Advances in Botanical Research, Vol. 14

Advances in

BOTANICAL RESEARCH VOLUME 14

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 E. G. CUTTER

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

John Innes Institute, Norwich, England Department of Biological Sciences, The University, Dundee, Scotland Department of Botany, University of Manchester, Manchester, England Department of Botany, Royal Holloway & Bedford New College, University of London, Egham Hill, Egham, Surrey, 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

VOLUME 14

1987

ACADEMIC PRESS Harcourt Brace Jovanovich, Publishers

London San Diego New York Boston Sydney Tokyo Toronto

ACADEMIC PRESS LIMITED 24/28 Oval Road, London NWl7DX

United States Edition published by ACADEMIC PRESS INC. San Diego, CA 92101

Copyright @ 1987 by ACADEMIC PRESS LIMITED Ail rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. British Library Cataloguing in Publication Data Advances in botanical research.-Vol. 14 1. Botany-Periodicals 581’.05 QK1 ISBN 0-12-005914-2

Printed by Galliards (Printers) Ltd Great Yarmouth, Norfolk

CONTRIBUTORS TO VOLUME 14

R. J. ELLIS, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U K C . ROBINSON, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK JOHN C . GRAY, Botany School, University of Cambridge, Downing Street, Cambridge CB2 3EA, U K M. GINZBURG, Botany Department, Institute of Life Sciences, The Hebrew University of Jerusalem, Israel

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One process fundamental to all cells is the transport of proteins from their sites of synthesis to regions of accumulation or secretion. This requires that proteins enter and cross membranes separating cell compartments, and the mechanisms responsible for this very specific and directional transport are now summed-up in the term “targeting”. In the first of the articles in this volume, Ellis and Robinson discuss the basic principles involved in targeting. Although inevitably much of the information is derived from animal and microbial research and has to be extrapolated to plants, it seems that enough has been done to confirm that some principles of the mechanisms of targeting are universal. A particularly intriguing section of this article speculates on the manipulation of targeting to redirect proteins from their normal destinations, thus creating organisms with novel properties of potential biotechnological value. Isoprenoid compounds play vital roles in a whole range of plant functions, including defence, as components of electron transport molecules, intermediates in polysaccharide synthesis, structural components of membranes, hormones, and many other activities. There are many reviews on the more chemical aspects of plant isoprenoids, but in the second article of this volume, Gray considers a rather neglected, and somewhat controversial aspect, the control of isoprenoid biosynthesis, in particular the role of individual enzymes and their subcellular location. Gray pinpoints the rather equivocal nature of much of the work in this area, discusses the most likely models, and identifies the areas of weakness and how these limitations may be overcome to provide a more comprehensive view of this facet of plant metabolism. Understanding of areas of plant metabolism such as this is likely to be vital if the full potential of gene manipulation in plants is to be realized. Dunaliella is one of a number of green algae that can withstand very high external concentrations of salt, and has achieved a certain prominence in biotechnological thinking because of this very property; the osmotic balance of the cells being maintained by the accumulation of the “compatible solute” glycerol which has a certain commodity value. However, it appears that this simple view of osmoregulation is misleading, and in her review Ginzburg considers a number of facets of the biology of Dunaliella vii

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Vlll

PREFACE

that seems in need of more thorough investigation, including its taxonomic status, concentrating on a detailed evaluation of studies on the physiology and biochemistry of salt tolerance. J . A. CALLOW

CONTENTS

CONTRIBUTORS TO VOLUME 14 . . . . . . . . . . PREFACE

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V

vii

Protein Targeting R . J . ELLIS and C . ROBINSON I.

I1 .

Introduction . . . . . . . . . . . . . . . . . Basic Principles . . A . Protein Targeting B . ProteinTargeting C . Protein Targeting

. . . . . . . . . . . . . . to the Nucleus . . . . . . . . . to the Mitochondrion . . . . . . to the Chloroplast . . . . . . . .

I11 .

Outstanding Problems . . . . . . . . A . Protein Conformation duringTransport B . The Origin of Receptors . . . . . . C. The Origin of Presequences . . . .

IV .

Opportunities for Genetic Engineering

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1

3 4 6 13

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19 19 20 20

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21

Control of Isoprenoid Biosynthesis in Higher Plants JOHN C . GRAY I.

The Pathways of Isoprenoid Metabolism . A . Formation of HMG-CoA . . . . B . Formation of Mevalonate . . . . C . FormationofIsopentenylDiphosphate D . Formation of Prenyl Diphosphates . E . Formation of Isoprenoid Compounds F . Mevalonate Shunt . . . . . . . G . Conclusions . . . . . . . . .

ix

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27 28 30 34 38 42 50 52

CONTENTS

X

I1.

Compartmentation of the Pathways of Isoprenoid Biosynthesis . . . . . . . . . . . . . . . . . A . Subcellular Location of Enzymes of Isoprenoid Biosynthesis . . . . . . . . . . . . . . . . B . Permeability of Membranes to Biosynthetic Intermediates . . . . . . . . . . . . . . . C . Conclusions . . . . . . . . . . . . . . . .

52

55 65 67

I11.

Control of the Isoprenoid Biosynthetic Pathway . . . . . . A . Fine Control . . . . . . . . . . . . . . . B . Coarse Control . . . . . . . . . . . . . . . C . Conclusions . . . . . . . . . . . . . . . .

68 69 80 85

1 v.

General Discussion . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

86 86

DunuZieZZu: A Green Alga Adapted to Salt M . GINZBURG I. I1.

Introduction . . . . . . . . . . . . . . . . . Taxonomy

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I11.

Distribution and Ecology . . . . . . . . . . . . .

IV .

Growth Conditions . . . . . . . . . . . . . A . Composition of Medium . . . . . . . . . . B . Total Salinity . . . . . . . . . . . . . C. EffectofNaClConcentrationonGrowth . . . . D . Effect of K+ . . . . . . . . . . . . . . E . Effect of Li+ . . . . . . . . . . . . . . F . Effect of S042- . . . . . . . . . . . . . G . Effect of Ca2+ . . . . . . . . . . . . . H . Effect of Mg2+ . . . . . . . . . . . . . I . Effect of P . . . . . . . . . . . . . . J . Trace Elements . . . . . . . . . . . . . K . Effect of N . . . . . . . . . . . . . . L . Effect of C Source . . . . . . . . . . . . M . Effect of Light . . . . . . . . . . . . . N . Effect of Temperature . . . . . . . . . . 0. Effects of Interactions Between Parameters on Growth P . Summary . . . . . . . . . . . . . . .

V.

Cell Anatomy . . . . . . . . . . . . . . A . Light Microscopy . . . . . . . . . . . B . Electron Microscopy . . . . . . . . . . C. Interspecific Anatomical Differences . . . . . D . Intergeneric Anatomical Differences . . . . . E . Effect of Salt on Cell Anatomy . . . . . . . F. Effect of Light Intensity and Temperature on Cell Anatomy . . . . . . . . . . . . . .

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95 95 98 101 101 101 103 107 107 108 108 108 108 109 109 112 112 115 116 117

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118 118 119 123 123 123

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125

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CONTENTS

VI .

VII .

VIII .

IX .

X.

Composition of Dunaliella Cells . . . . . . . . . A . Major Organic Compounds . . . . . . . . B . Protein . . . . . . . . . . . . . . C . Soluble Carbohydrates . . . . . . . . . D . Starch . . . . . . . . . . . . . . . E . Relation of Glycerol and Starch Contents to NaCl . F . Lipids and Carotenoids . . . . . . . . . G . Nucleic Acids . . . . . . . . . . . . H . Conclusions . . . . . . . . . . . . .

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

Enymes . . . . . . . . . . . . . . . . . A . Enzymes Concerned with Glycerol Metabolism . . B . Enzymes Concerned with Nitrate Reduction (Nitrate Reductase, Nitrite Reductase) . . . . . . . . C . Photosynthetic Enzymes . . . . . . . . . D . Enzymes Metabolizing Starch . . . . . . . . E . Phosphofructokinase . . . . . . . . . . . F . Effect of NaCl on Dunaliella Enzymes . . . . . G . Effectsof Glycerol on Dunaliella Enzymes . . . . H . SpecialFeaturesof DunaliellaEnzymes . . . . .

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Photosynthesis . . . . . . . . . . . . . . . A . Products of Photosynthesis . . . . . . . . . . B . EffectofLightIntensityon Photosynthesis . . . . . C . Photosynthetic Electron Flow . . . . . . . . . D . Rate of Photosynthesis After Long Period of Adjustment to a Given NaCl Concentration . . . . . . . . E . Effect of Change of NaCl Concentration on Photosynthesis . . . . . . . . . . . . . .

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126 126 128 128 130 130 131 133 133 134 134 137 139 140 140 141 143 143 143 146 148 148

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149

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149

Regulation of Cell Volume . . . . . . . . . . . . A . Immediate Effect of Change in Osmotic Pressure of Medium . . . . . . . . . . . . . . . . . B . Recovery of Original Cell Volume After Osmotic Shock . C . Measurements of Ion Concentrations (K', Na+. CI-) . . D . Can Osmoregulation be Accounted for Entirely in Terms of GlycerolSynthesisandDegradation? . . . . . . . E . Two-Compartment Hypothesis for the Regulation of Cell Volume . . . . . . . . . . . . . . .

153 153 160 167 173 174

Conclusions . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .

175 177 178

AUTHOR INDEX . . . . . . . . . . . . . .

185

SUBJECT INDEX . . . . . . . . . . . . . .

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Protein Targeting

R. J. ELLIS and C. ROBINSON

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U K

I. 11.

111.

IV.

Introduction . . . . . . . . . . . . . . . . . Basic Principles . . . . . . . . . . A. Protein Targeting to the Nucleus . . . B. ProteinTargeting to the Mitochondrion C. Protein Targeting to the Chloroplast . .

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. . . . . .

Outstanding Problems . . . . . . . . . . A. Protein Conformation during Transport . . B. The Origin of Receptors . . . . . . . . C. The Origin of Presequences . . . . . . Opportunities for Genetic Engineering

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1

3 4 6 13

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19 19 20 20

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21

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I. INTRODUCTION The dominant paradigm of current biological thought supposes that all the characteristics of living organisms are caused ultimately by the properties of the proteins that they contain. The extraordinary diversity of living organisms is mirrored by the plethora of different proteins that exist and by the complexity of the interactions that these proteins undergo. The large size and polymeric nature of protein molecules are responsible for the abundance of the reactions and structures that they create and control. Copyright 0 1987 Academic Press Limited All rights of reproduction in any form reserved.

Advances in Botanical Research Vol. 14

ISBN 0-12-005914-2

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R. J . ELLIS AND C. ROBINSON

A corollary to these conclusions is that proteins act in a highly structured cellular environment which is itself produced by the properties of proteins. This structural setting reaches its peak in eukaryotic plant cells, which each contain at least 15 distinct types of membrane, delimiting a corresponding number of hydrophilic subcellular compartments. Each type of membrane has a characteristic protein composition, as does each subcellular compartment, and it is this protein complement that enables each membrane and subcellular compartment to carry out its metabolic roles. With the exception of a small number of proteins that are synthesized inside mitochondria and chloroplasts, all these proteins are synthesized by cytoplasmic ribosomes. It follows that a fundamental process found in all cells is the transport of proteins into and across membranes. This process must be highIy specific so that a given protein accumulates in a single defined subcellular location; it is this specificity which ultimately generates and maintains the ultrastructure of cells. This transport specificity also applies to proteins which are secreted from the cell into the extracellular medium and to those proteins which are transported into the cell from the medium. In all cases, selectivity is maintained in the membranes which these proteins traverse. From these considerations it follows that a central problem for cell biologists is to unravel the mechanisms which ensure that proteins are directed from their site of synthesis to the site where they function. The term “targeting” is used to indicate the directional nature of these mechanisms. In the last 20 years much information has been published about the mechanisms of protein targeting. The pioneer work was carried out by George Palade on the secretion of proteins by pancreatic cells (Palade, 1975), and relied on the ingenious use of electron microscopic and autoradiographic techniques to follow the intracellular route of these proteins. Subsequent work concentrated on in vitro systems containing microsomal vesicles prepared from dog pancreas. Such cell-free preparations will synthesize and transport proteins across the microsomal membranes, and their use led to the formulation of hypotheses about the underlying mechanism (Blobel and Dobberstein, 1975a,b; Blobel, 1980; Wickner and Lodish, 1985). The study of protein transport in cell-free systems has been extended to chloroplasts (Highfield and Ellis, 1978; Cashmore et al., 1985; Schmidt and Mishkind, 1986), mitochondria (Hay et al., 1984; Douglas et al., 1986; Colman and Robinson, 1986; Hurt and van Loon, 1986) and bacteria (Muller and Blobel, 1984a,b). The in vitro approach has not as yet proved so useful for the study of protein targeting into the nucleus, but microinjection techniques applied to Xenopus oocytes and the transfection of cultured animal cells with mutated viral gene constructs has produced much detailed information (Dingwall and Laskey, 1986). A different type of analytical approach relies on the ease of classical

PROTEIN TARGETING

3

genetical studies in microorganisms such as Escherichia coli (Oliver, 1985; Benson et al., 1985; Pugsley and Schwartz, 1985) and yeast (Schekman, 1982; Novick, 1985). All these approaches have benefited from the increasing use of recombinant DNA techniques to analyse the precise nature of protein targeting information since these techniques allow the construction of fusion proteins composed of parts of two or more proteins that reside in different compartments. In the case of higher plants, these recombinant DNA techniques have been combined with Agrobacferiummediated transformation to achieve the targeting of foreign proteins into the chloroplasts of intact leaf cells and whole plants (Van den Broeck ef al., 1985; Schreier et al., 1985). It is not the intention of this chapter to provide an encyclopaedic or historical review of all this research. Instead our aim is to try to distil some basic principles of protein targeting from the current literature and to discuss selected examples which illustrate these principles. Most research on protein targeting has employed systems derived from animals and microorganisms, but what little has been carried out with plant cells suggests that the same principles apply to them also. Indeed, one of the most striking features of our understanding of protein targeting is the universality of its mechanisms, even between prokaryotes and eukaryotes. Thus we hope that this survey will stimulate the application of similar experimental approaches to the study of protein targeting in plant cells. In the final sections of this chapter the problems and possibilities presented by this field of research are discussed.

11.

BASIC PRINCIPLES

Present knowledge about the mechanism of protein targeting suggests four general conclusions: 1. Targeting information resides in the protein itself, in the form of discrete, relatively short aminoacyl sequences. 2. These targeting sequences can be located almost anywhere in the protein, but are often located at the aminoterminus in the form of transient presequences (also called prepieces, leader sequences, signal sequences, transit peptides or aminoterminal extensions), 3. Targeting sequences function by interacting with specific receptors located in the appropriate subcellular compartment. 4. Targeting sequences act in a relatively autonomous fashion in that the sequences on either side of them are not critical to their function; this is a most important property from the point of view of genetic engineers, since it allows protein retargeting, i.e. the localization of proteins in compartments where they do not normally occur.

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Some of the best evidence available to support these proposed principles will now be reviewed with respect to three organelles of eukaryotic cells, namely the nucleus, the mitochondrion and the chloroplast. Protein transport across the endoplasmic reticulum will not be considered here since it has been recently reviewed (Wickner and Lodish, 1985; Novick, 1985) but what is known about its mechanism is consistent with the basic principles suggested above. A.

PROTEIN TARGETING TO THE NUCLEUS

In most cells the nuclear envelope is dispersed into small vesicles during mitosis and subsequently reforms. Nuclear proteins are released into the cytosol at every mitosis and migrate back across the newly formed envelope membranes. It is thus not surprising to learn that all the experimental evidence on nuclear protein targeting indicates that the targeting information resides within the mature protein structure and not within presequences (Dingwall, 1985; Dingwall and Laskey, 1986). A feature of the nuclear envelope that distinguishes it from the bounding membranes of other organelles is the occurrence of structured pores (Unwin and Milligan, 1982). These pores present no barrier to the diffusion of ions or molecules up to the size of about 60 kDa for a globular protein (Bonner, 1978). However, proteins of larger size than about 60 kDa are either barred from or fail to accumulate in the nucleus unless they contain nuclear targeting information. The best-studied examples of such targeted nuclear proteins are nucleoplasmin and the large T antigen of the virus SV40. Nucleoplasmin is a large (M,= 110,000) soluble pentameric molecule present in high concentrations’in Xenopus oocyte nuclei, where it functions in nucleosome assembly as a molecular chaperone (Laskey and Earnshaw, 1980). When injected into the Xenopus oocyte cytoplasm, nucleoplasmin rapidly accumulates in the nucleus (Dingwall et al., 1982). Proteolytic digestion of the nucleoplasmin pentamer reveals that each subunit has two structural domains. The more protease-sensitive domain must remain intact in at least one subunit if the pentamer is to enter the nucleus. This so-called “tail” domain has been isolated and will itself accumulate in the nucleus after injection into the cytoplasm. In contrast, the “core” pentamer lacking all tails fails to enter the nucleus after injection into the cytoplasm. These results demonstrate that the targeting information in nucleoplasmin resides in a discrete polypeptide domain of the protein present in the third of the chain near the carboxyterminus. Much more incisive information about nuclear targeting has been provided by the study of the accumulation of the SV40 large T antigen in the nuclei of cultured animal cells and Xenopus oocytes. This protein is a tetramer with subunits of M,=90,000, and the isolation of its encoding gene

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5

has allowed the production of mutant forms in which particular aminoacyl residues have been altered, deleted or transferred by gene fusion to other proteins (Smith et al., 1985a). The precise requirement for the nuclear targeting of the large T antigen has been shown to be the presence of a seven aminoacyl sequence, namely Pr~'~~-Lys-Lys-Lys-Arg-Lys-Val'~~ (Kalderon et al., 1984a,b). Mutation of Lys12' to threonine abolishes the targeting function of this sequence, but mutation of either Lys12' or L Y S ' ~ ~ to threonine does not. Moreover, this sequence will function as a nuclear location signal when transferred to other proteins which do not normally accumulate in the nucleus, such as pyruvate kinase, or when transferred to the aminoterminus of a mutated large T antigen which cannot itself enter the nucleus. Recent work on nucleoplasmin has revealed the existence of two regions of homology to the targeting sequence of the large T antigen; these regions lie in the short carboxyterminal domain, which is known to contain the information for targeting nucleoplasmin to the nucleus (Dingwall et al., 1987). The most remarkable example of the autonomous nature of the large T antigen targeting sequence has been provided by Goldfarb et al. (1986). These authors constructed a synthetic dodecapeptide which contains the large T antigen targeting sequence, and chemically crosslinked it to either bovine serum albumin or immunoglobulin, neither of which accumulates in the nucleus in unmodified form. The crosslinks are formed between a carboxyterminal cysteine residue in the peptide and amino groups in the protein; each molecule of bovine serum albumin is crosslinked to 10-20 peptide molecules. The resulting peptide-protein products accumulate in the nucleus of Xenopus oocytes when microinjected into the cytoplasm; when L Y S 'is~replaced ~ by threonine, nuclear accumulation is reduced sixto seven-fold. Moreover, the rate of accumulation of the peptide-protein in the nucleus shows saturation kinetics, and is markedly reduced by the injection of free peptide, suggesting the existence of a receptor-mediated transport process. Whether this presumed receptor is located in the cytosol or in the nuclear pore complex is unknown. Antisera raised against the dodecapeptide recognize several nuclear proteins, suggesting that a family of nuclear proteins may possess large T antigen-like targeting sequences. The major unresolved question in the study of nuclear protein targeting is how the sequence information is recognized and utilized. A remarkable feature of the SV40 large T antigen targeting sequence is its ability to function in very different protein environments; this ability suggests that the sequence need not adopt a fixed secondary structure but perhaps adopts a particular configuration on binding to a receptor. If this idea is correct, there may be several classes of nuclear receptors since studies on the targeting sequences of nuclear proteins other than nucleoplasmin and the large T antigen have not revealed a single consensus sequence (Dingwall and Laskey, 1986).

