehman, R. C., 4, 5, 42, 68 Lekkerkerker, H., 5 1, 67 Levin, W. B., 86, 98, 99, 100, 118 Li, T., 8, 68 Lienard, B., 129 Lindberg, B., 127, 153 Ling, G. N., 4, 68 Liu, T. Y.,34, 66 Lively, J. S., 72, 118 Lloyd, N. D. H., 79, 80, 96, 97, 98, 99, 100, 121

Loomis, W. D., 89, 121 Lorimer, G. H . , 77, 90, 91, 93, 98, 117, 118, 121. 179, 190 Love, A. H . G., 61, 66 Ludtke, M., 129, 154 Ludwig, L. J., 96, 121 Luning, K . , 114, 121, 122 Lutes, R . , 105, 106, 107, 119 Liittge, U., 166, 190

M

120, 123

Kiippers, U., 86, 87, 88, 89, 90, 92, 110, 111, 114, 120, 121, 123

L

McCleary, B. V., 130, 139, 143, 154 McClendon, J. H., 143, 154 McCully, M. E., 80, 121 McFadden, B. A , , 90, 121, 122 MacLachlan, J. L., 79, 80, 96, 97, 98, 99, 100, 121

Lactsch, W. M., 186, 190 Lai, C.-C., 49, 68 Laing, W. A , , 99, 121 Lambers, M. H. R., 57, 68 Lanaras, T., 90, 121 Lange, 0. L., 175, 176, 190 Langevin, D., 46, 68 Langford, G. M., 43, 69 Langley, K. H . , 43, 55, 56, 57, 58, 68, 69 Lanni, F., 5, 69 Larher, F., 169, 189 Larkum, A . W. D., 79, 99, 119 Latzko, E., 190

McLeod, G. C., 86, 119 McNeil, P. L., 5, 69 Maeba, P., 11 1, 122 Maeda, T., 25, 67 Manton, I., 94, I21 Mango, G., 166, 190 Markgraf, F., 159, 190 Marloth, R . , 148, 154 Marlow, J., 61, 67 Marsden, W. J. N., 89, 121 Marsh, B., 160, 190 Maskell, E. J., 79, 121 Matheson, N. K., 146, 154, 155

198

AUTHOR INDEX

Matsui, H., 89, 120 Matsumoto, G., 50, 69 Mayne, B. C., 186, 190 Meier, H.,126, 129, 130, 132, 134, 136, 137, 139, 141, 1.54 Melling, A., 61, 65 Menzel, D., 91, 92, 118 Mercer, E. I., 187, 190 Meyer, A. O., 190 Miller, A. G., 81, 121 Mishina, H., 33, 61, 68 Mitchell, 84, 121 Montgomery, R., 130, 155 Mook, W. G., 101, 121 Moon, R., 81, 119 Moore, L. C., 5 , 68 Moms, I., 72, 86, 87, 89, 90, 93, 99, 100, 113, 118. 119, 121 Morrison, A., 130, 149, 154 Mukerji, D., 86, 87, 89, 90, 93, 99, 100, 113, 118, 121 Mukherjee, A. K., 129, 130, 154 Munns, R., 169, 190 Munme, G., 53, 67 Mure, A., 53, 54, 65 Mustacich, R. V., 5 5 , 56, 57, 58, 59, 68

N Nadelmann, H., 125, 133, 146, 148, 150, 1.54 Nagai, R., 7, 68 Nash, L. J., 162, 189 Neuman, R. D., 46, 67 Nevins, D. J., 127, 153 Newton, S. A,, 58, 68 Nickel, B., 51, 55, 66 Nickel, B. G., 5 5 , 69 Nieuwenhuysen, P., 38, 68 Nikaido, H., 75, 121 Nilsson, G. E., 61, 68 Nisizawa, K., 86, 87, 88, 91, 92, 109, 110, I l l , 115, 118, 120. 121, 123 Nolan, W. G., 143, 154 Nordhom, G., 90, 92, 123 Northam, M., 105, 106, 107, 119 Nossal, R., 3, 49, 50, 61, 65, 66, 68 Nothnagel, E. A,, 58, 68 Numberg, E., 130, 154

0

Oberg, P. A., 61, 68 Odeblad, E., 50, 68 Ogden, J., 105, 106, 107, 119 Ogren, W. C., 99, 121 O'Leary, M. H., 74, 102, 103, 119, 120, 121 Oliver, C. J., 22, 27, 67, 68 Orgenigo, M., 80, 119 Osmond, B., 86, 119 Osmond, C. B . , 79, 86, 91, 92, 99, 101, 102, 103, 118, 119, 120, 121, 180, 190 Overton, J. D., 130, 153