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R. J. ELLIS AND C. ROBINSON

The microinjection of colloidal gold particles coated with nucleoplasmin into Xenopus oocyte cytoplasm has provided strong evidence that at least in this case entry is via the centre of nuclear pores (Feldherr et al., 1984; Fig. l ) , while Newmeyer et al. (1986) have shown that ATP is required for the transport of nucleoplasmin both into the nuclei of Xenopus eggs in vivo and into synthetic nuclei in vitro. Synthetic nuclei are made by adding either Xenopus or bacteriophage lambda DNA to cell-free homogenates of Xenopus eggs; double envelopes containing pores assemble around chromatin to form nuclear-like structures. The fact that these structures assemble from bacteriophage DNA, and accumulate nucleoplasmin but not bovine serum albumin, implies that assembly of a functional nuclear envelope requires no specific eukaryotic DNA sequences. Nucleoplasmin is accumulated by synthetic nuclei only if ATP is present at 1 - 2 m ~ . Lowering of the endogenous ATP pool in intact Xenopus eggs by injection of ATPase completely prevents the nuclear accumulation of nucleoplasmin. Whether the ATP is required by an active transport mechanism or permits the pore complex to widen to allow facilitated diffusion has not been established, but the ability to construct synthetic nuclei should allow the dissection of nuclear protein targeting into its component steps. B. PROTEIN TARGETING TO THE MITOCHONDRION

About 90% of the several hundred proteins found in mitochondria are encoded in nuclear genes and synthesized by cytosolic ribosomes. Extensive transport of proteins into the mitochondrial compartment is thus a major feature of mitochondrial biogenesis (Schatz and Mason, 1974). Proteins are transported into each of the four structural phases of the mitochondrion, i.e. the outer and inner membranes, the intermembrane space and the matrix. Much information is now available about the nature of this transport process, derived largely from the elegant studies conducted on yeast in the laboratory of G. Schatz (Basle) and on Neurospora in the laboratory of W. Neupert (Munich). Several recent reviews discuss this information (Hay et al., 1984; Colman and Robinson, 1986; Hurt and van Loon, 1986; Douglas et a f . , 1986). By contrast, there is no published information on the transport of proteins into the mitochondria of higher plants. Hay et a f . (1984) suggest that the transport of proteins into mitochondria can be divided conceptually into four steps and this proposal will be followed here. 1. Synthesis, Usually as a Larger Precursor

All imported mitochondrial proteins that have been studied are synthesized by free cytosolic ribosomes and are released into the cytosol in

* N

v c Fig. 1. Transport of nucleoplasmin through nuclear pores. Colloidal gold particles (15 nm diameter) were coated with purified nucleoplasmin and injected into the cytoplasm of Xenopus oocytes. After incubation for either 1.5 h ( A ) or 4 h (B), the oocytes were fixed and sectioned for transmission electron microscopy. Symhols: N , nucleus; M, mitochondrion; C, cytoplasm; Y, yolk platelet. The arrows indicate nuclear pores. Pictures kindly supplied by A . D . Mills and C. Dingwall of the CRC Molecular Embryology Group, Department of Zoology, University of Cambridge.

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R.J. ELLIS AND C. ROBINSON

soluble form prior to transport; protein transport into mitochondria is thus described as post-translational. The binding of polysomes synthesizing mitochondrial proteins to the outer mitochondrial membrane is seen in yeast cells after treatment with cycloheximide (Ades and Butow, 1980), but there is no evidence to suggest that the import of proteins into mitochondria is obligatorily co-translational under normal conditions (Suissa and Schatz, 1982). In many cases, but not all, these imported proteins are synthesized as larger precursors with an aminoacyl extension, or presequence, at the aminoterminus. The apparent molecular sizes of these precursors are between 0.5 and 10 kDa larger than the mature proteins. There is no obvious correlation between the size of the presequence and the final location of the mature protein, nor do precursors to different subunits of the same enzyme complex possess a standard size of presequence. The known primary structures of presequences do not show extensive homology but they do share similar overall characteristics in that they are relatively rich in basic residues (lysine and arginine) and in the hydroxylated amino acid residues serine and threonine. Unlike the presequences of secreted proteins, which contain an aminoterminal cluster of basic residues flanking a membrane-spanning hydrophobic core sequence, the presequences of mitochondrial proteins are punctuated by basic amino acids about every four to eight residues (Hurt and van Loon, 1986). Presequences of mitochondrial proteins whose final location is either in or exposed t o the intermembrane space contain a long stretch of uncharged amino acids adjacent to an aminoterminal sequence punctuated with basic and hydroxylated residues. Proteins of the outer mitochondrial membrane are synthesized without prepieces, but their aminoterminal sequences nevertheless resemble those of intermembrane precursors in that the basic aminoterminus is followed by a stretch of uncharged residues. The sequence of only one nuclear-encoded mitochondrial protein from a higher plant has been published. The p subunit of the ATP synthase complex of Nicotiana plumbaginofolia is encoded by two nuclear genes; the sequence contains an aminoterminal extension rich in basic aminoacyl and serine residues (Boutry and Chua, 1985). Present evidence suggests that the presequences of mitochondrial proteins serve three functions. Firstly, they confer solubility in the cytosol on proteins which are very hydrophobic when mature, e.g. subunit IV of yeast cytochrome c oxidase or the proteolipid of Neurospora ATP synthase. Secondly, they carry mitochondrial targeting information; gene fusion studies have demonstrated that cytosolic proteins can be retargeted to the mitochondrial compartment by addition of the appropriate presequences (or portions thereof) to their aminotermini. For example, mouse cytosolic dihydrofolate reductase will enter yeast mitochondria both in vivo and in vitro if the first 12 of the 25 amino acid residues of the presequence of

PROTEIN TARGETING

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cytochrome c oxidase subunit IV are fused to its aminoterminus (Hurt et al., 1985b). The targeting information in those mitochondrial proteins which lack presequences similarly resides at the aminoterminus; thus the targeting information for the 70 kDa protein of the outer mitochondrial membrane resides in the first 41 amino acid residues of the mature protein (Hase et al., 1984). The third function of mitochondrial presequences is to direct the protein to the correct subcompartment or membrane of the mitochondrion. For example, the presequence of cytochrome c I ,which is an inner membrane protein exposed to the intermembrane space, directs attached dihydrofolate reductase into the intermembrane space (van Loon et al., 1986). The three properties of presequences suggest not only that a precursor protein adopts a different conformation to that of the mature protein, but also that the presequence folds and acts independently of the attached sequence. 2. Binding of the Precursor to the Outer Mitochondria1 Membrane The next step after synthesis of the precursor is its interaction with the cytoplasmic face of the outer mitochondrial membrane. This interaction could involve either specific receptors or partitioning into the lipid bilayer or a combination of both. The available evidence is scanty and supports the possible involvement of both types of interaction. Binding of apocytochrome c , which has no presequence, to Neurospora mitochondria involves a limited number of binding sites and is not inhibited by holocytochrome c (Zimmerman et al., 1981). Excess apocytochrome c does not inhibit the import of precursors to other mitochondrial proteins. Treatment of isolated mitochondria with low concentrations of trypsin (1Opg ml-') prevents the subsequent binding of added precursor proteins (Riezman et al., 1983). Transport of several different precursor proteins into isolated rat heart mitochondria is prevented by addition of a synthetic presequence based on the presequence of ornithine carbamyltransferase; this inhibition is overcome by addition of excess precursor protein (Gillespie et al., 1985). Such observations are consistent with the involvement of specific receptors, presumably proteinaceous in nature. However, no mitochondrial receptor protein has yet been purified, despite vigorous efforts. Moreover, synthetic presequences have amphiphilic properties and will insert into a variety of artificial and biological membranes; synthetic presequences will insert spontaneously into lipid monolayers containing no protein, lyse lipid vesicles, and uncouple energized mitochondria (Roise et al., 1986). It has been proposed that amphiphilic helicity is a general feature of mitochondrial presequences and that this feature is important in the initiation of protein transport, but that no proteinaceous receptors may be involved (Roise et al., 1986). A similar model for the import of apocytochrome c

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R.J. ELLIS AND C. ROBINSON

across the outer mitochondrial membrane stresses the role of phospholipids in this process (Rietveld and de Kruijff, 1986). Thus the question of the existence of specific outer membrane receptors for mitochondrial precursor proteins is unresolved.

3. Translocation and the Requirement for.Energy Bound mitochondrial precursor proteins have four possible fates; they may either enter the outer membrane and remain there, cross the outer membrane and reside in either the intermembrane space or in the inner membrane, o r cross both membranes and enter the matrix. The current evidence suggests that different pathways exist for proteins destined for different mitochondrial subcompartments and that the selection of these pathways is mediated by targeting information present in the aminotermina1 sequence in the form of two structural and functional domains. Hurt and van Loon (1986) propose that targeting information in imported mitochondrial proteins consists of a linear array of short matrixtargeting domains, stop-transport (or membrane-anchor) domains, and proteolytic cleavage sites. A stop-transport domain consists of a stretch of uncharged amino acid residues that anchors the protein in a membrane. All imported mitochondrial proteins, regardless of their final location, contain a matrix-targeting domain at their aminoterminus, but not all contain a stop-transport sequence or cleavage sites. Different combinations of the two types of targeting domain and of cleavage sites can in principle account for the targeting of different proteins to all four structural phases of the mitochondrion (Figs 2 and 3). The best evidence for this model comes from gene fusion studies in yeast. For example, the first 12 amino acid residues of the 70 kDa protein of the outer membrane, which has no presequence, will transport a cytosolic protein to the matrix if fused to its aminoterminus (Hurt et al., 1985a). However, the 70 kDa protein itself does not enter the matrix because the stop-transport domain that follows the matrix-targeting domain anchors the outer protein in the outer membrane. The most complex pathway is found for proteins that reside in the intermembrane space, such as cytochrome c l . The matrix-targeting domain of the precursor to cytochrome c1 is removed by a specific matrix protease as soon as it enters the matrix; however, the remainder of the protein does not enter the matrix because the stop-transport domain anchors it in the inner membrane. A second protease located in the intermembrane space is then postulated to release the protein into this space by cleavage on the other side of the stop-transport domain (Fig. 3). It is not yet understood how the stop-transport domain distinguishes between the outer and inner membranes. This distinction may depend on the relative length, strength or spacing of the stop-transport and matrix-targeting domains. The matrix-targeting domain does not invariably reside at the amino-

11

PROTEIN TARGETING

Arrangement of targeting domoins

Final location of protein Matrix

‘++++’4 N

I+++-

Outer membrane

N

I ++++plplldpllllllt

Inner membrane

4 N

I++++!-

4

lntermembrane space

4

Fig. 2. Arrangement of targeting domains and cleavage sites in imported mitochondrial proteins. All imported mitochondria1 proteins contain a basically charged aminoterminal sequence that targets to the matrix. A second targeting domain, consisting of a membranespanning stretch of uncharged amino acid residues, prevents entry into the matrix in those proteins destined for other mitochondrial phases. The relative position of the matrix-targeting domain to the stop-transport domain and to cleavage sites determines the final location of the protein. Symbols: + + +, matrix-targeting domain; M, stop-transport domain; , proteolytic cleavage site. Based on Fig. 2 of Douglas et (I/. (1986).

+

lNTERMEMBRANE SPACE TARGETING

MATRIX-TARGETING

Fig. 3. A model for intramitochondrial protein sorting. Imported proteins are composed of a transported “passenger” protein (stippled circle) with an attached matrix-targeting presequence (open box with positive charges), intermembrane space targeting presequence (open box with positive charges followed by a coil) or outer membrdne targeting sequence (open box with positive charges followed by a coil). The sitcs where the presequences are cleaved are indicated by arrows. Proteins enter the mitochondrion at “contact sites” between the outer and inner membranes. Reprinted with kind permission from Hurt and van Loon ( 1986).

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R.J. ELLIS AND C . ROBINSON

terminus in all mitochondrial precursor proteins. The presequence of human ornithine transcarbamylase contains 32 amino acid residues. Removal of residues 2-7 or residues 2 6 3 1 has little effect on import of the precursor by isolated rat liver mitochondria, but deletion of residues 8-22 prevents import completely. Substitution of arginine 23 by glycine also totally inhibits import, while the effect of additional substitutions suggests a critical role for arginine 23 in a local secondary structure essential for import (Horwich et al., 1986). The transport of proteins into mitochondria requires energy except for outer membrane proteins and some intermembrane space proteins such as cytochrome c . This energy requirement is commonly met in experiments with isolated mitochondria by the addition of ATP, but the use of specific inhibitors has established that the critical requirement is not for ATP per se, or even a proton gradient, but the establishment of a potential across the inner membrane (Pfaller and Neupert, 1985). It has been shown that this membrane potential is required to import the aminoterminal domain of the precursor of the p subunit of the ATP synthase complex, but is not required for the transport of the remainder of the protein into the matrix of isolated Neurospora mitochondria (Schleyer and Neupert, 1985). These authors found that when this precursor is bound to energized mitochondria at 4”C, the aminoterminal domain enters the matrix and is cleaved by the matrix protease, while the remainder of the protein is still accessible to antibodies or proteases on the cytoplasmic face of the outer membrane. When these mitochondria are warmed to 24”C, the remainder of the precursor molecule enters the matrix, even in the presence of ionophores which discharge the membrane potential. These important studies demonstrate that the membrane potential is required only to initiate transport into the matrix, not to complete it, and in addition show that the imported protein spans both membranes at the same time during transport. Thus precursor proteins may enter the mitochondria through “contact sites” where the outer and inner membranes are close together (Fig. 3). Whether the membrane potential is required to form contact sites or acts in some other way is not established.

Proteolytic Cleavage of Imported Precursors Imported mitochondrial proteins that contain presequences undergo either one or two proteolytic cleavages (sometimes called processing steps). The first of these cleavages is mediated by a matrix-located metalloendoprotease which itself is imported from the cytoplasm. This protease has been partially purified from yeast and rat liver mitochondria (Bohni et al., 1983; Miura et al., 1982). It is highly specific for mitochondrial precursor proteins and will not attack either non-mitochondria1 proteins or denatured mitochondrial precursor proteins. The enzyme does not require ATP and is insensitive to serine protease inhibitors and sulphydryl reagents; it is, 4.

PROTEIN TARGETING

13

however, inhibited by metal-chelating agents but the identity of the metal is still unclear. The matrix protease exhibits little species specificity, since precursors from different species can be imported and cleaved by heterologous mitochondria. What determines the position of cleavage is unknown, but it appears to involve a particular secondary or tertiary structure rather than a specific sequence around the cleavage site. Mitochondria1 proteins that either reside in or are exposed to the intermembrane space are processed in two sequential steps (Figs 2 and 3). The first cleavage is carried out by the matrix protease and produces a precursor of intermediate size bound to the inner mitochondria1 membrane; these intermediate precursors have been detected in intact yeast cells and spheroplasts as well as in isolated mitochondria (Gasser et al., 1982). The second cleavage converts these intermediates to the mature size and takes place on the outer surface of the inner membrane. This second protease is distinct from the one located in the matrix and has so far resisted purification. There is genetic evidence that more than one protease is responsible for processing different precursors exposed to the intermembrane space in yeast (Pratje and Guiard, 1986). In the case of cytochrome cl, covalent haem attachment is required before cleavage can occur (Ohashi et al., 1982). The use of selective inhibitors has established that processing is not required for the energy-dependent transport of protein to occur across the inner membrane (Reid et al., 1982). Recently it was reported that both cleavages of the precursor of subunit 9 of the ATP synthase complex of Neurospora crassa are catalysed by the matrix processing activity. The significance of two-step processing in this case is unknown but may be related to the unusually large size of the presequence of this precursor (Schmidt et al., 1984). C. PROTEIN TARGETING TO THE CHLOROPLAST

The chloroplast is the best-studied organelle of plants with respect to protein targeting. Indeed, the first report of protein transport into isolated chloroplasts (Highfield and Ellis, 1978) appeared only one year later than the first report of protein transport into isolated mitochondria (Harmey et al., 1977). However, the absence of an organism with which it is possible both to isolate intact chloroplasts capable of protein transport and to carry o u t genetic studies has impeded the progress of research on protein transport into chloroplasts compared to that into mitochondria. This problem is compounded by the relative lack of interest of biochemists in plants, The chloroplast is the most complex organelle known, in structural terms, since it contains three distinct types of membrane (the outer and inner envelope membranes and the thylakoid membrane) enclosing three

14

R. J . ELLIS AND C. ROBINSON

sub’compartments(the interenvelope membrane space, the stroma and the thylakoid lumen). All six phases are known to possess different complements of proteins, with the exception of the interenvelope space, which has so far defied analysis. The chloroplast is also complex in metabolic terms, since as well as housing the entire photosynthetic machinery this organelle carries out several other vital metabolic processes such as the assimilation of inorganic nitrogen and sulphate, the synthesis of amino acids, fatty acids, chlorophylls and carotenoids, and the interconversion of intermediates by enzymes of the glycolytic and oxidative pentose phosphate pathways. As with mitochondria, the vast majority of chloroplast proteins are encoded in nuclear genes and synthesized by cytosolic ribosomes. Thus the biogenesis of chloroplasts from proplastids depends upon a massive import of several hundred different types of protein molecule into all the structural phases of the organelle. It follows that efficient sorting mechanisms must exist to direct imported proteins to their correct locations within the chloroplast. The basic features of chloroplast protein transport resemble those of mitochondrial protein transport in many, but not all, respects (Ellis, 1981a; Ellis and Robinson, 1985; Cashmore et al., 1985; Schmidt and Mishkind, 1986). The same division of the process into four steps that was used to describe mitochondrial protein transport will be used to summarize what is known about protein transport into chloroplasts.

1. Synthesis of Imported Chloroplast Proteins as Precursors All imported chloroplast proteins studied to date are initially synthesized on free cytosolic ribosomes as larger precursors containing aminoterminal presequences. The sizes of these presequences vary from about 2 to 16 kDa, but most are around 5 to 8 kDa. These precursors are imported into the chloroplast after synthesis is complete, i.e. by a post-translational mechanism (Highfield and Ellis, 1978; Chua and Schmidt, 1979); there is no microscopic evidence to suggest that cytosolic ribosomes are normally bound to the chloroplast envelope during the development of chloroplasts. Chloroplast protein transport thus resembles mitochondrial and nuclear protein transport in its post-translational nature, and differs from the co-translational transport of proteins into the endoplasmic reticulum. The primary structures of the presequences of four different chloroplast proteins have been published (Smeekens et al., 1985; Karlin-Neumann and Tobin, 1986; Nagy et al., 1986). These sequences show no extensive homology to one another and lack long hydrophobic stretches, but like the presequences of mitochondrial proteins they are rich in basic residues and seines. Furthermore, the presequences of the same protein in different species are less well conserved than are the sequences of the mature protein. It has been proposed that the known presequences of four imported chloroplast proteins share “three major blocks of homology”

PROTEIN TARGETING

15

which form a common framework, mediating functions shared by each presequence (Karlin-Neumann and Tobin, 1986). However, to these reviewers the blocks of homology appear to be minor rather than major, and confirmation of this interesting suggestion awaits the determination of presequences for more chloroplast proteins. The current evidence suggests that targeting information in chloroplast precursor proteins is unlikely to consist of specific amino acid sequences, but that aspects of threedimensional structure and charge distribution of the presequence are recognized by the importing machinery of the chloroplast. There is evidence that the presequences of chloroplast proteins serve the same three functions as do those of mitochondria1 proteins. The major protein of the thylakoid membrane is termed the light-harvesting chlorophyll alb-binding protein. The mature binding protein is very hydrophobic, being one of the few thylakoid proteins that are soluble in chloroformmethanol; nevertheless, this protein is synthesized in the cytoplasm as a precursor which is soluble in aqueous media. The chloroplast-targeting function of the presequence of the small subunit of ribulose bisphosphate carboxylase has been demonstrated by gene fusion experiments in which a bacterial protein was shown to enter the stromal subcompartment both in vivo in transgenic plants and in vitro with isolated chloroplasts (Van den Broeck et al., 1985; Cashmore et a l . , 1985; Schreier et al., 1985; Schreier and Schell, 1986). The two-domain model proposed to describe the targeting of proteins to the subcompartments of the mitochondrion (Figs 2 and 3) has been extended to chloroplasts to explain how plastocyanin originates in the cytosol but accumulates in the thylakoid lumen (Smeekens et al., 1986).

2. Binding of Precursors to the Outer Chloroplast Envelope Membrane Precursors of the small subunit of ribulose bisphosphate carboxylase and the chlorophyll alb-binding protein bind in an energy-independent manner to the outer surface of intact isolated chloroplasts. Binding occurs even when import is prevented by the uncoupler nigericin, but the bound precursors subsequently enter the chloroplast if the nigericin is removed and ATP is added (Cline et al., 1985). These precursors also bind to isolated chloroplast envelopes, whereas mature carboxylase small subunit and other proteins synthesized by a wheatgerm extract programmed with poly(A)-containing RNA do not bind, suggesting some specificity in the interaction (Ellis, 1981b, 1983a; Pfisterer et a f . , 1982). The bound precursors are not processed by isolated envelopes since the processing activity is soluble and is removed during the preparation of envelopes. Precursors bound to isolated envelopes are digested by low concentrations of trypsin, while attempts to demonstrate transport into isolated envelope vesicles have been unsuccessful so far. Pfisterer el af. (1982) reported that chloroplast protein precursors do not bind to isolated thylakoids, but in our

16

R.J. ELLIS AND C . ROBINSON

laboratory binding to both isolated thylakoid and leaf mitochondrial membranes has been observed, even when precautions are taken to avoid artefacts caused by aggregation of the precursors. Both laboratories have observed that chloroplast precursor proteins will bind to the surface of plastic tubes, so glass tubes and controlled washing procedures must be used to avoid erroneous interpretations. It is now possible to separate the outer and inner membranes of the chloroplast envelope (Cline et a f . , 1981) but experiments to determine whether precursors bind only to the outer envelope have not yet been reported. Treatment of intact isolated chloroplasts with low concentrations of thermolysin reduces subsequent binding of pure in vitro synthesized chloroplast protein precursors. Control experiments have shown that this reduction is not due to the destruction of the precursors by residual amounts of the protease, while thermolysin is known to digest only a subset of the outer envelope membrane polypeptides (Cline et a f . , 1985). In this latter work, thermolysin treatment did not abolish binding completely, suggesting that there may be a nonspecific binding component which must be taken into account in any attempt to isolate specific receptors. As with mitochondrial protein targeting, nothing is known of the number, structure, specificity or mode of action of the import receptors that are presumed to exist in the outer chloroplast envelope, but a recent report (Bitsch and Kloppstech, 1986) points the way to their future characterization.

3. Transport and Energy Dependence Very little information is available about the mechanism by which proteins are transported across the chloroplast envelope or thylakoid membranes. The only solid data concern the energy dependence of the transport step; Grossman et al. (1980) demonstrated by the use of uncouplers that ATP per se, and not an energized thylakoid membrane, is required for protein transport into intact isolated chloroplasts. Thus protein transport can be driven by exogenous ATP in vitro even in the presence of uncouplers or in the dark. This finding highlights one of the major differences between chloroplast and mitochondrial protein transport. The reason for this difference is not clear, but it may reflect the fact that the chloroplast inner envelope membrane has no energy-transducing functions, unlike the mitochondrial inner membrane. Several studies have been carried out using gene fusion techniques to determine how alterations in the precursor of the small subunit of ribulose bisphosphate carboxylase affect transport into the chloroplast (Schreier and Schell, 1986). When the presequence of the carboxylase small subunit is fused in frame with the aminoterminus of the bacterial enzyme neomycin phosphotransferase, the resulting fusion protein is imported into intact isolated chloroplasts from Pisum sativum and correctly processed (Van den

PROTEIN TARGETING

17

Broeck et al., 1985). Although the efficiency of import of this fusion protein by isolated chloroplasts is low compared to that of the authentic small subunit precursor, the efficiency is greatly improved if an additional stretch of 23 amino acids from the aminoterminus of the mature small subunit is included in the fusion protein (Wasmann et at., 1986). This observation suggests that domains within the mature part of the small subunit precursor contribute to the efficiency of import, as suggested previously on theoretical grounds (Ellis, 1984). Despite these observations, the fusion protein containing the additional stretch of 23 amino acids accumulates within the chloroplasts of transgenic plants to a lower degree than the fusion protein lacking this stretch (Kuntz et al., 1986). One suggested explanation is that the additional stretch renders the precursor more susceptible to cytosolic proteases, a factor not present in experiments with isolated chloroplasts. This observation indicates that in vitro experiments alone may not accurately predict the transport efficiency of a fusion protein in transgenic plants. In view of the overall similarities between the presequences of imported chloroplast and mitochondrial proteins, gene fusion experiments have been carried out to determine their functional interchangeability. The aminoterminal 31 residues of the 45-residue presequence of the carboxylase small subunit from Chlamydomonas will target attached proteins to the mitochondrial matrix in yeast cells (Hurt et al., 1986). The efficiency of this targeting is low compared to that of authentic mitochondrial precursor proteins, but this surprising result raises the question as to how well the targeting sequences in a plant cell discriminate between the chloroplasts and mitochondria. Is it possible that the discrimination between the two types of organelle is not absolute, but that a proportion of imported proteins ends up in the wrong compartment and is there destroyed by proteases? If this is the case, how do the presumed selective proteases accumulate only in the correct compartment in the first place? The answers to these intriguing questions will come only when experiments have been done which compare directly the discrimination shown by the presequences of chloroplast and mitochondria1 proteins from the same species. Processing of Imported Precursors Imported stromal proteins are processed to their mature size by a highly specific protease located in the stromal fraction. This protease has been partially purified from Pisum sativum (Robinson and Ellis, 1984a). The enzyme has an M , of about 180,000 as determined by gel filtration and is inhibited by metal-chelating agents such as EDTA and 1,lOphenanthroline; its activity does not require the addition of ATP. At least one precursor, that of the small subunit of ribulose bisphosphate carboxylase, is processed by highly purified preparations of this protease in two steps (Robinson and Ellis, 1984b). Since the mature protein derived from 4.

18

R. J. ELLIS AND C. ROBINSON

this precursor resides in the stroma, the significance of this two-step processing is unknown. Inhibitor experiments show that at least the first cleavage is not required for the transport of the small subunit precursor across the chloroplast envelope, while experiments with precursors containing amino acid analogues suggest that the processing enzyme recognizes specific structural features of the precursor other than, or in addition to, the residues at which cleavage takes place (Robinson and Ellis, 1985). The partially purified stromal protease also cleaves the precursor of plastocyanin, a soluble protein located in the thylakoid lumen. The biogenesis of plastocyanin is of especial interest because three membranes lie between the sites of synthesis and function of this protein. Preplastocyanin is processed to an intermediate form in the stroma, with about two-thirds of the presequence being removed. The intermediate precursor thus generated is subsequently transported into the thylakoid lumen, where maturation is completed by a second processing protease located in the lumen (Hageman et al., 1986; Smeekens et al., 1986). Figure 4 illustrates the processing of preplastocyanin by the two proteases. It seems likely from these observations that the presequence of plastocyanin contains two targeting domains; the first directs the protein into the stroma, while the second directs the intermediate form into the thylakoid lumen. The second processing site in the plastocyanin precursor is preceded by a region 20 amino acid residues in length which is rich in valine and alanine (Smeekens et al., 1985); this region might form a membranespanning domain which targets the intermediate to the thylakoid membrane. The tertiary structures of these domains, and the ways in which they interact with the presumed import receptors and processing enzymes, are unknown.

1

2

3

4

pre-PC>

Fig. 4. Two-step processing of in vitro-synthesized preplastocyanin from Silene pratensis. Radioactively labelled preplastocyanin was made in v i m and incubated successively with stromal protease and a thylakoid extract prepared from Pisurn sarivum. The figure shows a fluorograph of an SDS polyacrylamide gel. Lane 1, preplastocyanin; lane 2, preplastocyanin incubated with stromal protease; lane 3 , preplastocyanin incubated with stromal protease and then with detergent-solubilized thylakoids; lane 4, preplastocyanin incubated with intact isolated chloroplasts. Symbols: prePC, preplastocyanin; intPC, intermediate form of precursor; PC, mature plastocyanin. Reprinted with kind permission from Hageman et al. (1986).

PROTEIN TARGETING

19

111. OUTSTANDING PROBLEMS Of the many interesting questions that remain unanswered about protein targeting, three are outstanding: A. What is the conformation of a protein as it traverses a membrane? B. What determines that specific receptors for imported proteins are located in the correct subcellular compartment? C . Where do presequences come from? Each of these questions will now be discussed. A.

PROTEIN CONFORMATION DURING TRANSPORT

Due to the lack of appropriate techniques, almost nothing is known about the structure of a protein as it traverses a membrane during transport. Nor is it known whether transport is directly through a lipid bilayer or through a proteinaceous pore or through some combination of both. It is especially difficult to envisage how a very hydrophobic protein, such as the chlorophyll alh-binding protein of the chloroplast thylakoid, can traverse the chloroplast envelope membranes and enter the hydrophilic stroma before encountering the thylakoid membrane. Recognition of a stop-transport sequence might require an appropriate protein in the membrane, so that stretches of hydrophobic amino acids are not in themselves sufficient to cause transport to stop. Alternatively, the hydrophobic stretches of the imported protein may be folded so as to shield them from interaction with the lipid bilayer during transport. Two reports are consistent with the view that proteins must be at least partially unfolded before they can traverse a membrane. The transport of mouse dihydrofolate reductase into isolated yeast mitochondria is dependent on the addition of a presequence from a normally imported mitochondria] protein. The transport is blocked by methotrexate, which binds to the active site of the reductase, but both binding of the fusion protein to the mitochondria] surface and processing by the purified matrix protease are only slightly reduced (Eilers and Schatz, 1986). These observations suggest that methotrexate inhibits transport by preventing unfolding of the protein rather than by masking the presequence. The demonstration by Schleyer and Neupert (1985) that the precursor to the /3 subunit of the ATP synthase complex spans both mitochondrial membranes during transport also suggests that at least the aminoterminal region of the protein is unfolded during transport. Neither of these reports indicate how extensive unfolding must be to allow movement of a protein across a membrane.

20

R. J. ELLIS AND C. ROBINSON

B. THE ORIGIN OF RECEPTORS

Crucial to the operation of the basic principles of protein targeting suggested in Section I1 is the prior location of specific receptors in the appropriate subcompartment. But how are these receptors targeted to the correct location in the first place? Consideration of this problem suggests that the answer must lie not in ontogeny but in phylogeny, and is bound up with the evolutionary origin of membrane-limited compartments. The conclusion appears inevitable that each cell inherits from its parent cell a representative piece of each type of membrane that it requires. This piece of membrane can then be extended by protein targeting but it cannot be created de nuvo. It follows that if a cell ever lost all of one type of membrane it could not rebuild this membrane, even though the genetic information for all the components of this membrane is present. The interesting paper of Blobel (1980) presents speculations about the evolutionary origin of membrane-limited compartments and suggests that Virchow’s famous doctrine “omnis cellula e cellula” should be extended to include “omnis membrana e membrana”. C. THE ORIGIN OF PRESEQUENCES

It is now generally accepted that chloroplasts and mitochondria arose from free-living prokaryotic cells by endosymbiosis (Gray and Doolittle, 1982; Ellis, 1983b). A consequence of this view is that a massive transfer of genes from symbiont to nucleus must have occurred during evolution to produce the present dominance of the nucleus over organelle biogenesis. Presumably, duplicate copies of the transferred genes were retained within the organelle until the nuclear sequences became fully functional by the addition of information encoding the presequences of organellar proteins. But where did these presequences come from? Two compatible ways to explain the origin of presequences have been proposed (Baker and Schatz, 1987). The first supposes that presequences arose by point mutations in the 5 ’ untranslated sequences of the transferred genes. The second supposes that presequences were created by DNA rearrangements which added already encoded additional blocks of amino acids to the aminotermini of organellar proteins. Since the function of organellar targeting sequences does not depend on a specific primary structure but on overall composition and distribution of charged residues, it is possible that such sequences occur normally as parts of cytosolic proteins. In support of this hypothesis, sequences which are capable of restoring mitochondria1 targeting to a truncated precursor for subunit IV of cytochrome c oxidase have been found frequently in both the genome of Escherichia coli and within the gene for mouse dihydrofolate reductase

PROTEIN TARGETING

21

(Baker and Schatz, 1987; Hurt and Schatz, 1987). Normally these targeting sequences are cryptic since they are buried within the structure of the cytosolic proteins, but their targeting potential is realized when they are fused to the aminoterminus of mature organellar proteins. These gene fusion experiments may thus be re-enacting events that took place during the evolution of mitochondria and chloroplasts from prokaryotic symbionts. This hypothesis also explains why targeting sequences are often located at the aminoterminus of the imported protein; in this position the targeting sequence can fold into an independent domain without affecting the folding of the attached protein.

IV. OPPORTUNITIES FOR GENETIC ENGINEERING The relatively autonomous nature of protein targeting information suggests that it should be possible in principle to use recombinant DNA technology to direct a protein derived from any source into any structural phase of a transformed cell. There are already published reports describing how foreign proteins have been inserted into the nucleus, mitochondrion, chloroplast, endoplasmic reticulum and vacuole of intact cells. This ability to retarget proteins is an exciting development since it opens up a number of possibilities by which genetic engineering techniques could be used to create organisms with novel and useful properties (Ellis, 1985). One possibility that has already been realized is that targeting sequences which direct proteins to the endoplasmic reticulum can be attached to proteins of medical and industrial interest so that these products are secreted from the cell into the medium. For example, yeast cells will secrete human epidermal growth factor into the medium if the presequence for the yeast mating a factor is fused to the sequence for the growth factor (Brake et al., 1984). Recent work has shown that the a factor presequence directs the secretion of accurately and completely processed proteins in several other cases (Zsebo et a f . , 1986). Since yeast cells can be grown on a large scale and release relatively little endogenous protein to the medium, this technique permits the production of heterologous proteins in a readily purifiable form (Smith et af., 1985b). A more speculative possibility is that the ability to retarget proteins will allow the directed modification of metabolic pathways in the subcellular compartments of eukaryotic cells. For example, it is now feasible to consider how one might alter the properties of the chloroplast compartment by the addition of novel proteins that normally occur only in animal or bacterial cells. Could one improve the efficiency of a metabolic process in the chloroplast by adding foreign enzymes which short-circuit the normal pathway, use ATP and NADPH more effectively, or cause intermediates to be pumped across the chloroplast envelope? We suspect

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that the limit to what is possible by this approach is not set by the technical difficulties, but by the mental and physical isolation of those researchers skilled in recombinant DNA technology from those skilled in studying intermediary metabolism. The two types of researcher tend to occupy different laboratories and certainly work in different intellectual climates. We suggest that great advances could be made if the two groups could be brought into effective interaction, not only in creating novel organisms with commercially valuable properties, but also in advancing our understanding of metabolic processes.

ACKNOWLEDGEMENTS We thank R . A . Laskey, G.Schatz, H. J. Bohnert and P.Schreier for access to data before publication. R. J. E. wishes to acknowledge financial support from the Science and Engineering Research Council.

REFERENCES Ades, I. Z. and Butow, R. A. (1980). J. Biol. Chem. 255, 9918-9924. Baker, A. and Schatz. G. (1987). Proc. Natl Acad. Sci. USA 84.3117-3121. Benson, S. A . , Hal1,’M. N. and Silhavy, T. J. (1985). Ann. Rev. Biochem. 54, 101-134. Bitsch, A. and Kloppstech, K. (1986). Eur. J. Cell Biol. 40, 160-166. Blobel, G. (1980). Proc. Nail Acad. Sci. USA 77, 14961500. Blobel, G. and Dobberstein, B. (1975a). J. Cell Biol. 67, 835-851. Blobel, G. and Dobberstein, B. (1975b). J. Cell Biol. 67, 852-862. Bohni, P. C., Daum, G. and Schatz, G. (1983). J. Biol. Chem. 258,4937-4943. Bonner, W. M. (1978). In “The Cell Nucleus” (H. Bausch, ed.), Vol. 6, part C, pp. 97-148. Academic Press, New York. Boutry, M. and Chua, N-H. (1985). EMBO J. 4, 2159-2165. Brake, A. J., Merryweather, J. P., Coit, D. G., Heberlein, V. A., Masiarz, F. R., Mullenbach, G. T., Urdea, M.S., Valenzuela, P. and Barr, P. J. (1984). Proc. Natl Acad. Sci. USA 81,4642-4646. Cashmore, A. Szabo, L., Timko, M., Kausch, A., Van den Broeck, G., Schreier, P., Bohnert, H. J., Herrera-Estrella, L., Van Montagu, M. and Schell, J. (1985). Biotechnology 3, 803-808. Chua, N-H. and Schmidt, G. W. (1979). J. Cell Biol. 81,461483. Cline, K., Andrews, J., Mersey, B., Newcomb, E. H. and Keegstra, K. (1981). Proc. Natl Acad. Sci. USA 78,3595-3599. Cline, K., Werner-Washburne, M., Lubben, T. H. and Keegstra, K. (1985). J. Biol. Chem. 260,3691-3696. Colman, A. and Robinson, C. (1986). Cell 46, 321-322. Dingwall, C. (1985). Trends in Biochem. Sci 10, 64-66. Dingwall, C. and Laskey, R. A. (1986). Ann. Rev. Cell Biol. 2,365-388. Dingwall, C., Sharnick, S. V. and Laskey, R. A. (1982). Cell 30,449-458. Dingwall, C., Dilworth, S. M., Black, S. J., Kearsey, S. E., Cox, L. S. and Laskey, R. A. (1987). EMBO J. 6,69-74.

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23

Douglas, M. G., McCammon, M. T. and Vassarotti, A. (1986). Microbiol. Rev. 50, 166-178. Eilers, M. and Schatz, G. (1986). Nature Lond. 322, 228-232. Ellis, R. J. (1981a). Ann. Rev. Plant Physiol. 32, 111-137. Ellis, R. J. (1981b). Biochem. SOC. Symp. 46,223-234. Ellis, R. J . (1983a). Subcell. Biochem. 9 , 237-261. Ellis, R. J. (1983b). Nature Lond. 304, 308-309. Ellis, R. J. (1984). Sci. Prog. Oxf. 69, 129-142. Ellis, R. J. (1985). Nature Lond. 313, 353-354. Ellis, R. J. and Robinson, C. (1985). In “The Enzymology of Post-Translational Modification of Proteins” (R. B. Freedman, ed.), Vol. 2, pp. 25-39. Academic Press, London. Feldherr, C. M., Kallenbach, E. and Schultz, N. (1984). J. Cell Biol. 99, 22 16-2222. Gasser, S. M., Ohashi, A., Daum, G., Bohni, P. C., Gibson, J., Reid, G. A,, Yonetani, T. and Schatz, G. (1982). Proc. Nut1 Acad. Sci. USA 79,267-271. Gillespie, L. L., Argan, C., Tanela, A. T., Hodges, R. S., Freeman, K. D. and Shore, G. C. (1985). J . Biol. Chem. 260, 16045-16048. Goldfarb, D. S., Gariepy, J., Schoolnik, G. and Kornberg, R. D. (1986). Nature Lond. 322,641-644. Gray, M. W. and Doolittle, W. F. (1982). Microbiol. Rev. 46, 1 4 2 . Grossman, A., Bartlett, S. and Chua, N-H. (1980). Nature Lond. 285, 625-628. Hageman, J., Robinson, C., Smeekens, S. and Weisbeek, P. (1986). Nature Lond. 324,567-569. Harmey, M. A., Hallermayer, G., Korb, H. and Neupert, W. (1977). Eur. J . Biochem. 81,533-544. Hase, T., Muller, U., Riezman, H. and Schatz, G. (1984). EMBO J. 3,3157-3164. Hay, R., Bohni, P. and Gasser, S. (1984). Biochim. Biophys. Acta 779, 65-87. Highfield, P. E. and Ellis, R. J. (1978). Narure Lond. 271, 420-424. Horwich, A. L., Kalousek, F., Fenton, W. A., Pollock, R. A. and Rosenberg, L. E. (1986). Cell 44,451-459. Hurt, E. C. and Schatz, G. (1987). Nature Lond. 325,499-503. Hurt, E. C. and van Loon, A. P. G. M. (1986). Trends in Biochem. Sci. 5,204-207. Hurt, E. C., Muller, U. and Schatz, G. (1985a). EMBO J . 4, 3509-3518. Hurt, E. C., Pesold-Hurt, B., Suda, K., Oppligeler, W. and Schatz, G. (1985b). EMBO J . 4, 2061-2068. Hurt, E. C., Soltanifar, N., Goldschmidt-Clermont, M., Rochaix, J-D. and Schatz, G. (1986). EMBO J . 5 , 1343-1350. Kalderon D., Richardson, W. D., Markham, A. T. and Smith A. E. (1984a). Nature Lond. 311, 33-38. Kalderon, D., Roberts, B. L., Richardson, W. D. and Smith, A. E. (1984b). Cell 39,499-509. Karlin-Neumann, G. A. and Tobin, E. M. (1986). EMBO J. 5 , 9-13. Kuntz, M., Simons, A., Schell, J. and Schreier, P. H. (1986). Mol. Gen. Genet. 205,454-460. Laskey, R . A. and Earnshaw, W. C. (1980). Nature Lond. 286, 763-767. Miura, S., Mori, M., Amaya, Y. and Tatibana, M. (1982). Eur. J . Biochem. 122, 641-677. Muller, M. and Blobel, G. (1984a). Proc. Natl Acad. Sci. USA 81, 7421-7425. Muller, M. and Blobel, G. (1984b). Proc. Nut1 Acad. Sci. USA 81, 7737-7741. Nagy, F., Fluhr, R., Morelli, G., Kuhlemeier, C., Poulsen, C., Keith, B., Boutry, M. and Chua, N-H. (1986). Phil. Trans. R. SOC. Lond. B313,409417.