P Paine, P. L., 5 , 68 Palmer, G. R., 43, 44, 61, 67, 68. 69 Panyonis, W. J., 11, 66 Pardue, J. W., 104, 121 Parker, M. L., 148, 154 Parker, P. C., 104, 106, 107, 118, 121 Parker, R. L., 105, 106, 107, 119 Parker, T. G . , 42, 68 Patin, D. L., 129, 132, 155 Peetermans, J., 51, 67 Peisker, M., 103, 121, 186, 189 Peoples, M. B., 89, 121 Percheron, P., 129, 155 Percival, E. G. V., 129, 153 Peterson, B. J., 72, 121 Petracchi, D., 53, 54, 65 Pickard, W. F., 57, 68 Picton, J. M., 39, 40, 42, 68. 69 Piddington, R. W., 38, 5 5 , 68 Piez, K. A , , 43, 67 Pike, E. R., 3, 22, 66, 67 Pollard, E. C., 4, 5 , 42, 68 Popp, M., 169, 189 Porter, K. R., 4, 69 Possingham, J. V., 94, 119 Pravencher, S. W., 26, 27, 33, 40, 68, 69 Purohit, K., 90, 122 Pusey, P. N., 3, 7, 16, 67, 69

R Racey, T. J., 51, 55, 66, 69 Rackham, 0.. 74, 122

AUTHOR INDEX

Radford, E. P . , Jr., 74, 122 Ragan, M . A . , 89, 122 Ragoni-Kiibbeler, M . , 87, 114, 123 Randall, D. D., 95, 122 Rao, C. V. N., 129, 154 Rashbrook, R . B . , 129, 153 Raven, J . A., 72, 74, 75, 77, 79, 80, 81, 82, 83, 84, 86, 95, 96, 97, 98, 99, 100, 101, 102, 103, 109, 115, 116, 118. 120, 122 Ray, T. B . , 86, 87, 122, 186, 190 Rees, D. A . , 146, 148, 154 Reese, E. T., 139, 154 Reid, J . S. G., 126, 130, 131, 132, 133, 134, 136, 137, 138. 139, 141, 146, 148, 149, 150, 151, 152, 153, 153, 154 Reinhold, L., 84, 120 Reiss, R., 125, 126. 144, /.54 Rettig, E.. 130, 154 Rhodes, M. J . C.. 89, 122 Richter, H., 162. 190 Riva, C. E., 61, 67 Robbins, J . B., 34, 66 Robic, D., 129, 131, 153, 155 Rodin, R. I . , 162, 190 Roksandie, Z . , 188, 190 Rosenberg, E. Y . , 75, 121 Ross, D., 5 5 , 68 Ross, D. A . , 29, 31. 32, 67 Rott, J . , 79, IIY Rupley, J. A , , 4, 69

S Sachs, J . , 145, 155 Sackett, W. H., 104, 105, 119 Sackett. W. M., 104, 105, 108, 123 Saini, H . S., 146, 154, 155 Salenid, E. G . , 61. 68 Salisbury, J . L., 90, 93, 122 Sallem, M. A . E . , 129, 154 Saltman, P . , 87. 120 Sanwal, B . D., I l l . 112, 119, 122 Sato, M.. 4, 6 9 Sattelle, D. B.. 38, 43, 44, 55, 56, 57, 58. 61. 67. 68. 69 Saunders, R. E., 90, 122 Saxena, V. K., 130, 155 Sayre, R. T., 186. I90 Scalar, R. S., 104, I21

199

Schaeffer, D. W., 15, 69 Scheitler, B., 166, 171, 172, 180, 186, 190, 191 Schlegel, R. A., 5 , 69 Schleiden, M. J . . 126, 155 Schmidt, H. L., 188, 190 Schmitz, K., 87, 114, 115, 121. 122, 123 Schneerson, R., 34, 66 Schulze, E., 131, 146, 155 Schulze, E.-D., 175, 176, 178, 179, 180, 188, 190, 191 Seiler, A , , 142, 155 Sellen. D. B., 5, 69 Serres, C., 50, 66, 67 Seybold, A., 81, 122 Shackel, K . A , , 162, 190 Sheetz, M. P., 58, 69 Shepherd, D. C., %, 98, 99, 100, 118 Shibata, Y . , 139, 154 Shimizu, H., 50, 69 Shively. J . M . , 90, 122 Shuh, S. W . , 94, 118. 119 Siegel, D. P., 37, 69 Sikes, C. S., 108, 122 Siniakov, M. S., 38, 68 Skirrow, G . , 73, 122 Small, I . V . , 4, 69 Smart, C . L., 130, 155 Smillie, R. M., 75, 119 Smith, A. M . , 79, 118 Smith, B. N., 105, 106, 107, 122, 188, 190 Smith, F., 130, f55 Smith, F. A,, 77, 79, 81, 82, 103, 122. 123 Smith, J . A. C., 166, 190 Smith. W. W., 94, 119 Sprey, B . , 94, 122 Spudich, J . A , , 58, 69 Staverman, W. M., 101, 121 Steeman Nielson, E., 122 Steer, M. W . , 3, 16, 29, 30, 38, 39, 42, 63, 64, 67. 68. 69 Steiger, E., 131, 146, 155 Steiner, R . . 51, 69 Stepanenko, B. N., 130, 155 Stewart, G. R., 169, 170, 189, 190 Stewart. W. D. P., 90, 119 Stichler. W., 179, 190 Stocker. O., 176, 190 Strdub, P. W . , 44, 69 Sturgeon, R. J . , 127, 153