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Newmeyer, D. D., Lincocq, J. M., Burglin, T. R. and de Robertis, E. M. (1986). EMBO J . 5,501-510. Novick P. (1985). Trends in Biochem. Sci. 10,432-434. Ohashi, A., Gibson, J., Gregor, 1. and Schatz, G. (1982). J. Biol. Chem. 257, 13042-13047. Oliver, D. (1985). Ann. Rev. Microbiol. 39, 615-648. Palade, G. (1975). Science 189,347-358. Pfaller, N. and Neupert, W. (1985). EMBO 3. 4,2819-2825. Pfisterer, J., Lachmann, P. and Kloppstech, K. (1982). Eur. J. Biochem. 126, 143-148. Pratje, E. and Guiard, B. (1986). EMBO J. 5, 1313-1317. Pugsley. A. P. and Schwartz, M. (1985). FEMS Microbiol. Revs 32,3-38. Reid. G. A., Yonetani, T. and Schatz, G . (1982). J. Biol. Chem. 257,13068-13074. Rietveld, A. and de Kruijff, B. (1986). Bioscience Reports 6, 775-782. Riezman, H., Hay, R., Witte, C., Nelson, N. and Schatz, G. (1983). EMBO J . 2, 1113-1118. Robinson, C. and Ellis, R. J. (1984a). Eur. J . Biochem. 142, 337-342. Robinson, C. and Ellis, R. J. (1984b). Eur. J . Biochem. 142, 343-346. Robinson, C. and Ellis, R. J. (1985). Eur. J . Biochem. 152, 67-73. Roise, D., Horvath, S. J., Richards, J. H., Tomich, J. M. and Schatz, G . (1986). EMBO J . 5, 1327-1334. Schatz, G. and Mason, T. L. (1974). Annu. Rev. Biochem. 43, 51-87. Schekman, R. (1982). Trends in Biochem. Sci. 7,243-246. Schleyer, M. and Neupert, W. (1985). Cell 43,339-350. Schmidt, B., Wachter, E., Sebald, W. and Neupert, W. (1984). Eur. J. Biochem. 144,581-588. Schmidt, G. W. and Mishkind, M. (1986). Ann. Rev. Biochem. 55,879-912. Schreier, P. H. and Schell, J. (1986). Phil. Trans. R. SOC. Lond. B313,429-432. Schreier, P . H., Seftor, E. A., Schell, J. and Bohnert, H. J. (1985). EMBOJ. 4, 25-32. Smeekens, S., de Groot, M., van Binsbergen, J. and Weisbeek, P. (1985). Nature Lond. 317,456-458. Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K. and Weisbeek, P. (1986). Cell 46, 365-375. Smith, A. E., Kalderon, D., Roberts, B. L., Colledge, W. H. and Edge, M. (1985a). Proc. R. SOC.Lond. B226,43-58. Smith, R. A., Duncan, M. J. and Moir, D. T. (1985b). Science 229, 1219-1223. Suissa, M. and Schatz, G . (1982). J. Biol. Chem. 257, 13048-13055. Unwin, P. N. T. and Milligan, R. (1982). J. Cell Biol. 93,63-75. Van den Broeck, G., Timko, M. P., Kausch, A. P., Cashmore, A, R., Van Montagu, M. and Herrera-Estrella, L. (1985). Nature Lond. 313, 358-363. van Loon, A. P. G. M., Brandli, A. and Schatz, G. (1986). Cell 44, 801-812. Wasmann, C. C., Reiss, B., Bartlett, S. G. and Bohnert, H. J. (1986). Mol. Gen. Genet. 205,446453. Wickner, W. T. and Lodish, H. F. (1985). Science 230, 4 W 0 7 . Zimmermann, R . , Hennig, B. and Neupert, W. (1981). Eur. J. Biochem. 116, 455460. Zsebo, K. M., Lu, H-S., Fieschko, J. C., Goldstein, L., Davis, J., Duker, K., Suggs, S. V., Lai, P-H., and Bitter, G. A. (1986). J. Biol. Chem. 261, 5858-5864.

Control of Isoprenoid Biosynthesis in Higher Plants

JOHN C.GRAY

Botany School, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK

I.

The Pathways of Isoprenoid Metabolism . A. Formation of HMG-CoA . . . . B. Formation of Mevalonate . . . . C. FormationofIsopentenylDiphosphate D. Formation of Prenyl Diphosphates . E. Formation of Isoprenoid Compounds F. Mevalonate Shunt . . . . . . . G. Conclusions . . . . . . . . .

. . . . . . .

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

. . . . . . .

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

27 28 30 34 38 42 50 52

Compartmentation of the Pathways of Isoprenoid Biosynthesis . . . . . . . . . . . . . . . . . A. Subcellular Location of Enzymes of Isoprenoid Biosynthesis . . . . . . . . . . . . . . . . B. Permeability of Membranes to Biosynthetic Intermediates . . . . . . . . . . . . . . . C. Conclusions . . . . . . . . . . . . . . . .

65 67

111.

Control of the Isoprenoid Biosynthetic Pathway . . . . . . A. Fine Control . . . . . . . . . . . . . . . B. Coarse Control . . . . . . . . . . . . . . . C. Conclusions . . . . . . . . . . . . . . . .

68 69 80 85

IV.

General Discussion . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . .

86 86

11.

52 55

Copyright @ 1987 Academic Press Limited All rights of reproduction in any form reserved.

Advances in Botanical Research Vol. 14 ISBN 0-12-005914-2

25

JOHN C . GRAY

26

Higher plants contain a bewildering array of isoprenoid compounds with a wide variety of structures and functions. The presence of high concentrations of some of these compounds in turpentine oil, derived by steam distillation of wood and resin, has given rise to an alternative generic name, the terpenoid compounds. All of these compounds have a structural motif based on the isoprene (I) carbon skeleton and all are derived biosynthetically from isopentenyl diphosphate (11), the “active isoprene” compound whose existence was predicted on the basis of chemical structural studies on a large number of natural products.

i“‘ H2cRc\

CH3

I

HCH2H2C ~ c \ c / c \ o p p

C I

H

I

H\ /H

/ \

H

H

II

In higher plants many of these isoprenoid compounds play vital roles in the metabolism and development of the plant. The plant growth regulators, abscisic acid and gibberellins, are isoprenoid compounds, and many cytokinins contain an isoprenoid side chain. Isoprenoid side chains are also found in many other biologically active molecules, including chlorophylls, plastoquinone and other prenylquinones in chloroplasts, where together with carotenoids they are involved in photosynthesis. Isoprenoid side chains are also found in the mitochondria1 electron transfer chain components, ubiquinone and haem a of cytochrome oxidase. Long-chain prenyl phosphates are involved in polysaccharide and oligosaccharide synthesis, and sterols are components of biological membranes where they may modulate membrane fluidity. In addition, individual groups of plants have evolved specific isoprenoid compounds for specific functions, such as attractants for insect pollination, or as phytoalexins for defence against fungal or bacterial infection. Plants must be able to produce this wide range of isoprenoid compounds in different amounts in different parts of the plant at different stages of growth and development, As all these compounds are produced by a common biosynthetic pathway, the plant must have exquisite control mechanisms to ensure the synthesis of the necessary compounds in the right place at the right time. The aim of this chapter is to consider our knowledge of the control of isoprenoid biosynthesis in higher plants, particularly with respect to the properties and subcellular locations of the enzymes involved. This aspect of isoprenoid biosynthesis has been strangely neglected in recent books and reviews, and there has been a widespread

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

27

uncritical acceptance of certain models for the regulation of isoprenoid biosynthesis in plants. In this article the experimental evidence for postulated regulatory mechanisms will be critically assessed and an attempt will be made to provide a conceptual framework for further experimentation.

I. THE PATHWAYS OF ISOPRENOID METABOLISM The biosynthetic pathway for the formation of isoprenoid compounds in plants is based firmly on the pathway of sterol synthesis worked out over many years in animals and yeast. In plants, isoprenoid biosynthesis may be viewed as a main pathway from acetyl-CoA via mevalonate and isopentenyl diphosphate (IPP) to long-chain prenyl diphosphates, with a large number of branch points leading to the individual isoprenoid compounds. This is shown schematically in Fig. 1, where the main classes of isoprenoid compounds found in plants are included. The central importance of IPP as a precursor of isoprenoid compounds must be emphasized, and the subcellular organization of the pathways utilizing this acetyl CoA

t

acetoacetyl CoA

t

HMG CoA

f

mevalonate

cytokinin sidechains

monoterpenes

i

4

* rubber

Gr

* sterols

1

+

haem a sidechains

IPP

-

FPP

J

phytyl sidechains gibberellins

GGPP

-

-

carotenoids

t

plastoquinone sidechains

nonaprenyl PP

ubquinone sidechains

decaprenyl PP

f

Fig. 1. Scheme for the biosynthesis of isoprenoid compounds from a common pathway.

28

JOHN C. GRAY

intermediate is a vital factor in the regulation of isoprenoid metabolism in plan& An understanding of the metabolic pathways and the properties of the enzymes involved is essential for any discussion of the regulation of isoprenoid metabolism, particularly of any reactions that might divert intermediates away from isoprenoid biosynthesis. A. FORMATION O F HMG-CoA

The routes for the formation of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) in higher plants have been much neglected. Two pathways have been established in animals and fungi. The first involves the condensation of two molecules of acetyl-CoA to produce acetoacetyl-CoA, which is then condensed with another molecule of acetyl-CoA to give HMG-CoA (Fig. 2). The presence of such a pathway in plants was suggested by the experiments of Johnston et al. (1954) who demonstrated t

O H3CKSCoA ocetyl-CoA CoASH

0

4 1

0

+

CoASXCH3

o H3CKSCoA acetyl-CoA

0

0

H $ ~ S C O A

acetyl- CoA

0

0

+

HOMCH3

acetwcrtyl-CoA

H$&CoA

acetoacrtata

acetyl -CoA

-

HMO CoA

CH3H NH2

0

CH3 0

-

a ketoiaocaproak

u scoA

-

leucine

HsC-COOH

CH3O

m

H E uCOOH

3 methylglutaconyl-CoA

*CH3 0

CoASH

H3ChKSCaA

co2

+

2H

irovaleryl-CoA

A

%+

CH3 0

H 3 C U S C o A

FADH2

dimethylacrylyl-COP.

Fig. 2. Scheme for the synthesis of HMG-CoA from acetyl-CoA, showing the intermediates of the leucine degradation pathway. The enzymes catalysing the reactions are numbered. 1, acetoacetyl-CoA thiolase; 2, HMG-CoA synthase; 3, HMG-CoA lyase; 4, leucine aminotransferase; 5 , 2-oxoisocaproate dehydrogenase; 6, isovaleryl-CoA dehydrogenase; 7, 3methylcrotonyl-CoA carboxylase; 8, 3-methylglutaconyl-CoA hydratase.

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

29

the incorporation of [14C]acetateinto HMG by extracts of flax seedlings. The incorporation of acetate was stimulated by added ATP and CoA. The synthesis of a derivative of HMG, presumably the CoA ester, from acetyl-CoA by extracts of sweet potato tissue has been demonstrated by Oshima and Uritani (1967). Support for this pathway in plants is also provided by chemical degradation studies on p-carotene synthesized in vivo from [ 114C]acetateor [214C]acetate(Braithwaite and Goodwin, 1960). Distribution of label in the individual isoprene units is as expected if the intermediates were synthesized via acetoacetyl-CoA and HMG-CoA. The equilibrium of the first step in this scheme, the formation of acetoacetylCoA from two acetyl-CoA molecules, is such that the reverse reaction is favoured (Hartmann and Lynen, 1961) and it has been suggested that the condensation of acetyl-CoA and malonyl-CoA, with the production of acetoacetyl-CoA and the release of C 0 2 , would give an essentially irreversible first step to the pathway (Brodie et al., 1963). The incorporation of [214C]malonyl-CoAinto rubber in the latex of Hevea brasiliensis has been observed, but can probably be accounted for by the prior decarboxylation of the malonyl-CoA to acetyl-CoA (Higgins and Kekwick, 1973). The enzymes involved in the formation of HMG-CoA from acetyl-CoA in plants have not been characterized. Acetoacetyl-CoA thiolase (EC 2.3.1.9) has not been purified from any plant source and its properties are essentially unknown. The formation of acetoacetyl-CoA from acetyl-CoA in extracts of spinach leaves is optimal at pH 8.8 in the presence of 50 mM Mg2+ (Schulze-Siebert and Schultz, 1985). HMG-CoA synthase (EC 4.1.3.5) has similarly not been purified from any plant source and its presence is largely inferred from the formation of HMG-CoA from acetyl-CoA in plant extracts. As HMG-CoA synthesis has been reported to contribute to the control of cholesterol synthesis in animal cells (White and Rudney, 1970), further information on the nature of this enzyme in plants is urgently needed. A second pathway for the formation of HMG-CoA has been established in bacteria, animals and fungi. In this pathway, HMG-CoA is an intermediate in the degradation of the branched-chain amino acid leucine, leading ultimately to acetyl-CoA and acetoacetate (Fig. 2). Labelled leucine has been shown to be incorporated into carotenoids in barley leaf pieces (Ehrenberg and Daniel, 1962), into squalene and P-amyrin in germinating pea seeds (Suga et al., 1980) and into sterols and sesquiterpenoids in tissue cultures (Ryder and Goad, 1980; Anastasis et a f . , 1983, 1985). However, detailed examination by "C-nmr spectroscopy of the labelling patterns of the isoprenoids synthesized from [''CC]leucine in tissue cultures of Androgruphis paniculuta indicated that the leucine was degraded to acetyl-CoA and acetoacetate before incorporation into the isoprenoid compounds (Anastasis ef a f . , 1985). The conversion of

30

JOHN C. GRAY

3,3-dimethylacrylic acid (p-methylcrotonic acid) to HMG, a key step in the leucine degradation pathway, has been demonstrated in extracts of flax seedlings (Johnston et al., 1954) and the limited incorporation of [13C]dimethylacrylic acid into sterols by tissue cultures of Andrographis paniculata has also been shown (Anastasis et al., 1985). However, as with leucine, the labelling pattern of the sterols produced was consistent with the degradation to acetyl-CoA and acetoacetate before incorporation into isoprenoids. It therefore seems probable that the HMG-CoA derived from leucine is not converted directly to mevalonate, but is degraded to acetyl-CoA and acetoacetate, which may then serve as precursors for isoprenoid biosynthesis. HMG-CoA lyase (EC 4.1.3.4), the enzyme responsible for the breakdown of HMG-CoA to acetoacetate and acetylCoA in other organisms, has been detected in the latex of Heveu brasiliensis (Hepper and Audley, 1969), but has not been characterized from higher plants. B. FORMATION OF MEVALONATE

The formation of mevalonate from HMG-CoA is catalysed by the enzyme HMG-CoA reductase (EC 1.1.1.34). The enzyme catalyses the two-step reduction of the thioester group of HMG-CoA to the 5’ hydroxyl of mevalonate using two molecules of NADPH (Fig. 3). This reaction is the major rate-controlling step in isoprenoid biosynthesis in animals. The enzyme has been detected in the latex of Hevea brasiliensis (Hepper and Audley, 1969; Lynen, 1969; Sipat, 1982a) and in extracts of pea seedlings (Brooker and Russell, 1975a), sweet potato roots (Suzuki et al., 1975), radish seedlings (Bach et al., 1980) and leaves of Nepetu cataria (Arebalo and Mitchell, 1984). In all cases the enzyme activity was membrane bound and could be pelleted by centrifugation. The major subcellular location appears to be the endoplasmic reticulum, but enzyme activity has also been reported to be associated with mitochondria (Brooker and Russell, 1975b; Suzuki and Uritani, 1976; Bach et al., 1980) and plastids (Brooker and Russell, 1975b; Arebalo and Mitchell, 1984). The subcellular localization of the enzyme is of considerable importance to our understanding of the control of isoprenoid metabolism and will be discussed in more detail in Section II.A.2. The properties of HMG-CoA reductase have, until recently, been determined from assays on membrane preparations. The use of membrane preparations for this purpose has its limitations, particularly if the fractionation procedures employed fail to separate the different membranes of the tissue. This is especially dangerous in the case of the enzyme from Hevea latex where the membrane-bound enzyme is sedimented in a fraction containing hydrolytic enzymes (Sipat, 1982b). The action of these

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS r

HO

1

CH3

\ /

1

H?

COOH

,CH3

I 1 NADPH

CHOl

mevaldate

HMG-COA

31

HO

CH3

\/

COOH

&,OH

mevalonate

HO

\/”

mevinolin Fig. 3. The conversion of HMG-CoA to mevalonate, showing the structure of mevinolin, a potent inhibitor of HMG-CoA reductase.

enzymes, proteolytic or lipolytic, may well modify the HMG-CoA reductase or its membrane environment, thus changing its properties. The enhanced HMG-CoA reductase activity in sweet potato membranes in the presence of bovine serum albumin may be due to effects on such hydrolytic processes (Suzuki and Uritani, 1976). The serum albumin may act as an alternative substrate for proteolytic enzymes, thus relieving proteolytic degradation of HMG-CoA reductase, or as a protein capable of binding free fatty acids of endogenous origin or formed by the action of lipolytic enzymes. From studies on HMG-CoA reductase in membrane preparations of pea seedlings it is clear that the enzyme uses NADPH, rather than NADH, as the source of reducing power (Brooker and Russell, 1975a). The enzyme activity appears to require the presence of free thiol group(s), as the enzyme is activated in the presence of thiol compounds such as cysteine, glutathione, 2-mercaptoethanol and dithiothreitol and is inhibited by thiol reagents such as iodoacetamide (Suzuki et al., 1975), p-chloromercuribenzoate (Sipat, 1982a) and DTNB (5,5’-dithiobis (2-

32

JOHN C. GRAY

nitrobenzoate)) (Bach and Lichtenthaler, 1983a). Some of the kinetic parameters of the enzymes from different sources are presented in Table I. The pH optimum of the enzyme activity differs between the different preparations; the enzymes from pea microsomal membranes and Hevea latex show optimal activity at pH 6.8 and pH 6.6-6.9 respectively, whereas higher pH optima have been reported for membrane preparations from radish microsomes (pH 7 . 9 , sweet potato membranes (pH 7.3-7.5) and pea plastid membranes (pH 7.9). It is not yet clear if these differences are due to inherent differences in the enzymes, or are due to extraneous factors in the different membrane preparations. The reported values of K, for RS-HMG-CoA vary over two orders of magnitude from 7.7X lo-’ M for pea plastid membranes to l x M for pea microsomal membranes. The possible significance of such a wide variation in K , is not clear. The K, for NADPH has not been reported for any of the membrane preparations, although Bach et al. (1980) reported sigmoidal kinetics with the radish microsomal preparation, obtaining half-maximal activity at approximately 7 x M NADPH. Solubilization of HMG-CoA reductase from membrane preparations using detergents has been attempted but only limited solubilization (up to 33% of activity solubilized) of enzyme activity from Hevea latex bottom fraction was obtained with Triton X-100 (Sipat, 1982a). However, Bach er al. (1986) reported that the enzyme in radish membranes could be solubilized by incubation in Brij W-1 and that in the presence of glycerol an increase in enzyme activity was obtained. The solubilized radish enzyme was subsequently purified .by ammonium sulphate precipitation and chromatography on DEAE-Sephadex, Blue dextran-agarose and HMG-CoAhexane-agarose. The enzyme behaved as a protein of M , 180,000 on sucrose density gradient centrifugation in the presence of Triton X- 100, and as a polypeptide of M , 45,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate, suggesting a tetrameric structure for the enzyme (Bach et al., 1986). However, unlike the reference marker proteins, the enzyme would be expected to be associated with a Triton X-100 micelle during sucrose gradient centrifugation and the micelle would contribute approximately 90,000 to the relative molecular mass of the complex (Helenius and Simons, 1975). This would suggest that the purified HMG-CoA reductase may be dimeric. The purified radish enzyme has been used for kinetic studies to establish an enzyme mechanism. Values of K , of 3 X 1 0 - 6 ~for RS-HMG-CoA and 2 . 7 ~ 1 0 for - ~ NADPH ~ were obtained. Inhibition of the enzyme reaction by the products, CoA and NADP+, was observed. CoA showed competitive inhibition with respect to HMG-CoA and noncompetitive inhibition with respect to NADPH; Ki values of 2X10-4 M and 9x M CoA were obtained for the competitive and noncompetitive inhibitions respectively. NADP+ inhibition was competitive with respect to NADPH and noncompetitive with respect to

TABLE I Kinetic properties of HMG-CoA reductase Km

Source of enzyme

pH optimum

Radish seedlings 16,000g membranes Purified enzyme 105,OOOgmembranes

7.5

Hevea brasiliensis latex 40,OOOg membranes

6.6-6.9

Pea seedlings 3000g membranes 50,OOOg membranes

7.9 6.9

Sweet potato roots, infected 105,000g membranes

-

7.57.5

RS-HMG-CoA

NADPH

5 . 5 ~ 1 0 - ~ ~ 3.0~ M 2.7x 4.75~ M a 5.6~

M

7 . 7 ~ 1 0M ~’ 1 ~ 1 0 - ~ ~ -

1.3x 4.2~

“Sigmoidalkinetics reported by Bach er af. (1980). M. bKm value at RS-HMG-CoAconcentrations less than 1 X M. ‘K, value at RS-HMG-CoAconcentrations greater than 1 x

Mb M‘

M

Reference Bach and Lichtenthaler (1984) Bach etal. (1986) Bath and Lichtenthaler (1984) Sipat (1982a) Wong et al. (1982) Brooker and Russell (1975a) Suzuki et al. (1975)

34

JOHN C. GRAY

HMG-CoA; Ki values of 6 . 5 ~ 1 0 - ' ~and l . 2 X 1 0 - 3 ~were obtained. These data are consistent with an ordered enzyme mechanism in which HMG-CoA is the first substrate bound to the'enzyme (Bach et al., 1984). Mevalonate, the other product of the reaction, does not appear to be an inhibitor of the membrane-bound enzyme at concentrations up to l ~ l O M- ~(Brooker and Russell, 1975a; Sipat, 1982a; Bach and Lichtenthaler, 1984), but Bach et al. (1986) have reported that mevalonate is a competitive inhibitor with respect to HMG-CoA of the purified radish enzyme. A Ki value of 0.99 M mevalonate was obtained. The radish microsomal HMG-CoA reductase has been shown to be severely inhibited by mevinolin (Bach and Lichtenthaler, 1982), a metabolite isolated from Aspergillus terreus (Alberts et al., 1980). Mevinolin is one of a group of related compounds, isolated from ascomycetes, which show hypocholesterolaemic activity in mammals due to potent competitive inhibition of HMG-CoA reductase (Alberts et al., 1980). The structure of part of these compounds resembles HMG and mevalonate (see Fig. 3). With the radish microsomal enzyme, mevinolin inhibits competitively with respect to HMG-CoA and has a Kiof 2 . 2 ~ l O M - ~(Bach and Lichtenthaler, 1983a). At high concentrations of NADPH, mevinolin acts as a noncompetitive inhibitor of the enzyme with respect to NADPH, but at lower NADPH concentrations ( C 1 . 6 lo-' ~ M) a variety of kinetic responses of the enzyme to mevinolin were obtained (Bach and Lichtenthaler, 1983a). Mevinolin and compactin (ML-236B) have been shown to be potent inhibitors of sterol synthesis in cell suspension cultures of Acer pseudoplatanus (Ryder and Goad, 1980) and Silybum marianum (Doll et a f . ,1984) and in radish seedlings (Bach and Lichtenthaler, 1983b; Schindler et a f . , 1985). Differences in the inhibition of accumulation of different isoprenoid compounds by mevinolin in Silybum marianum (Doll et al., 1984) and in radish seedlings (Bach and Lichtenthaler, 1983b; Schindler et al., 1985) have been interpreted as evidence for HMG-CoA reductase in several subcellular locations in these plants. This will be discussed in Section II.A.2. C. FORMATION OF ISOPENTENYL DIPHOSPHATE

Mevalonate is converted to IPP by the sequential action of three enzymes, each using one molecule of ATP per molecule of substrate (Fig. 4). This is a well-characterized part of the isoprenoid biosynthetic pathway in plants, due mainly to the longstanding commercial availability of radioactively labelled mevalonate. Incubation of [2I4C]-or [3H]rnevalonatewith extracts of numerous plants in the presence of ATP results in the formation of mevalonate 5-phosphate, rnevalonate 5-diphosphate and IPP, which may be separated from each other and from mevalonate by a variety of

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

I

35

H H

COOH mevalonate

mevalonaie-5- phosphate

CH20PP H2C

3 7 p

RC\/ I\ H H

ADP

t Pi

isopentenyl disphosphate

Cop

iH'

" HJc

1

CHpOPP

/c\c/

coon

/\

H H

-

rnevalonate- 5 diphosphote

Fig. 4. Scheme for the synthesis of IPP from mevalonate. The enzymes are: 1 , mevalonate kinase; 2, mevalonate 5-phosphate kinase; 3, mevalonate 5-diphosphate decarboxylase.

chromatographic methods. The formation of these compounds from mevalonate is therefore relatively easy to demonstrate. Quantitation of the reactions in plant extracts is more difficult because of the presence of phosphatase activity. In extracts of cotyledons of Phaseolus vulgaris, mevalonate 5-phosphate, mevalonate 5-diphosphate and IPP were hydrolysed at rates in excess of the phosphorylation of mevalonate (Table 11). Hydrolysis of mevalonate 5-phosphate by extracts of Phaseolus cotyledons was also demonstrated by Hill and Rogers (1974), and Oshima-Oba and Uritani (1969) reported the active hydrolysis of IPP to isopentenyl monophosphate and isopentenol by extracts of sweet potatoes. Fluoride, an inhibitor of phosphatase activity, has often been reported to increase TABLE I1 Phosphatase activity of extracts of Phaseolus vulgaris cotyledons" Substrate 1X M Mevalonate 5-phosphate 1x M Mevalonate 5-diphosphate 1x M Isopentenyl diphosphate ~x~O-~MADP 5x M ATP

Phosphatase activity nmoI min-' (mg protein)-'

0.62 3.18 11.85 600 1350

"The extracts displayed a mevalonate kinase activity of 0.37 nmol min-' (mg protein)- '.

36

JOHN C. GRAY

the production of phosphorylated compounds from mevalonate in extracts of higher plants (Beytia et al., 1969; Potty and Bruemmer, 1970; Shewry and Stobart, 1973). The three enzymes involved in the conversion of mevalonate to IPP have been partially purified from plant sources and some of their properties have been determined. Perhaps the major point of controversy is the subcellular location of these enzymes in plants. This will be discussed in fuller detail in Section II.A.3. Mevalonate kinase (EC 2.7.1.36) catalyses the phosphorylation of mevalonate in the presence of ATP and M 8 + . The activity of this enzyme has been demonstrated in extracts of a large number of plants. However, because of the presence of phosphatase activity in these extracts much of the published information on the characteristics of mevalonate kinase is misleading. This is particularly the case with the pH optimum of the reaction. A wide range of pH optima, from 5.5 to 8.0, has been reported for mevalonate kinase in extracts of various plants. However, with purified preparations of mevalonate kinase from Hevea latex (Williamson and Kekwick, 1965), cotyledons and leaves of Phaseolus vulgaris (Gray and Kekwick, 1973a) and leaves of Agave americana (Suarez and GarciaPeregrin, 1977), the pH optima were all in the range 7.0-7.9. Acidic pH optima for kinase reactions are unlikely on theoretical grounds because of protonation of ATP phosphate groups and the consequent decrease in the concentration of the Mg-ATP substrate complex. For the partially purified preparations of the enzyme from Hevea, Phaseolus and Agave, K, values - ~ RS-mevalonate and of 1 . 3 ~ 1 0 M, - ~ 4 . 5 ~ 1 0 -M~ and 5 . 0 ~ 1 0 M 2X10-3 M, 1 . 5 ~ 1 0 M - ~and 1 . 4 ~ 1 0 M- ~ATP were obtained respectively. The enzyme from leaves of Phaseolus was inhibited 40% by the product mevalonate 5-phosphate at 3 x M, but was not inhibited by ADP or IPP. Potent inhibition of the enzymes from Phaseolus vulgaris cotyledons, Hevea latex and Cucumis melo cotyledons by prenyl diphosphates was observed by Gray and Kekwick (1972, 1973a). Geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP) and phytyl diphosphate all inhibited competitively with respect to ATP. The Kifor FPP was in the range 5.2-10.8~ M for mevalonate kinase from the three plants. This is similar to the inhibition observed with mevalonate kinase from animals (Dorsey and Porter, 1968; Flint, 1970). A possible physiological role for such inhibition will be discussed in Section 1II.A. The relative molecular mass of mevalonate kinase from Hevea, Phaseolus and Cucumis has been estimated from gel filtration and sucrose density gradient centrifugation to be approximately 100,000 (Gray and Kekwick, 1973b), the same size as the enzyme from animals and yeast. However, two forms of mevalonate kinase differing in relative molecular mass have been separated by gel filtration from Pinus pinaster and Agave americana (Garcia-Peregrin et al., 1973b). Although no values of M , were

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

37

reported, the larger form eluted from Sephadex G-100 approximately in the position expected for a protein of M, 100,000. The other form was eluted in a larger volume and therefore corresponds to a protein of lower M,. The origin of this lower M , form is not known and it has not been observed in other plants. It may have been a proteolytically degraded form of mevalonate kinase that retained enzyme activity. Treatment of Phaseolus leaf mevalonate kinase with papain produced altered molecular forms of the protein which retained enzyme activity (Gray and Kekwick, 1973a). Two forms of mevalonate kinase separable by sucrose density gradient centrifugation have been reported in extracts of Nepeta cataria leaves (Arebalo and Mitchell, 1984). One form sedimented at the same rate as mevalonate kinase from pig liver and yeast and therefore presumably corresponds to the M, 100,000 form; the other form sedimented further into the gradient, indicating that it was either a higher M, form or associated with membranes. Unfortunately, neither the centrifugation time nor the position of marker proteins or membranes in the gradient were given, and hence it is not possible to assess the significance of this second form of the enzyme. Membrane-bound mevalonate kinase activity has not been reported for other plants. Although the activity of mevalonate 5-phosphate kinase (EC 2.7.4.2) has been detected by the formation of mevalonate 5-diphosphate when extracts of various plants were incubated with mevalonate and ATP, specific assays for enzyme activity have rarely been carried out. Quantitative estimates of mevalonate 5-phosphate kinase activity have been made only for the latex of Hevea brasiliensis (Lynen, 1969; Skilleter and Kekwick, 1971) and for extracts of germinating pea seeds (Green and Baisted, 1972). The enzyme has been partially purified from Hevea latex and shows optimal activity at pH 7.2. The K, values for mevalonate 5-phosphate and - ~ respectively. The equilibrium ATP were 4 . 2 ~ 1 0 -M~ and 1 . 9 ~ 1 0 M constant for the reaction was estimated to be 0.34. The enzyme appears to require free thiol group(s) for activity because enzyme activity was lost in the absence of N-acetylcysteine (Skilleter and Kekwick, 1971). The enzyme mevalonate 5-diphosphate decarboxylase (EC 4.1.1.33) catalyses the decarboxylation and dehydration of mevalonate 5diphosphate to yield IPP. ATP is required for the reaction and it has been suggested that mevalonate 3-phosphate-5-diphosphateis formed as a transitory intermediate. The enzyme has been specifically assayed only in Hevea latex (Skilleter and Kekwick, 1971) and extracts of sweet potato roots (Oba et al., 1976), although the activity of the enzyme has been detected by the formation of IPP when extracts of various plants were incubated with mevalonate and ATP. The enzyme has been partially purified only from Hevea latex and shows optimal activity at pH 5.5-6.5. The K, values for mevalonate 5-diphosphate and ATP were 4X 10-' M and 1 . 2 ~ 1 0 -M~ respectively. The enzyme activity was inhibited by the

38

JOHN C. GRAY

products IPP and ADP; 50% inhibition was observed with 1 - 2 m ~IPP and with 5 mM ADP. D. FORMATION OF PRENYL DIPHOSPHATES

Isopentenyl diphosphate is the key intermediate in the formation of isoprenoid compounds. For most isoprenoid compounds, polymerization of C5 units is required to produce longer chain prenyl diphosphates which are the substrates for various enzymes at the branch points leading to the synthesis of the wide range of isoprenoid compounds (see Fig. 1). Labelled IPP has been shown to be an excellent precursor of many isoprenoid compounds in extracts of various plants. Studies with yeast and animals established that the first step in the formation of higher prenyl diphosphates was the isomerization of IPP to dimethylallyl diphosphate (DMAPP). The DMAPP then acted as the prenyl donor to a molecule of IPP, producing geranyl diphosphate, itself an allylic diphosphate which can act as a prenyl donor to another molecule of IPP, producing farnesyl diphosphate. This condensation of allylic diphosphates with IPP produces higher prenyl diphosphates (Fig. 5). The reactions are catalysed by IPP isomerase (EC 5.3.3.2) and prenyltransferases (EC 2.5.1.1). In the main isoprenoid biosynthetic pathway, these enzymes produce all-trans prenyl diphosphates. Isopentenyl diphosphate isomerase catalyses the isomerization of IPP and DMAPP. The presence of the enzyme in plant extracts is usually inferred from the incorporation of labelled IPP into higher prenyl diphosphates, and only rarely is the presence of DMAPP demonstrated (GeorgeNascimento and Cori, 1971; Gleizes et af., 1983a). A direct assay, quantitatively measuring the formation of DMAPP from IPP, can be used only in the absence of prenyl transferase, and this has been achieved only for pumpkin fruit (Ogura et af., 1968, 1969, 1971), cotton root (Widmaier et af., 1980) and tomato fruit preparations (Spurgeon et af., 1984). The enzyme has been partially purified from each of these sources (Ogura et af., 1968; Widmaier et af., 1980; Spurgeon et af., 1984). The cotton root enzyme shows optimal activity in the pH range 7.4-8.0 in the presence of 2 m Mg2+. ~ The K , value of the cotton root enzyme for IPP was ~ M and 2 . 2 lo-’ ~ M for two forms 7 . 4 lops ~ M,similar to values of 4 . 5 lo-’ of the enzyme separated by hydroxylapatite chromatography of pumpkin fruit extracts (Ogura et af., 1971), but rather higher than the K, value of 5.7X10p6M reported for the tomato fruit enzyme (Spurgeon et af., 1984). The enzyme from all sources is substantially inhibited by iodoacetamide (Ogura et af., 1971; Jedlicki et af., 1972; Spurgeon et af., 1984), suggesting that thiol groups are required for enzyme activity. Both forms of the pumpkin enzyme are inhibited by inorganic pyrophosphate, with approx-

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

L C H z O P P

1

39

L C H z O P P

dimethylollyl diphoaphate

lsopentenyl diphwphate

Y

CHZOPP

1

geranyl diphosphate

/LCHzOPP

I PP

fornesyl diphosphate

geronylgeranyl diphosphate

Fig. 5. Scheme for the polymerization of IPP to higher prenyl diphosphates. The enzymes are: 1, IPP isomerase; 2, prenyltransferase.

imately 50% inhibition being obtained with 1 X M pyrophosphate (Ogura et al., 1969, 1971). Prenyl diphosphates have been reported to inhibit the pumpkin and tomato enzymes. Dimethylallyl diphosphate ( 8 X 1 0 - ’ ~ ) gave 40% inhibition of the tomato enzyme. whereas 60% inhibition was produced by 8 ~ 1 M0geranyl ~ ~ diphosphate (Spurgeon et al., 1984). Geranyl diphosphate ( 1 x lop4M) produced 75% inhibition of the pumpkin enzyme (Ogura et al., 1969). The M, of the enzyme from tomato fruits has been estimated to be 34,000 by gel filtration on Sephadex G-100 (Spurgeon et al., 1984). This is considerably smaller than the size suggested by Widmaier et al. (1980) for the cotton root enzyme. The enzyme in cotton root extracts was associated with an electrophoretically separated protein fraction which migrated as a native protein of M , 92,000 and as a polypeptide of M , 98,000 in the presence of sodium dodecylsulphate. However, it is not clear if these are

40

JOHN C . GRAY

true estimates of the M , of the isomerase because the protein fraction was only 95% pure and the isomerase may have been a contaminating activity of the main protein fraction (Widmaier et al., 1980). The prenyltransferases are a group of enzymes which catalyse the condensation of IPP with allylic diphosphates. Enzyme fractionation studies indicate that there may be some specificity in the end-product produced by different prenyltransferases. Farnesyl diphosphate synthetase activity and geranylgeranyl diphosphate synthetase activities have been separated by ion-exchange chromatography on DEAE-Sephadex of extracts of pumpkin fruit (Ogura et al., 1972) and germinating castor beans (Green et al., 1975). Farnesyl diphosphate synthetase has been purified from pumpkin fruit (Ogura et al., 1968), cotton roots (Adams and Heinstein, 1973; Widmaier et al., 1980), germinating castor beans (Green and West, 1974), pea seedlings (Allen and Banthorpe, 1980) and orange rind (De La Funte etaf., 1981). Two forms of the enzyme from castor beans were separated by ion-exchange chromatography on QAE-Sephadex, but they had similar M , values, estimated at 72,500k3000 by gel filtration on Sephadex G-100. This value is slightly lower than the value of 96,OoOk4oOO reported for the apparently homogeneous enzyme from pea seedlings (Allen and Banthorpe, 1980). Gel electrophoresis of the pea enzyme in the presence of sodium dodecylsulphate gave a single polypeptide of M , 45,000f.3000, suggesting that the enzyme is a dimer of two similar sized subunits. The enzyme activity appears to be optimal at about pH 7.5 in extracts of pumpkin fruit, pea seedlings and orange rind, although a slightly lower optimum (pH 6.8) was reported from castor bean endosperm, and the cotton root preparations showed optimal activity at pH 7.6-8.5. The presence of Mg2+ was required for activity and 2 mM Mg2+ was optimal for the castor bean and orange rind preparations. Magnesium ions are probably required to produce a complex with the diphosphate moiety of the substrates of the enzyme. The enzymes from all sources were able to catalyse the condensation of IPP with DMAPP and GPP, but could not use FPP as the allylic substrate. The orange rind enzyme had K , values of 7.3X10-7 M GPP and 4.9~10-’M IPP, whereas the pumpkin enzyme had a K, of 1 . 3 ~ 1 0 M- ~GPP and the pea enzyme had a K , of 1 . 4 lo-’ ~ M IPP. The enzymes from pumpkin fruit and orange rind have been reported to be inhibited by several prenyl diphosphates and monophosphates, and to a limited extent by inorganic pyrophosphate (Ogura et al., 1969; De La Funte et af., 1981). Neryl diphosphate produced 85% inhibition of the pumpkin enzyme at 1x M, and 50% inhibition of the orange enzyme at M. Citronellyl diphosphate gave 50% inhibition of the orange 1.8x M, and geranyl monophosphate gave 50% inhibition of enzyme at 8x M and 3.6X M,respectivethe pumpkin and orange enzyme at 1x ly. Inhibition by inorganic pyrophosphate was obtained at relatively high concentrations: 1 . 8 lo-’ ~ M pyrophosphate was required to produce 50%