200

AUTHOR INDEX

Swinney, H. L., 8, 66 Szarek, S. R., 180, 190

T Tabita, F. R., 90, 91, 92, 93, 102, 105, 119, 122 Taguchi, M., 111, 122 Takaki, M., 132, 155 Tanaka, T., 61, 69 Tanner, J. E., 5 , 69 Tartaglia, P., 51, 66 Tattersfield, D., 99, 119 Taylor, D. L., 5 , 69 Tazawa, M., 6, 57, 69 Tenland, T., 61, 68 Terborgh, J., 86, 119 Tersky, B., 87, 114, 123 Thomas, D. A., 178, 180, 188, 190, 191 Thomas, E. A., 79, 99, 122 Thompson, J. L., 129, 155 Thompson, W., 53, 67 Thorpe, T., 132, 154 Ticha, I., 186, 189 Ting, I. P., 86, 120, 180, 190 Titus, J. S . , 89, 120 Tolbert, N. E., 72, 95, 99, 122 Tominaga, Y.,6, 69 Tosteson, D. C., 74, 80, 81, 84, 119 Traub, A , , 53, 67 Tregunna, E. B., 79, 81, 99, 118, 122 Treichel, S . , 171, 180, 190, 191 Troughton, J. H., 101, 122 Truby, E., 81, 119 Tschirch, A , , 125, 155 Turner, J. S . , 99, 122

U Utter, M. F., 111, 122

V van Baalen, C., 91, 92, 93, 102, 104, 105, 119. 121 Vamer, J. E., 141, 155 Vaughan, J. M . , 3, 7, 16, 67, 69 Vesk, M., 80, 118

Villa, M., 4, 65, 69, Vogel, T., 125, 155 Volk, R. J., 96, 97, 120 Volochine, B., 15, 25, 50, 65, 66, 67, 69 von Cammerer, S., 188, 190 von Willert, D. J., 166, 171, 172, 173, 174, 178, 179, 180, 181, 182, 184, 186, 188, 189, 190, 191 von Willert, K., 162, 180, 190

W Walder, W. O., 132, 154 Walker, N. A., 79, 122 Walker, N. W., 74, 81, 103, 123 Walter, H., 162, 163, 169, 172, 177, 179, 191 Walz, H., 175, 176, 190 Wmg, Y.-L., 5, 69 Ware, B. R., 5, 14, 37, 38, 55, 56, 57, 58, 59, 68, 69 Webb, W. W., 58, 68 Weidner, M., 88, 90, 92, 110, 111, 114, 120, 121, 123 Wenzler, H.F., 143, 154 Westhead, E. W., 37, 69 Whatley, J. M . , 186, 191 Wheeler, W. N . , 79, 103, 123 Whistler, R. L., 126, 127, 128, 130, 155 Whitelaw, J. H., 61, 65 Whittingham, C. P., 79, 120 Wiebe, H. H., 151, 155 Wight, N. J., 146, 148, 154 Wilber, K. M., 108, 122 Willenbrink, J . , 72, 86, 87, 88, 111, 114, 115, 120, 121, 122, 123 Williamson, I . R., 129, 153 Wilson, A. T., 85, 118 Wilson, M. C., 52, 69 Wiltzius, P., 44, 69 Winkler, F. J., 188, 190 Winterstein, E., 126, 155 Wirth, J., 190 Wittenbach, V. A., 89, 123 Wohlfarth-Bottermann, K. E., 6, 60, 69 Wojcieszyn, J. W., 5 , 69 Wolfrom, M . L., 126, 127, 128, 129, 132, 155 Wolosewick, J. J., 4, 69 Wong, C. S . , 102, 123