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

41

inhibition of the orange rind enzyme, and l x M pyrophosphate gave only 17% inhibition of the pumpkin enzyme. The activity of the orange rind enzyme was inhibited by p-chloromercuribenzoate (PCMB) and dithiobis (2-nitrobenzoic acid) (DTNB) and this inhibition was prevented by incubation of the enzyme with GPP and Mg2+ (De La Funte et al., 1981). This suggests the presence of a thiol group at the active site, although activity was not inhibited by iodoacetamide (Adams and Heinstein, 1973; Jedlicki et al., 1972). The enzyme preparations from cotton roots (Widmaier et al., 1980) and orange rind (De La Funte et al., 1981) both produced other isomers of FPP in addition to the all-trans isomer. All four isomers of FPP were produced by the cotton root preparations. This appears to be due to the presence of other enzyme activities in the enzyme preparations, although it is not clear if the contaminating activities are isomerases or cis-specific prenyltransferases. The cotton root preparation was able to condense IPP and neryl diphosphate to produce cis,& FPP, suggesting the presence of a cis-specific prenyltransferase, but the orange rind preparation could not use neryl diphosphate. Some resolution of the all-trans FPP synthetase from contaminating activities was obtained for both the cotton root and orange rind preparations. A d a m and Heinstein (1973) were able to achieve some resolution of transferase activities using GPP, and of neryl diphosphate by ion-exchange chromatography on DEAE-cellulose, but complete separation of the activities was not possible. The two activities also appeared to have different stabilities. Activity with GPP was lost in the presence of Tris buffer whereas activity with neryl diphosphate was enhanced (Adams and Heinstein, 1973). The activities in orange rind also showed different stabilities; the activity producing alltrans FPP was stable to heating at 60°C for 10 min in the presence of GPP and Mg2+, whereas the activity producing cis FPP was inactivated. Similar inactivation of the czs-forming activity on storage at 4°C for 1 month was observed, whereas the trans-forming activity was retained (De La Funte et al.. 1981). Geranylgeranyl diphosphate synthetase activity has been partially purified from extracts of carrot roots (Nandi and Porter, 1964), pumpkin fruit (Ogura et al., 1972) and tomato fruit chromoplasts (Spurgeon et a l . , 1984). The carrot root enzyme was shown to catalyse the condensation of FPP and IPP (Nandi and Porter, 1964), whereas the pumpkin enzyme was able to carry out three sequential reactions using DMAPP, GPP and FPP as prenyl donors. The product of the reaction appeared to be proteinbound (Ogura et al., 1972), although it is not clear if this was to the enzyme or to other proteins in the extract. The GGPP synthetase activity in carrot root and pumpkin fruit extracts showed greater activity in the presence of Mn2+, rather than Mg2+. However, a similar effect was not observed with the tomato fruit enzyme; Mn2+ was not necessary for activity although in its absence optimal activity required 45 mM Mg2+. The K , values for the

42

JOHN C. GRAY

carrot root enzyme were estimated to be 1X lo-’ M IPP and 1X M FPP, although high concentrations of FPP were inhibitory. The carrot root enzyme was inhibited 94% by 3 X M FPP and 76% by 5 X loV3M pyrophosphate (Nandi and Porter, 1964). A stimulation of approximately 40% of the activity of the tomato fruit enzyme was obtained in the presence of 0.6% Tween 80 (Spurgeon et al., 1984). The GGPP synthetase activity of tomato fruits was estimated, by gel filtration on Sephadex G-100, to have a relative molecular mass of 64,000 (Spurgeon et al., 1984), somewhat smaller than the FPP synthetases of castor beans (Green and West, 1974) and pea seedlings (Allen and Banthorpe, 1980). Geranyl diphosphate synthetase activity has been reported in extracts of Citrofortunella mitis peel (Gleizes et al., 1983b) and sage (Salvia officinalis) leaves (Croteau and Johnson, 1985), where its product would be required for monoterpene synthesis. No details of the properties of this enzyme have been reported. The possibility that the IPP isomerase and prenyltransferase activities are closely associated in the cell has been raised by the co-purification of these activities from tomato fruit chromoplasts (Maudinas et al., 1977) and cotton roots (Widmaier et al., 1980). With cotton root extracts, a protein fraction catalysing the synthesis of FPP from IPP was eluted from Sepharose 6B in the position expected for a globular protein of M , 258,000, which was larger than the size of the separate electrophoretically separated protein fractions containing the IPP isomerase and FPP synthetase activities. A fraction catalysing the synthesis of acid-labile products from IPP has been described from tomato fruit (Maudinas et al., 1977). This protein fraction was further purified by ion-exchange chromatography and was eluted from Sephadex G-200 in the position expected for a globular protein of M , 40,000 (Islam et al., 1977). However, subsequently Spurgeon et al. (1984) have been able to separate IPP isomerase and prenyltransferase activities in tomato fruit extracts and have shown that the enzymes had M , values of 34,000 and 64,000 respectively. It seems probable that incomplete separation of the enzyme activities would result in fractions containing both activities and this might be interpreted as a complex of intermediate M , capable of converting IPP to higher prenyldiphosphates. The subcellular locations of the IPP isomerase and prenyltransferase activities will be discussed in Section II.A.4. E. FORMATION OF ISOPRENOID COMPOUNDS

The whole range of isoprenoid compounds is produced by biosynthetic pathways branching from the main biosynthetic pathway discussed above (Fig. 1). An understanding of the nature of these pathways and in particular the properties of the enzymes at the branch points is needed for

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

43

any discussion of the regulation of isoprenoid biosynthesis. In most cases the pathways have been resolved but there is little detailed information on the enzymes involved. The discussion below is therefore limited to the enzymes at the branch points of the pathways leading to the main isoprenoid compounds present in all plants. Details of the enzymes involved in the synthesis of exotic isoprenoid compounds, such as rubber and essential oils, are not included, nor is there any discussion of the synthesis of abscisic acid, which remains obscure.

I . Cytokinins Many cytokinins contain a modified isoprenoid residue, and the first step in the biosynthetic pathway appears to be the condensation of DMAPP with 5’-AMP. Chen and Melitz (1979) showed the presence in habituated tobacco callus tissue of an enzyme activity which catalysed the transfer of the dimethylallyl group from DMAPP to 5’-AMP to give dimethylallyl adenosine 5’-phosphate or N6-(A’-isopentenyl) adenosine 5’-phosphate. The activity was partially purified by ammonium sulphate precipitation and by gel filtration on Sephadex G-100. The enzyme activity was eluted from Sephadex G-100 after the peak of IPP isomerase activity. A similar enzyme activity is present in crown gall tumours incited by Agrobacterium fumefaciens and has been partially purified by Hommes ef al. (1985). The enzyme uses DMAPP, but not IPP, as the side chain donor, and 5’-AMP, but not adenine or adenosine, as acceptor. The enzyme is encoded in the fmr locus of the T-DNA which is transferred into the plant cell from A. fumefaciens. The nucleotide sequence of the fmr locus from a number of Ti plasmids has been determined (Heidekamp et al., 1983; Goldberg ef al., 1984) and this suggests a protein composed of 241 amino acid residues and an M , of approximately 26,000. This is similar in size to the enzyme from habituated tobacco callus, but it is clear from DNA hybridization studies that the gene for the endogenous enzyme is not related to the gene transferred into the plant from A . fumefaciens. There are, as yet, no reports of properties of the enzyme which might suggest a regulatory role. 2. Sterols The first step in the synthesis of sterols and pentacyclic triterpenes, such as P-amyrin, is the condensation of two molecules of FPP to form squalene. In animals and yeast, this has been shown to take place in two steps, probably catalysed by the same enzyme (Qureshi et al., 1973). The initial condensation produces presqualene diphosphate which is then reduced with NADPH to give squalene (Fig. 6). These partial reactions have not been assayed in plant extracts. The enzymes necessary for the synthesis of squalene from [214C]mevalonate have been shown to be present in extracts of developing or germinating pea seeds (Graebe, 1967; Green and Baisted, 1971) and Green and Baisted (1972) have shown that extracts of pea

44

JOHN C . GRAY

\

\

fornesyl diphosphate

CH$PP+

PPOHzC

1

"f"r"( farnesyl diphosphate

CHZOPP

I

presqualene diphosphate

squalene

Fig. 6 . Scheme for the synthesis of squalene from FPP, via presqualene diphosphate.

seedlings are able to catalyse the synthesis of squalene from [4,8, 12''C]FPP, in the presence of NADPH. However, the enzyme activity has not been characterized further.

3. Carotenoids The first step specific for the synthesis of carotenoids is the condensation of two molecules of GGPP to give the C40 hydrocarbon, cis-phytoene. The reaction is catalysed by phytoene synthetase and proceeds via the intermediate prephytoene diphosphate (Fig. 7). It has not been established if a single enzyme is responsible both for the formation of prephytoene diphosphate and for its conversion to cis-phytoene. Phytoene synthetase activity, assayed by the formation of phytoene from GGPP, has been demonstrated in extracts of chromoplasts from tomato fruits (Shah et af., 1968; Maudinas et al., 1977) and daffodil flowers (Kreuz et al., 1982). The conversion of GGPP to cis-phytoene by extracts of tomato fruits required Mg2+,but not Mn2+,and was optimal in the pH range 7.6-7.8 (Shah et af., 1968). Dithiothreitol stimulated activity, suggesting that thiol group(s) were necessary for activity. Neither NAD+ nor NADP+ were required for activity. A two-fold stimulation of activity in the presence of 5 0 m ~ phosphate was also reported (Shah et af., 1968) and may be due to an

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

geranylgeronyl dipphote

45

geranylgemnyl diphosphate

prephytoene diphosphate

CIS-

phytoene

Fig. 7. Scheme for the synthesis of phytoene from GGPP, via prephytoene diphosphate.

inhibition of phosphatase activity hydrolysing the substrate and intermediate of the reaction. Attempts to purify phytoene synthetase have resulted in protein fractions containing in addition IPP isomerase and prenyltransferase activities. It has been suggested that these enzymes form a high molecular weight complex capable of converting IPP to phytoene (Maudinas et al., 1977). The molecular weight of this complex has been estimated to be 166,000 in tomato fruit extracts (Maudinas et al., 1977) and 190,000 in pepper (Capsicum unnuum) chromoplasts (Camara, 1985). Further attempts to purify the tomato complex were unsuccessful, and phytoene synthetase activity was lost on ion-exchange chromatography. However, IPP isomerase and prenyltransferase activities were recovered and could be separated and further purified (Spurgeon et al., 1984). It seems probable that the association of these three enzyme activities was fortuitous, and it may have been caused by the acetone treatment of the tomato plastid preparation. The conversion of prephytoene diphosphate to cis-phytoene has been demonstrated in extracts of wheat leaves and in plastids from green and orange pepper fruits (Camara, 1984), but the enzyme has not been purified further. 4. Phylloquinone and Tocopherols Phylloquinone (vitamin K,) and tocopherols contain a phytyl side chain on an aromatic ring system (Pennock, 1983). Enzyme activities carrying

46

JOHN C. GRAY

out the prenylation reaction, using phytyl diphosphate, on 1,4dihydroxynaphthoate, leading to phylloquinone, have been demonstrated in spinach chloroplasts (Soll et al., 1980) and in pepper chloroplasts and chromoplasts (Gaudillikre et al., 1984). A similar activity catalysing the prenylation of homogentisate using phytyl diphosphate has been demonstrated in spinach chloroplasts (Soll et al., 1980). These activities have been localized to the inner membrane of spinach chloroplast envelopes (Soll et al., 1985). The chloroplast envelope preparation was reported to be unable to use GGPP for the prenylation reactions (Soll et al., 1980). However, further information on the properties or specificity of the polyprenyltransferase activities is not available. The phytyl diphosphate needed for tocopherol and phylloquinone synthesis is probably obtained by reduction of GGPP within the chloroplasts. Sol1 et al. (1983) have demonstrated the presence of GGPP reductase activity in broken spinach chloroplasts. The reductase activity showed optimal activity at pH 7.6 and appeared to be located predominantly in the chloroplast envelope fraction. NADPH is most probably the source of reducing power; omitting NADPH from the enzyme assays resulted in a sharp decrease in phytyl diphosphate formation (Soll et al., 1983). The enzyme system has not been characterized further and it is not clear if a single enzyme is able to carry out the reduction of the three double bonds, or if three separate enzymes are required.

5. Chlorophyll Chlorophyll a and b in mature plant tissues contain a phytol side chain esterified to the propionic acid residue on ring IV of the tetrapyrrole. However, in greening oats seedlings chlorophyll is initially esterified with geranylgeraniol, and this side chain is reduced via dihydrogeranylgeraniol and tetrahydrogeranylgeraniolto phytol (Schoch et al., 1977). The esterification of chlorophyllide with prenyl diphosphates is catalysed by the enzyme chlorophyll synthetase (Rudiger et al., 1980). Chlorophyll synthetase activity has been detected in etioplasts from oats (Rudiger et al., 1980), in chloroplasts from spinach (Soll et al., 1983) and pepper (Dogbo et al., 1984), and in chromoplasts from daffodil flowers (Kreuz and Kleinig, 1981b) and pepper (Dogbo et al., 1984). The enzyme has not been purified and its properties have been deduced from incubations with plastid membrane preparations. The enzyme is able to use GGPP, phytyl diphosphate and FPP as substrates in vitro (Rudiger et al., 1980; Soll et al., 1983). There appear to be some differences in the utilization of these prenyl diphosphates by membrane preparations from oat etioplasts and spinach chloroplasts, but it is not clear if these are due to inherent differences in the chlorophyll synthetase activities, or to differences in competing activities in the membrane preparations. Rudiger et al. (1980) incubated mixtures of prenyl diphosphates with oat etioplast membranes and then measured the

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

47

ratios of the prenols in the esterified tetrapyrroles. This showed that incubations containing equal amounts of GGPP and phytyl diphosphate produced tetrapyrroles containing twice as much geranylgeraniol as phytol. This was taken to indicate that phytyl diphosphate is esterified with chlorophyllide only half as effectively as GGPP. Similar experiments with mixtures of FPP and GGPP gave a ratio of 0.16 for farnesoV geranylgeraniol in the pigment (Rudiger er al., 1980). A different specificity of chlorophyll synthetase for GGPP and phytyl diphosphate has been suggested from experiments with broken spinach chloroplasts (Soll er ai., 1983). The rate of incorporation of phytyl diphosphate was five-fold higher than the rate of incorporation of GGPP in separate incubations containing 1.2x low3M prenyl diphosphates. These broken chloroplast preparations also used FPP as a substrate for chlorophyll synthetase at rates less than that for GGPP (Soll et al., 1983). Membrane fractions from chloroplasts and chromoplasts of pepper fruits also used [13H]GGPP at greater rates than [13H]FPP for the esterification of chlorophyllide (Dogbo et al., 1984). The esterification of chlorophyllide with GGPP by broken spinach chloroplasts was optimal at pH 7.1 (Soll et al., 1983). It seems unlikely that the specificity of chlorophyll synthetase for prenyl diphosphates will be different in different plants, and the most likely explanation of the different results with oats and spinach is differences in the levels of competing activities in the membrane preparations.

6. Gibberellins The first step in the synthesis of gibberellins is the two-stage cyclization of GGPP to ent-kaurene. The bicyclic compound copalyl diphosphate is an intermediate (Fig. 8) in the reaction catalysed by kaurene synthetase. However, enzyme activities catalysing the two individual steps have been separated by ion-exchange chromatography on QAE-Sephadex of extracts from the endosperm of Marah macrocarpus seeds (Duncan and West, 1981). The two enzymes have similar M , values of 82,000 estimated by gel filtration, andrare believed to interact during the production of ent-kaurene from GGPP. Copalyl diphosphate formed from ['4C]GGPP in situ was utilized 13 times more efficiently for ent-kaurene synthesis than a pool of exogenously supplied [3H]copalyldiphosphate (Duncan and West, 1981).

geranylgerany I diphasphate

capalyl disphosphaie

-

eni kaurene

Fig. 8. Scheme for the synthesis of kaurene from GGPP, via copalyl diphosphate.

48

JOHN C. GRAY

Kaurene synthesis from copalyl diphosphate has been demonstrated in extracts of several plants (West et al., 1982), but the overall conversion of GGPP to kaurene is much more difficult to measure. High levels of kaurene synthetase activity have been demonstrated only in extracts of Marah macrocarpus endosperm and Ricinus communis seedlings (West et al., 1982). The presence of inhibitory factors in extracts of sunflower (Helianthus annuus) seedlings (Shen-Miller and West, 1982) may account for the inability to detect kaurene synthetase activity in this, and other, tissues. However, there is little information on the nature of these factors and it is not clear if they have any physiological relevance to the regulation of enzyme activity. A large number of compounds have been examined for their effect on the activity of kaurene synthetase in extracts of Marah macrocarpus endosperm (Frost and West, 1977). This study showed that neither abscisic acid nor a number of gibberellins had any direct effect on kaurene synthetase activity. The substrate, GGPP, was, however, inhibitory at M, but the effect of the product, concentrations greater than 1x ent-kaurene, was not examined. 7. Plustoquinone The isoprenoid side chain of plastoquinone is formed by the prenylation of homogentisate by solanesyl (C44 diphosphate. Subsequent methylation of the product 2-methyl-6-solanesylquinol with S-adenosylmethionine yields plastoquinol (Fig. 9). The enzyme(s) responsible for the prenylation of homogentisate has been localized to the inner membrane of the spinach chloroplast envelope (Soll et al., 1985) although some activity may be present in the chloroplast thylakoid membranes (Soll et al., 1980). Nothing is known of the properties of the enzyme system; it is not clear if the enzyme responsible for the prenylation reaction is also able to carry out the decarboxylation reaction (see Fig. 9), or if these reactions are carried out by two enzymes. The source of the solanesyl diphosphate has not been established. This nonaprenyl diphosphate is presumably formed by successive prenylation reactions from GGPP, but the nature of the prenyltransferase responsible is not known. Lutke-Brinkhaus et al. (1985) have shown that Cz0and C45 polyprenols are the main products formed on incubation of spinach chloroplast stroma with [1'4C]IPP. The polyprenyl diphosphates did not accumulate, presumably because of the presence of phosphatase activity. However, this experiment suggests that spinach chloroplasts are able to synthesize the C45polyprenol required for plastoquinone synthesis. 8. Ubiquinone The isoprenoid side chain of ubiquinone is formed by the prenylation of 4-hydroxybenzoate by long-chain prenyl diphosphates (Fig. 9). The chain

*i OH

6

OH

COOH

H2C

OH

coon

t,4 -dihydroxy 2 - nophthoate

OH

-

homogentisote

phytyl PP

OH

I

CO,

+ PPi

OH

4 hydroxybenzwte decoprenyl PP

OH

COOH

I

I

OH 2-phytyl 1,4- nophthoquinol

I I

OH

I I

I I

I OH

I

J.

V OH

HJCO 10

OH

OH

phylloquinol Fig. 9.

a -tocopherd

plostoquind

Scheme for the prenylation of aromatic intermediates in the synthesis of prenylquinones.

OH ubquinol

50

JOHN C. GRAY

length of the prenyl side chain of ubiquinone in different plants ranges from 6 to 10 isoprene units (Schindler and Lichtenthaler, 1982; Schindler et al., 1985). The synthesis of 4-hydroxypolyprenyl benzoates from 4hydroxybenzoate by mitochondria isolated from potato tubers, spinach leaves and daffodil flowers, has been demonstrated by Lutke-Brinkhaus et al. (1984). The nature of the enzyme catalysing the prenylation reaction is unknown. The prenyl side chains of the ubiquinone precursors are formed within the mitochondria from IPP, but not from mevalonate 5-diphosphate (Lutke-Brinkhaus et af., 1984). This indicates that prenyltransferase activity necessary to form all the homologues up to decaprenyl diphosphate is present in the mitochondria. The characteristics of this enzyme system have not been studied. 9. Long-chain prenob The enzymes for the synthesis of long-chain prenols, such as dolichol and other polyprenols involved in oligosaccharide synthesis, have not been characterized. Daleo and Pont Lezica (1977) described the incorporation of ['4C]IPP into dolichol phosphate by a cell-free extract from pea seedlings, but the enzymes responsible have not been studied further. It seems probable that a cis-specific prenyltransferase is responsible for the addition of prenyl residues to all trans-FPP. F. MEVALONATE SHUNT

Studies with animals have shown that intermediates of the isoprenoid biosynthetic pathway can be diverted from this pathway and used for the synthesis of non-isoprenoid lipids (Edmond and Popjak, 1974). In mammals, the mevalonate shunt may direct as much as 50% of the mevalonate away from sterol production (Wiley et al., 1979). Recent studies have indicated that a similar pathway may be operating in higher plants. Nes and Bach (1985) demonstrated that in primary leaf tissue of wheat seedlings [23H]mevalonate was incorporated into long-chain fatty alcohols as efficiently as into sterols, whereas [53H]mevalonate was metabolized only to sterol. The pathway of the mevalonate shunt proposed for animal tissues is shown in Fig. 10. Mevalonate is metabolized to IPP and DMAPP as described in Sections 1.C and I.D, but then the DMAPP is dephosphorylated to give A'-isopentenol, which is subsequently oxidized by aldehyde dehydrogenase to produce dimethylacrylic acid. The CoA ester of dimethylacrylic acid then joins the leucine degradation pathway via HMG-CoA. The end products are acetoacetate and acetyl CoA. For incorporation of 3H from [23H]mevalonate into long-chain fatty alcohols, the acetoacetate must be converted to a precursor for fatty acid synthesis. The pathway for this conversion has not been examined in plants.

CONTROL OF ISOPRENOID BIOSYNTHESISIN HIGHER PLANTS

O

+

0

HO-CH3

51

0

H3CASCoA acetyl- CoA

acetoacetate

4 0

CH3 0

HO

11

*

SCoA

HO

SCoA

-

3-methyl glutaconyl-CoA

HMG COA

lo

t HO

CH3 0

CH3

HO &CHzOH H C U S C o A

-

dimethylacrylyl CoA

mevalonate

.t 19 CH3 0 H3C

OH

dimethylacrylate

~2

- isopentenal

~2

- isopentenol

DMAPP

Fig. 10. Scheme for the proposed reactions of the mevalonate shunt. The enzymes involved are numbered: 1 , HMG-CoA reductase; 2, rnevalonate kinase; 3, mevalonate 5-phosphate kinase; 4, mevalonate 5-diphosphate decarboxylase; 5 , IPP isomcrase; 6, phosphatase; 7, alcohol dehydrogenase; 8, aldehyde dehydrogenase; 9, 3-rnethylcrotonylCoA synthetase; 10, 3-methylcrotonyl-CoA carboxylase; 11, 3-methylglutaconyl-CoA hydratase; 12, HMG-CoA lyase.

The absence of 3H from long-chain fatty alcohols synthesized in wheat leaves supplied with [53H]mevalonate is consistent with the above pathway, but label would also be lost by a direct reversal of the HMG-CoA reductase reaction. Nes and Bach (1985) used mevinolin to inhibit HMGCoA reductase (as shown by the inhibition of incorporation of [214C]acetate into sterols) and showed that label from [23H]mevalonate

52

JOHN C. GRAY

was still incorporated into long-chain fatty alcohols. However, considerable inhibition of the incorporation of label from [23H]mevalonate into long-chain fatty alcohols by mevinolin was observed in wheat leaves in the light (48% inhibition) and in the dark (72% inhibition). The significance of this observation for the pathway of mevalonate conversion to long-chain fatty alcohols is not clear. The pathway of the mevalonate shunt in plants deserves further study, particularly with respect to the effect it will have on the flux through the biosynthetic pathways of isoprenoid metabolism. G . CONCLUSIONS

The properties of the enzymes catalysing the steps from HMG-CoA to GGPP have been fairly well characterized, particularly with respect to fundamental kinetic parameters such as K , and pH optimum. However, the enzymes characterized are from a wide variety of plants and rarely have several enzymes of the biosynthetic pathway been characterized from a single plant tissue. A full characterization of all the enzymes of the biosynthetic pathway from a single plant tissue will be required for an understanding of the regulation of the pathway. This will require a much more detailed investigation of the enzymes of HMG-CoA synthesis and of the branch point enzymes than has been carried out to date. There is virtually no information on the properties of the enzymes of HMG-CoA synthesis and this is a major shortcoming for studies on isoprenoid biosynthesis. The enzymes catalysing the reactions at the branch points leading to the wide range of isoprenoid compounds are for the most part membrane-bound and this has hampered their characterization. However, with the recent success in the solubilization and purification of HMG-CoA reductase (Bach et al., 1986), it is to be hoped that other membrane-bound enzymes will soon be characterized. The properties of enzymes such as squalene synthetase, phytoene synthetase and those catalysing the reduction of GGPP to phytyl diphosphate are urgently required.

11. COMPARTMENTATION OF THE PATHWAYS OF ISOPRENOID BIOSYNTHESIS The subcellular location of the enzymes of isoprenoid biosynthesis is of crucial importance to our understanding of the regulation of isoprenoid biosynthesis. It is this area of research that has produced the most controversial claims and counterclaims in this field. At the present time there are two opposed hypotheses for the intracellular organization of isoprenoid biosynthetic pathways in higher plants (see Fig. 11). In the first hypothesis, based on the pioneering work of Goodwin and his group, completely separate biosynthetic pathways from acetyl-CoA to the

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

53

A

4

cytoplam

Pyruvate 7

Pyruvate Acelyl-COP,---

----Acetyl

CoA

t

Acetyl -COA

t

MVA

MVA

I PP

I PP

Sterols

Isoprenoids

t

t

Ubiquinone

t

t

mitochondrion

chloroplast

B Sucrose

+

co2

I

Pyruvote

cytoplasm

-

Pyruvate

Triose

Ubiquinone

-

IPP

P

-

COe

t

MVA

t

1PP

t

-

IPP -+ lsoprenoids

Sterols chloroplast

Fig. 11. Models for the compartmentation of the enzymes of isoprenoid biosynthesis. A: Model based on that originally proposed by Goodwin and Mercer (1963). B: Model based on the proposal of Kreuz and Kleinig (1984).

organelle-specific isoprenoid compounds are located in the cytosol, plastids and mitochondria. The original formulation of this hypothesis (Goodwin and Mercer, 1963; Goodwin, 1965) envisaged that the different compartments are separated by membranes impermeable to the intermediates of the pathways. A second hypothesis, based on the failure to detect the enzymes of IPP synthesis in plastids and mitochondria (Kreuz and Kleinig, 1981a), suggests that the formation of IPP occurs solely in the cytosol and that the IPP then enters the different subcellular compartments for the synthesis of specific isoprenoid compounds. In view of the great relevance of the compartmentation of the enzymes of isoprenoid biosynthesis to its

54

JOHN C. GRAY

regulation in plants, the evidence for these two hypotheses will be critically assessed with the aim of determining if the valid experimental evidence supports one hypothesis rather than the other. The original observations that led Goodwin and Mercer (1963) to their view of the compartmentation or “compartmentalization” of isoprenoid metabolism were made from experiments designed to label isoprenoid compounds in vivo with [214C]mevalonate and 14C02. When etiolated maize shoots were placed in a solution of [214C]mevalonate and illuminated for 24 hours, sterols and squalene became radioactively labelled but neither P-carotene nor the phytyl side chain of chlorophyll were significantly labelled, even though both compounds were rapidly synthesized during leaf greening. When the shoots were illuminated in the presence of 14C02, @-carotene and phytol were highly labelled, but there was no significant radioactivity in sterols or squalene. These observations have been extended to other plants, including barley, tobacco, mustard, oats and pea, and the labelling of a wide variety of isoprenoid compounds from 14C02 and [214C]mevalonatehas been examined (Treharne et al., 1966; Threlfall et al., 1967; Griffiths et al., 1968; Lichtenthaler et al., 1982; Sergeant and Britton, 1984). These studies indicate that chloroplast isoprenoid compounds, such as carotenoids and the prenyl side chains of chlorophyll, plastoquinone and phylloquinone, are preferentially labelled from I4CO2, whereas the “extrachloroplastidic” isoprenoid compounds, such as sterols and the prenyl side chain of ubiquinone, are preferentially labelled from [214C]mevalonate.These experimental observations appear sound; it is the explanation of the observations that has produced conflicting views. Goodwin and Mercer (1963) suggested that the observations could be explained by a process of “compartmentalization” of isoprenoid biosynthesis in which two subcellular sites of synthesis (chloroplastic and extrachloroplastic) are separated by a membrane impermeable to mevalonate. The two subcellular sites would each contain all the enzymes necessary for the conversion of acetyl-CoA to higher prenyl diphosphates, and each compartment would also contain enzymes for the formation of compartment-specific isoprenoid compounds. Thus the enzymes specific for the formation of carotenoids and the side chains of chlorophyll and plastid quinones would be present in the chloroplasts, whereas the enzymes of sterol synthesis would be present in the cytoplasm. The chloroplast would also contain the enzymes necessary for C 0 2 fixation and the formation of acetyl-CoA. The presence of ubiquinone in mitochondria and the observed incorporation of IPP into the side chain of ubiquinone by isolated mitochondria led to the inclusion of mitochondria as a third compartment containing enzymes of isoprenoid biosynthesis. In the compartmentalization hypothesis, it is envisaged that the mitochondria have all the enzymes necessary for the conversion of acetyl-CoA to IPP and its subsequent incorporation into side chains of ubiquinone and haem a of cytochrome

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

55

oxidase (Fig. 11A). From its inception this hypothesis suggested a large number of experimentally testable predictions and many of the early experiments were carried out in Goodwin’s laboratory. These experiments fall essentially into two groups: those concerned with the subcellular location of the enzymes of isoprenoid biosynthesis, and those concerned with the permeability of the organelle membranes to intermediates of the biosynthetic pathway. A. SUBCELLULAR LOCATION OF ENZYMES OF ISOPRENOID BIOSYNTHESIS

The two hypotheses for the compartmentation of isoprenoid biosynthesis make different predictions concerning the subcellular locations of certain key enzymes of the pathway (see Fig. 11).

I . Formation of Acetyl-CoA The compartmentalization model based on the ideas of Goodwin and Mercer (1963) requires the provision of acetyl-CoA as a precursor for isoprenoid biosynthesis in three separate compartments, plastids, mitochondria and cytosol. The hypothesis requires that chloroplasts are capable of the formation of acetyl-CoA from photosynthetically fixed C02. Two laboratories have reported that isolated intact spinach chloroplasts are able to incorporate 14C02 into p-carotene and plastoquinone (Bickel and Schultz, 1976; Grumbach and Forn, 1980), suggesting that spinach chloroplasts are able to synthesize acetyl-CoA and incorporate it into isoprenoid compounds. However, in these experiments the incorporation into isoprenoid compounds is so extremely low (0.008% of applied 14C02) that the presence of any cytoplasmic contamination would make unjustified any conclusions concerning the chloroplast location of the biosynthetic pathway. Bickel and Schultz (1976) stated that “the hexokinase test for adhering cytoplasm was negative” but the limits of detection of their hexokinase assay were not stated. The chloroplasts of Grumbach and Forn (1980) were prepared simply by differential centrifugation and would be expected to be contaminated by cytoplasmic enzymes, with perhaps as much as 0.2% of the total cellular activity (see Table 111). As discussed below (Section 11.A.4), it seems most likely that the enzymes for the conversion of mevalonate to IPP are present as cytoplasmic contamination. There is also considerable doubt that spinach chloroplasts have the enzyme complement needed to synthesize acetyl-CoA from photosynthetically fixed C 0 2 (Givan, 1983). The most likely route for the formation of acetyl-CoA is the conversion of glycerate 3-phosphate to pyruvate, via glycolytic enzymes, followed by the oxidative decarboxylation of pyruvate by pyruvate dehydrogenase. However, pyruvate dehydrogenase activity is most probably absent from spinach chloroplasts (Roughan et al., 1979a,b;

56

JOHN C. GRAY

Murphy and Stumpf, 1981). Roughan et al. (1979a) failed to detect pyruvate dehydrogenase activity in spinach chloroplasts and Murphy and Stumpf (1981) concluded that the very low levels of pyruvate dehydrogenase activity in their chloroplast preparations were due to mitochondrial contamination. They also showed that decarboxylation of [214C]pyruvatedid not produce acetyl-CoA but was most probably due to acetolactate synthetase activity, which is necessary for the synthesis of the branched-chain amino acids valine, leucine and isoleucine in chloroplasts (Miflin, 1974; Schulze-Siebert et al., 1984). Pea chloroplasts also appear unlikely to be able to synthesize acetyl-CoA from photosynthetically fixed COz. Although pea chloroplasts contain appreciable pyruvate dehydrogenase activity (Elias and Givan, 1979; Williams and Randall, 1979), they are deficient in phosphoglyceromutase activity (Stitt and ap Rees, 1979), and would therefore be unable to convert glycerate 3-phosphate to glycerate 2-phosphate in the chloroplasts. It has been suggested that acetyl-CoA for fatty acid synthesis in spinach chloroplasts is synthesized from acetate, derived from the mitochondria (Murphy and Stumpf, 1981; Liedvogel and Stumpf, 1982). Roughan et al. (1979a) showed that acetate was the preferred substrate for fatty acid synthesis in isolated spinach chloroplasts, and Kuhn et al. (1981) demonstrated that acetyl-CoA synthetase activity is essentially confined to chloroplasts prepared from protoplasts of spinach leaves. The possibility that acetate is the precursor of acetyl-CoA needed for the synthesis of chloroplast isoprenoids was suggested in the model of Goodwin (1965). However, acetate is a poor precursor of chloroplast isoprenoids in vivo (Shah and Rogers, 1969) and in isolated spinach chloroplasts (Grumbach and Forn, 1980). The specific radioactivity of p-carotene synthesized by the spinach chloroplast preparation from labelled acetate was approximately an order of magnitude less than that of @carotene synthesized from 14C02, although the specific radioactivities of the applied precursors were essentially the same (Grumbach and Forn, 1980). Although the sizes of any pools of unlabelled precursors in these chloroplast preparations are unknown, this result is in contrast to those of Roughan et al. (1979b), who showed that fatty acids synthesized from [214C]acetatehad a higher specific radioactivity than those synthesized from 14C02. It therefore seems probable that the biosynthetic origins of acetyl-CoA for isoprenoid and fatty acid synthesis are different, and that the pathways for acetyl-CoA synthesis in plastids are used exclusively for fatty acid synthesis. A separate subcellular location for the synthesis of acetyl-CoA for isoprenoid biosynthesis would simplify the control mechanisms needed to regulate the provision of acetyl-CoA for two separate biosynthetic pathways. The provision of acetyl-CoA for isoprenoid synthesis in the cytosol has received scant attention until recently. Direct movement of acetyl-CoA from the mitochondria or plastids is unlikely because of the impermeability

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

57

of the boundary membranes of these organelles to acetyl-CoA (Liedvogel and Stumpf, 1982). Kaethner and ap Rees (1985) have recently shown that ATP-citrate lyase (EC 4.1.3.8) is located almost exclusively in the cytosol of pea leaves, and has sufficient activity to provide enough acetyl-CoA for the measured rate of carotenoid accumulation in this tissue. The formation of acetyl-CoA in mitochondria has never been considered a problem because of the presence of pyruvate dehydrogenase activity and the activity of the citric acid cycle. However, whether this acetyl-CoA can be used directly for isoprenoid synthesis within the mitochondria has not been established. 2. Formation of Mevalonate The subcellular location of the enzymes utilizing acetyl-CoA for isoprenoid biosynthesis has not been established, although Schulze-Siebert and Schultz (1985) have briefly reported that isolated spinach chloroplasts are able to form acetoacetyl-CoA from acetyl-CoA. However, it has not been established if the acetoacetyl-CoA is used for isoprenoid biosynthesis, or if the enzyme is working in this direction in vivo. Full details of this work are eagerly awaited. HMG-CoA synthase, the next enzyme in the biosynthetic pathway, has not even been assayed directly in plant tissues and its subcellular location is completely unknown. There is an enormous gap in our knowledge of the subcellular location of HMG-CoA synthesis from acetyl-CoA in plants, and at the present time it seems unwise to be dogmatic about the presence of these enzymes in any subcellular compartment. HMG-CoA reductase has been reported to be present in microsomal, mitochondria1 and plastid fractions of plant homogenates. However, as with the other enzymes of isoprenoid biosynthesis, the experimental results of many of the fractionation studies are not conclusive. Brooker and Russell (1975b) showed that HMG-CoA reductase activity in pea seedlings was associated with microsomal membranes which were separated by discontinuous sucrose density gradient centrifugation and shown to be free of succinate dehydrogenase activity, indicating an absence of mitochondrial membranes, An association of HMG-CoA reductase activity with microsomal membranes in sweet potato roots infected with black rot fungus was reported by Susuki and Uritani (1976) but the membrane fractions separated by sucrose density gradient centrifugation showed cytochrome c oxidase activity, indicating contamination with mitochondrial membranes. The presence of HMG-CoA reductase activity in microsoma1 membranes from radish seedlings has been suggested on the basis of differential centrifugation (Bach et al., 1980) but possible contamination from other membranes has not been assessed. The presence of HMG-CoA reductase in plastid membranes of pea seedlings has been suggested by Brooker and Russell (1975b) on the basis

58

JOHN C . GRAY

of fractionation studies using differential centrifugation. HMG-CoA reductase activity was associated with crude chloroplast preparations and with chlorophyll-containing fractions from a discontinuous sucrose gradient. However, not all fractions of the gradient were analysed for HMG-CoA reductase activity, and the extent of contamination by other cell membranes was not assessed. As centrifugation through discontinuous density gradients is a very poor method of separating different cell membranes, these experiments do not constitute evidence for the existence of HMG-CoA reductase activity in chloroplast membranes. The activity associated with preparations of pea chloroplasts, obtained by differential centrifugation, does, however, show differences in kinetic properties to the activity in microsomal membranes (Wong et a f . ,1982), but it is not clear to what extent this reflects the inherent properties of the enzyme or the nature of the membrane preparations. Kreuz and Kleinig (1984) showed that HMG-CoA reductase activity was associated with spinach chloroplasts prepared by differential centrifugation, but that subsequent purification of the chloroplasts by centrifugation through Percoll gradients decreased this activity by 25%. NADPH-cytochrome c reductase activity, a marker for the endoplasmic reticulum, was also present in these chloroplast preparations and showed a similar decrease in activity on Percoll gradient centrifugation. This suggested that the HMG-CoA reductase activity associated with the chloroplast preparations was due to microsomal membrane contamination. A report o n the presence of HMG-CoA reductase activity in chloroplasts from leaves of Nepeta cataria (Arebalo and Mitchell, 1984) must be regarded as unsubstantiated in the absence of information on possible contamination of the organelle preparations. The presence of HMG-CoA reductase activity in chromoplasts of pepper is also difficult to assess because of the presence of NADH-cytochrome c reductase activity in the chromoplast preparations (Camara et af., 1983). The presence of HMG-CoA reductase activity in mitochondria1 membranes has also not been established satisfactorily. Brooker and Russell (1975b) originally reported the co-migration of HMG-CoA reductase activity and succinate dehydrogenase activity in a pea seedling mitochondrial preparation centrifuged through a linear sucrose density gradient. However, not all the fractions from the gradient were assayed for HMG-CoA reductase, although the microsomal marker, NADPHcytochrome c reductase, was reported to be absent. The assay of all fractions from a gradient is important to establish the true association of an enzyme activity with organelle membrane preparations. Thus with the particulate fraction pelleted at 16,000g for 4 h from etiolated radish seedlings, HMG-CoA reductase activity migrated to a similar position in Percoll gradients as cytochrome c oxidase, but the two activities did not exactly co-migrate (Bach et a f . , 1980). From the results presented, no justifiable conclusion concerning the subcellular location of HMG-CoA