20 1

AUTHOR INDEX

Wong, K. F., 1 1 I , 123 Wong, T . Z . , 4, 69 Wong, W . W . , 104, 105, 108, 123 Woo, K . C . , 121 Woolford, M. W.. 51, 67, 69 Wyn-Jones, R . G . , 169, 190

Y Yamada, T., 91, 92, 123 Yamaguchi, T., 86, 123

Yokohama, Y . , 86, 120 Yomo, H . , 141, 155 Yu, H., 38, 69

Z Zenvirth, D., 83, 84, 97, 120, 123 Ziegenfuss, E. M . , 130, 153 Ziegler, H . , 179, 187, 191

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SUBJECT INDEX A

Chiro-inositol, 169 Chlamydomonas, 55 Chloride, 162- 166 Chlorophytes, 75, 87, 91 Chondrus crispus, 80 Chrysophytes, 75 Coffea arabica (coffee), 129, 132 Coherence time, 8 Colutea brevialata, 133 CONTIN, 27, 40 Cryptophytes, 75 Codium fragile, 79 Cyanopsis psoruliodes (guar), 134, 139, 143 Cyclamen eruopaeum, 146 Cylindrotheca sp., 91, 93 C. closterium, 113 Cytoplasmic streaming, 6-7, 38, 57-60

Acacia erioloba, 176 Acetabularia mediterranea, 100 Amoeba proteus, 6, 10 Amphidinium carterae, 113 Amyloids, 126, 131, 144, 146 Anabaena variubilis, 83-84, 95 Annona muricata, 13I Arihraerua leubnitziae, 172 Ascophyllum nodosum, 81, 83.88, 99, 112, 115, 117 Asparagus officimlis, 129 Asterias, 51

B Bacillariophytes, 75 Blood flow, 60-62

Briggs-Haldane/Michaelis-Menten relationship, 76-77, 79 Brownian motion, 14 Bryopsis maxima, 91 C Calcium. 162-166 Carbohydrates low-molecular-weight, 169 in seeds, 125- I55 Carbon metabolism in marine algae, 71-123 P-Carboxylases, 109- 1 13 Carum carvi, 129 Caulerpa verricillata, 97 Cell wall storage carbohydrates in seeds, 125155

biological function, 148- 15 I structure, 126- 132 Ceratonia siliqua (carob), 126, 133, 141-143 Cercis siliquastrum (Judas tree), 130 Cervical mucus, 50-51 Chaetoceros sp.. 102 C. calcitrans, I 13 Chaetomorpha crassa. 99 Cham. 6, 55

D Dictyota dichotoma, 1 1 I Diffusion, 3-6, 14 Dinophytes, 75 DISCRETE, 33, 34, 36, 40 Doppler shift, 2, 13, 15, 19, 21 Dunaliellu salina, 83, 84 D. tertiolecta, 98, 100, 113

E Elodea, 6, 55, 60 Endymion nutans, 129 Enteromorpha intestinalis, 99 E. linza, 99 E. iubulosa, 86 Eremosphaera viridis. 93 Escherichia coli, 51, I 12 Euchuma, 99 Euglena gracilis, 53-55

F Fucus gardneii, 99

F. serratus. 89, 99

203

204

SUBJECT INDEX

G Galactan, 131-132, 146-148, 152 Galactomannan, 129-131, 133-143, 149, 153 hydrolysis, 143 Galactose, 126 GifSordia mirchellae. 91 Gigarrina larissima, 99 Glenodinium, 97 Glucomannan, 129- 13I , 149 Glucose, 126 Gonyaulux tamarensis, 100 Granularity, see Speckle Gymnodinium sp., 1 I3

H Haemococcus. 55 Halimeda cylindricea, 79, 91. 93 Halymenia durvillaei, 103 Hernicelluloses, 125-155 Heterodyne systems, 19, 21 Honkenya peploides, 169

L. angustifolius, 131, 146-148 L . lureus. 131, 146, 148

M Macrocystis, 103 Mannan, 129-131, 144, 149 Mannose, 126, 129 Marine algae, 7 I - I23 carbon fixation, 85-88 carbon isotope discrimination, 101- 108 carbon metabolism in, 7 1 - 123 Methylation analysis, 128, 129 Micromonas sqrramata, 93 Motility, 6-7, 48-62 algal, 53-55 spermatozoal, 49-53

N Nitella, 6, 55-57 N . flexilis, 55 N . opaca, 56 Nuclear magnetic resonance (NMR), 4, 5

I Impatiens balsamina, 131, 144 Inorganic carbon system, 72-75 Intensity fluctuation spectroscopy, 21 Iridaea cordata. 99 Iridophyccus flaccidum, 86 Iris ochroleuca, 129 I . sibirica, 129

L Lactuca sariva (lettuce), 132 Laminaria digitata, 89 L. hyperborea, 111, 114 Laser beams, properties of, 7-8 Laser Doppler microscopy, 3, 28-37 instrument design, 28-33 Laser light scattering, 1-69 biological applications, 37-62 blood flow, 60-62 conventional, 10- 12 dynamic, 12-15 membranes, 45-48 optical mixing spectroscopy, 2, 3, 16-25 particle characterization, 37-42 particle interaction, 42-45 principles of, 7-28 Limulus, 45 Lupinus albus, 131, 132, 146, 148

0 Optical mixing spectroscopy, 2, 3, 16-25

P Palmaria palmata, 99 Paragalaktan, 131 , 146 Pavlova lutheri, I 13 PCOC, see Photosynthetic carbon oxidation cycle PCRC, see Photosynthetic carbon reduction cycle Pelvetia canaliculara, 99 PEPC, see Phosphoenolpyruvate carboxylase pH drift, 99 Phaeodactylum tricornutum. 87, 89, 93, 113, 114 Phaeophytes, 75, 87, 88, 91, 117 C4 metabolism, 114- 1 15 Phaseolus vulgaris, 89 Phoenix dactylifera (date palm), 129, 144 Phosphoenolpyruvate carboxylase (PEPC), 87, 102, 111, 117, 179, 185, 187, 188 Photon correlation, 21-25 Photosynthetic carbon oxidation cycle (PCOC), 94-100, 117 Photosynthetic carbon reduction cycle (PCRC), 85-86

205

SUBJECT INDEX Photosynthesis, 179- 188 Physarum. 4, 6, 55, 58-60 P . polycephalum, 58 Phytelephas macrocarpa (ivory nut), 126, 129 Pilayella littoralis, 89, 91, 93 Plantago sp.. 169 Pol.weura lastissirna, 99 Polysaccharides distribution, 128- 132 hydrolysis, 127- 128 types, 128-132 Polystyrene latex microspheres, 33-37, 57 Porphyra umbicalis. 99 P . cruentum, 113 Porphyridium sp., 98 Potassium, 162- 167 Proline, 170- I72 F'rymnesophytes, 75

Synechoroccus spp., 80, 83, 84, 91, 93, 9495, 96

T Tamarindus indica, 13I Tannins, 89 Tetragonolobus purpureus, 133 Thallassiosira. 97 T.fluviatilis, I 0 0 T. pseudonama, 98, 113 Thiobacillus intermedius, 90 Tradescantia virginiuna. 6, 39 Translation flow, 30-32 Triglochin maritimum, 170 Trigonella foenumgraecum (fenugreek), 133141, 149-151, 153 Tropaeoium majus (nasturtium), 126, 131, 144, 146

R Rana, 5 Rayleigh criterion, 1 I Rhodophytes, 75, 87, 88 RhodospirifIum rubrurn, 90 Rhodymenia palmuta, 99 RUBISCO (ribulose-1.5-biphosphate carboxylase/oxygenase), 72, 79-80, 85, 87, 88-94, 116-117, 179, 187 RuBPo (ribulose biphosphate oxygenase), 72, 94-100, 116-1 17

s Saccharum oficinarum (sugarcane), 86 Sargassum muticum, 79, 81, 99, 100 Scilla nonscripta. 129 Seed gums, 125-155 Seeds cell wall storage carbohydrates in, 125-155 leguminous, 130, 133-143 Siegert relation, 18 Skeletonema costaturn. 99 S. lostatus, 87 Sodium, 162-166 Spatoglossum parifcum, 9 I Speckle, 8-9 Spectrum analysis, 3, 21 Spergularia media, 169 Spermatozoa, 15, 49-53 Streaming, see Cytoplasmic streaming Sucrose, 169 Symbiodinum sp., 99

U Ulva expansa, 99 U. lactuca. 99 Uronic acids, 127-128, 131

V Vesicles, 37-38, 41-42 Viruses, 38 Viscosity, 4, 41-42

W Warburg effect, 98 Welwitschia mirabilis, 157- 191 carbon balance, 179-188 chemical composition, 162-172 distribution, 157- 159 life cycle, 159-162 osmoregulation, 162- 172 photosynthesis, 179- 188 transpiration, 173-179, 189 water economy, 172-179

X Xyloglucan, 131, 144, 146, 149, 152 Xylose, 126

Y Yucca. 132

2 Zygophyllum stapfi. 172, 173

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