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

59

reductase in the large particulate fraction of radish seedlings is possible. Similar reservations must be made about the data presented for the subcellular location of HMG-CoA reductase in diseased sweet potato roots (Suzuki and Uritani, 1976). The migration of HMG-CoA reductase activity in mitochondrial preparations in linear sucrose density gradients was much more similar to that of the microsomal marker NADPH-cytochrome c reductase activity than to cytochrome c oxidase, the mitochondrial membrane marker. Only with mitochondrial preparations from fresh, uninfected sweet potato roots did the HMG-CoA reductase co-migrate exactly with cytochrome c oxidase activity, the mitochondrial marker (Suzuki and Uritani, 1976), but even then the mitochondrial preparation was contaminated with microsomal membranes, as indicated by the presence of NADPH-cytochrome c reductase activity. Changes in HMG-CoA reductase activity associated with different subcellular fractions of plant tissues following a variety of treatments have been interpreted as support for the presence of the enzyme in several subcellular compartments. Treatment of plants or plant tissues with light (Brooker and Russell, 1979; Wong et al., 1982; Bach et al., 1980), herbicides (Grumbach and Bach, 1979) and growth regulators (Russell and Davidson, 1982; Nishi and Tsuritani, 1983), and infection with fungi (Suzuki and Uritani, 1976), have all been reported to alter the HMG-CoA reductase activity associated with different subcellular fractions. It has been suggested that the activity associated with mitochondrial fractions represents a constitutive HMG-CoA reductase, whereas the activity in microsomal membranes is affected by a variety of treatments (Nishi and Tsuritani, 1983). However, as discussed above, the association of HMGCoA reductase with any of the organelle membranes has yet to be demonstrated unequivocally. In addition, none of the studies showing changes in enzyme distribution have investigated the effect of the treatment on the behaviour of the organelle membranes themselves. Extended treatment with light, herbicides and growth regulators may be expected to affect centrifugal behaviour of membranes by alterations in lipid and protein constituents. Fractionation studies using a full range of marker enzymes are required to substantiate claims of altered HMG-CoA reductase activity in different subcellular compartments during these treatments. Further support for the presence of HMG-CoA reductase in three subcellular locations is claimed from the results of experiments on the inhibition of accumulation of isoprenoid compounds by mevinolin, a potent competitive inhibitor of the enzyme (Bach and Lichtenthaler, 1983b; Schindler et al., 1985). These experiments showed that mevinolin inhibited sterol accumulation in radish seedlings to a much greater extent than that of carotenoids and other chloroplast prenyllipids. The inhibition of ubiquinone accumulation was different from that of sterol accumulation and of chloroplast prenyl lipid accumulation (Doll et al., 1984; Schindler et

60

JOHN C . GRAY

al., 1985), and this was interpreted as indicating three different subcellular sites of HMG-CoA reductase, each with a different accessibility to the applied mevinolin. However, in the absence of any knowledge of the flux through the isoprenoid pathway and of the activities of the individual enzymes of the pathway in these tissues, such conclusions are not justified. An alternative interpretation of these data requiring only a single HMGCoA reductase is possible, particularly if the activity of HMG-CoA reductase is greater than the flux through the pathway. Inhibition of a single HMG-CoA reductase by mevinolin might be expected to result in a decrease in the concentrations of the subsequent intermediates. The synthesis and accumulation of the individual isoprenoid compounds will depend on the relative affinities of enzymes, or carriers (see Section II.B), for these intermediates. For example, if three enzymes, or carriers, acting on an intermediate had different K, values and the concentration of the intermediate fell, then the effect on the activities of the three enzymes would be different. If the substrate concentration were very much larger than the K,, even after the concentration decrease caused by mevinolin, then there would be no apparent change in the rate of that enzyme and no change in the accumulation of subsequent products. It is possible that an enzyme, or carrier, specific to the synthesis of chloroplast isoprenoids has a very low K , for one of the intermediates of the biosynthetic pathway and its activity is not significantly altered by decreased concentrations of intermediates caused by mevinolin inhibition of HMG-CoA reductase. A prime candidate might be the IPP transporter in the chloroplast envelope (see Section 1I.B). The purification of HMG-CoA reductase from radish membranes (Bach et al., 1986) offers the possibility of clarifying the location of this enzyme in plant cells. Antibodies raised against the purified protein could be used in immunocytochemical studies to pinpoint the subcellular location of the enzyme. The HMG-CoA reductase was purified from a radish membrane preparation which was purported to consist of mitochondrial membranes (Bach et al., 1980) but which may contain a population of “heavy” microsomal membranes. Immunocytochemical localization should establish if the enzyme is located in mitochondrial membranes, or in other membrane systems.

3. Formation of lsopentenyl Diphosphate The first experiments designed to test the compartmentalization hypothesis of Goodwin and Mercer (1963) examined the subcellular location of the enzymes capable of acting on [214C]mevalonate. It was reported that disrupted isolated bean chloroplasts were capable of metabolizing [214C]mevalonate to mevalonate 5-phosphate and mevalonate 5diphosphate (Rogers et a l . , 1966a) and to geranylgeraniol and phytoene (Charlton et al., 1967; Buggy et al., 1969, 1974). Rogers et al. (1966b) also

CONTROL OF ISOPRENOD BIOSYNTHESIS IN HIGHER PLANTS

61

reported the presence of isoenzyme forms of mevalonate kinase differing in pH optima in leaves of Phaseofus vulgaris. Chloroplast preparations were reported to show optimal activity at pH 7.5, whereas mevalonate kinase in leaf extracts showed optima at pH 5.5 and pH 7.5. The enzyme activity at p H 5.5 was ascribed to the cystolic mevalonate kinase because of a similar pH optimum in extracts of etiolated bean cotyledons. However, these experiments showing the presence in bean leaf extracts of mevalonate kinase with a pH optimum at pH 5.5 could not be repeated (Gray and Kekwick, 1973a; Hill and Rogers, 1974). Gray and Kekwick (1973a) reported the presence of a single form of mevalonate kinase with a p H optimum of 7.0 in bean leaves and demonstrated that the enzyme activity behaved like cytosolic marker enzymes on tissue fractionation in aqueous media and in organic solvents. No evidence was found to suggest a chloroplast location for this enzyme. Subsequently a large number of reports have suggested the presence of mevalonate kinase activity in plastid preparations (Garcia-Peregrin et af., 1973a; Cooke, 1977; Camara and Brangeon, 1981; Arebalo and Mitchell, 1984) but in none of these reports has cytoplasmic contamination been assessed using suitable marker enzymes. Chloroplasts prepared by differential centrifugation and by discontinuous sucrose density gradient centrifugation show measurable mevalonate kinase activity but this can be completely ascribed to cytoplasmic contamination (see Table 111). An examination of the subcellular distribution of mevalonate kinase in pea leaves shows that the enzyme activity behaves like the cytoplasmic marker phosphoenolpyruvate carboxylase. No increase in mevalonate kinase activity in the chloroplast preparations was obtained by rupturing the chloroplast envelope by sonication, osmotic shock or detergents such as Triton X-100. The mevalonate kinase activity associated with these chloroplasts [0.002 nmol min-' (g fresh wt)-'] is not sufficient to account for the rate of carotenoid accumulation [0.7 nmol acetyl-CoA equivalent min-' (g fresh wt)-' (Kaethner and a p Rees, 19851 in this tissue. A similar conclusion concerning the location of mevalonate kinase has been drawn from studies with spinach chloroplasts purified by centrifugation on linear Percoll gradients (Kreuz and Kleinig, 1984). The chloroplast preparation showed no cytoplasmic contamination, as assessed by alcohol dehydrogenase activity, and no mevalonate kinase activity, even after French press treatment to disrupt the chloroplast envelope. This study also showed that purified spinach chloroplasts were unable to metabolize mevalonate 5-phosphate or mevalonate 5-diphosphate, indicating that the chloroplasts did not contain mevalonate 5-phosphate kinase or mevalonate 5-diphosphate decarboxylase (Kreuz and Kleinig, 1984). These activities were, however, detected in the post-chloroplast supernatant, suggesting their presence in the cytosol. It therefore appears that chloroplasts are unable to synthesize IPP from mevalonate, although the necessary enzymes are present in the cytosol.

TABLE 111 Subcellular localization of mevalonate kinase in pea shoots Enzyme activity nmol mil.-' (mg chlorophyll)-' Enzyme

% recovery in

No. of Chloroplasts+ preparations Homogenate Chloroplasts Chloroplasts supernatant

Mevalonate kinase

3

54f

5

Phosphoenolpyruvatecarboxylase

6

198f

42

0.3f0.05

0.3f0.03

104f5

3.0f1.2

0.6f0.01

102f3

Phosphoglyceratemutase

6

10900k2010

138f42

0.5f 0.08

101f5

Ribulose bisphosphate carboxylase

3

342f 108

378f180

30.1k2.72

Glyceraldehyde3-phosphate dehydrogenase (NADP)

5

3000f 318

2418f408

28.2f1.40

123f5 92f5

Enzyme assays were carried out on a homogenate of pea shoots, and on the chloroplast pellet and supematant obtained by rapid centrifugation to 6O0Og. Mevalonate kinase assays were carried out on exactly the same fractions prepared by M. Stitt, as described in Stitt and ap Rees (1979). Phosphoenolpyruvate carboxylase and phosphoglycerate mutase are regarded as marker enzymes for the cytosol, whereas ribulose bisphosphate carboxylase and NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase are regarded as chloroplast marker enzymes.

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

63

The presence of all three enzymes in plant mitochondria has not been examined, although Lutke-Brinkhaus et al. (1984) have shown that purified potato mitochondria are unable to utilize mevalonate 5-diphosphate for isoprenoid biosynthesis, even after disruption of the mitochondrial membranes. This observation, together with the cytosolic location of mevalonate kinase in pea leaves (Table HI), suggests that mitochondria do not have the capacity for the synthesis of IPP from mevalonate. 4. Formation of Prenyl Diphosphates In contrast to the controversy surrounding the subcellular location of the enzymes for the synthesis of IPP, there appears to be a consensus that the enzymes utilizing IPP are distributed in three subcellular sites. The presence of IPP isomerase and prenyltransferase in chloroplasts, chromoplasts and leucoplasts is suggested by the rapid incorporation of IPP into isoprenoid compounds in preparations of isolated plastids (Block et al., 1980; Beyer et al., 1980; Camara et al., 1982; Gleizes et al., 1983b; Kreuz and Kleinig, 1984), although the levels of cytoplasmic contamination in most of these plastid preparations have not been assessed. Green et al. (1975) showed the presence of IPP isomerase in a proplastid fraction from developing castor bean endosperm. The enzyme activity exactly co-migrated in a discontinuous sucrose gradient with triose phosphate isomerase activity, a marker for proplastids. The choice of triose phosphate isomerase as a marker for proplastids is not ideal because it is also a cytoplasmic enzyme, and discontinuous sucrose density gradient centrifugation may not completely remove cytoplasmic contamination from organelle preparations. These experiments also showed the presence of IPP isomerase activity in mitochondrial fractions from the same sucrose gradient. However, the IPP isomerase activity was not exactly coincident with the succinate dehydrogenase activity, and triose phosphate isomerase was also present in these fractions. Definite conclusions on the location of IPP isomerase in mitochondria from this experiment would be unjustified. However, the presence of IPP isomerase activity in mitochondria is indicated by the ability of isolated mitochondria from potato tubers, spinach leaves and daffodil flowers to incorporate ['4C]IPP into intermediates of the ubiquinone biosynthesis pathway (Lutke-Brinkhaus et al., 1984). The possibility of cytoplasmic contamination was excluded because of the absence of glucose 6-phosphate dehydrogenase activity and the inability of the mitochondrial preparations to metabolize [214C]mevalonate 5-diphosphate. These experiments with isolated mitochondria also indicate the presence of prenyltransferase activity capable of producing decaprenyl derivatives (Lutke-Brinkhaus et al., 1984). Whether this is due to the presence of a single prenyltransferase enzyme is not clear. The ability of isolated chloroplasts and chromoplasts to produce labelled GGPP and geranylgeraniol derivatives from [14C]IPPsuggests that these

64

JOHN C. GRAY

organelles possess GGPP synthetase activity. This activity is also associated with the proplastid fraction from developing castor bean endosperm (Green et al., 1975). Gleizes et al. (1983b) have suggested that GPP synthetase activity is located in a leucoplast preparation from peel of Citrofortuneffamitis, but the level of cytoplasmic contamination has not been assessed. The absence of substantial FPP synthetase activity from organelle preparations suggests that this activity is principally located in the cytosol, where it would be required for sterol synthesis. However, this has not been substantiated experimentally. 5. Formation of Isoprenoid Compounds The subcellular locations of the enzymes catalysing the final stages of the synthesis of isoprenoid compounds are not, in general, a point of contention. There is good evidence that the later stages of the synthesis of chloroplast isoprenoid compounds, such as carotenoids and the side chains of plastoquinone, phylloquinone, tocopherols and chlorophyll, are located within the chloroplasts. Each of these compounds is synthesized from GGPP, and there appears to be some compartmentation of the enzymes using GGPP within the chloroplasts. Esterification of chlorophyllide with GGPP, catalysed by chlorophyll synthetase, takes place in the chloroplast thylakoid membrane (Soll et al., 1983) although a soluble chlorophyll synthetase activity has been described in chromoplasts from daffodil (Kreuz and Kleinig, 1981b) and pepper (Dogbo etal., 1984). The formation and utilization of phytyl diphosphate for phylloquinone and tocopherol synthesis is localized in the inner membrane of the chloroplast envelope (Soll et al., 1980, 1985). Phytoene synthetase appears to be associated with chloroplast envelope membranes (Lutke-Brinkhaus et a f . ,1982), although a soluble activity has been demonstrated in chromoplasts of tomato fruits (Shah et af., 1968) and pepper fruits (Camara, 1985). Kaurene synthetase activity has also been demonstrated in plastid preparations from castor bean seedlings (Simcox et al., 1975) and from pea and barley leaves (Railton et al., 1984), but it is not clear if this is the only subcellular site of synthesis of ent-kaurene. The later stages of ubiquinone synthesis have been demonstrated in isolated mitochondria from potato tubers, spinach leaves and daffodil flowers (Lutke-Brinkhaus et al., 1984); isolated mitochondria were able to incorporate label from ['4C]IPP into the polyprenyl side chain of ubiquinone precursors. Squalene synthetase has been reported to be present in a microsomal preparation from spinach leaves and daffodil flowers (Lutke-Brinkhaus et af.,1984) but is not present in mitochondria isolated from the same tissues. There are reports of slight squalene synthetase activity present in isolated chloroplast preparations (Charlton et af., 1967), but the extent of con-

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

65

tamination with microsomal membranes was not determined. The synthesis of cytokinins and dolichols is likely to occur in the cytosol, although no definitive experiments have been carried out to determine the subcellular location of the biosynthetic enzymes. B. PERMEABILITY OF MEMBRANES TO BIOSYNTHETIC INTERMEDIATES

The original hypothesis to explain the labelling of isoprenoid compounds from [214C]mevalonate and 14C02 was that the mevalonate could not penetrate the chloroplast envelope to reach the chloroplast site of isoprenoid biosynthesis (Goodwin and Mercer, 1963). This idea was examined by incubating chloroplasts, prepared from freeze-dried bean ( P h a s e o h vulgaris) leaves in organic solvents, with [214C]mevalonate; label was reported to be incorporated into phosphorylated intermediates (Rogers et al., 1966a) and into geranylgeraniol and phytoene (Charlton ef al., 1967; Buggy et al., 1974) only if the chloroplast preparations were sonicated. Plastids prepared from Agave americana and etiolated wheat shoots in aqueous media were reported to show increased incorporation of [214C]mevalonate into phosphorylated intermediates after sonication (Garcia-Peregrin et al., 1973a; Cooke, 1977). These experiments were interpreted as indicating the presence of a membrane barrier to mevalonate, but no evidence was presented to show that the plastid envelope was originally intact and was disrupted by the sonication treatment, or that the sonication treatment was not inactivating competing activities such as phosphatases. A direct investigation of mevalonate uptake by plastids from oat seedlings using silicone-oil centrifugation filtration indicated that mature chloroplasts did take up [214C]mevalonate(Wellburn and Hampp, 1976) although similar studies with pepper fruit chloroplasts showed no uptake of [214C]mevalonate(Schneider ef al., 1977). However, in both studies the permeability of the plastid envelope to mevalonate was shown to change during plastid development. Plastids from etiolated oat leaves actively accumulated mevalonate but the permeability of plastids isolated from leaves greened for different times progressively decreased after 1-2 h illumination (Wellburn and Hampp, 1976). Permeability changes during plastid development had previously been suggested by the altered distribution of label from [43H]mevalonate in different subcellular compartments of greening oat seedlings detected by electron microscope autoradiography (Cockburn and Wellburn, 1974), but there was very wide variation in the labelling of the tissue during the 24 h incubation with mevalonate and this precludes any realistic interpretation of the data. Increased permeability of plastid membranes to mevalonate during the transformation of chloroplasts into chromoplasts of pepper fruit was indicated by the silicone oil centrifugation method (Schneider el al., 1977). These changes in envelope

66

JOHN C. GRAY

permeability were suggested to be necessary to provide mevalonate for isoprenoid biosynthesis in plastids that were not photosynthetically competent and would therefore not be able to synthesize mevalonate. However, this view no longer seems tenable in view of the subcellular location of mevalonate-activating enzymes (see Section II.A.3). Another feature of the compartmentalization hypothesis, although not stated explicitly in the original formulation, is that the plastid envelope should be impermeable to all the common intermediates of the isoprenoid biosynthetic pathway, to prevent movement of intermediates between the subcellular sites of synthesis. However, it is now clear that plastid and mitochondrial membranes are relatively permeable to IPP. Intact spinach chloroplasts incubated with [114C]IPP actively incorporate label into geranylgeraniol and chlorophyll side chains (Kreuz and Kleinig, 1981a). Similar results were obtained with pepper fruit chloroplasts (Dogbo et al., 1984). The rate of incorporation of [1’4C]IPPby intact spinach chloroplasts was greater than that by chloroplasts disrupted by a French press treatment, suggesting that the chloroplast envelope is permeable to IPP. Similarly, the plastid envelope does not appear to be a barrier to IPP in proplastids from developing castor bean endosperm (Green et af., 1975). Intact chromoplasts from daffodil flowers and pepper fruits also incorporate [ 1’4C]IPPinto isoprenoid compounds, indicating that the chromoplast envelope is permeable to IPP (Beyer et af., 1980; Camara et al., 1982). However, it appears that the chromoplast membrane may be a barrier to the free movement of IPP because disrupted Narcissus chromoplasts gave a “considerably increased” rate of incorporation compared with intact chromoplasts (Beyer et al., 1980). Further studies on the movement of IPP across plastid envelope membranes and particularly the characterization of the IPP transporter are urgently required. The membranes of mitochondria from potato tubers have also been shown to be permeable to IPP (Lutke-Brinkhaus et af., 1984). Intact and disrupted potato mitochondria showed the same total incorporation of [1’4C]IPP into isoprenoid compounds, although there were differences in the products made in the two incubations. Disrupted mitochondria incorporated label predominantly into 4-hydroxydecaprenyl benzoic acid, and produced some free farnesol, whereas intact mitochondria produced a wider range of polyprenylphenols which were further along the biosynthetic pathway to ubiquinone. Intact mitochondria did not accumulate labelled 4-hydroxydecaprenyl benzoic acid and produced only minor amounts of free farnesol (Lutke-Brinkhaus et al., 1984). The permeability of plastid and mitochondrial membranes to one of the intermediates of the isoprenoid biosynthetic pathway implies that the hypothesized membrane impermeability is not an adequate explanation of the differential labelling of isoprenoid compounds from 14C02 and [2’4C]mevalonate.

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

67

C. CONCLUSIONS

The evidence outlined in this section appears to fit the hypothesis of Kreuz and Kleinig (1984) (Fig. 11B) rather better than the modified compartmentalization hypothesis (Fig. 11A). However, it is clear that more definitive experiments need to be carried out on the subcellular location of the enzymes required for the synthesis of mevalonate. The subcellular location of HMG-CoA reductase activity in particular needs careful study because, at the present time, it is difficult to understand why the enzyme should be present in plastids and mitochondria if the enzymes needed for the metabolism of mevalonate are not also present in these organelles. However, there seems to be no doubt that the mevalonate-activating enzymes are not present in chloroplasts from pea shoots and spinach leaves (Section II.A.3). The possibility that different tissues of plants show differences in compartmentation of isoprenoid biosynthetic pathways has not been sufficiently explored. Differences in the location of phytoene synthetase and chlorophyll synthetase in chloroplasts and chromoplasts (Section II.A.5) indicate that significant differences may exist between tissues predominantly synthesizing carotenoids in large amounts. The explanation of the original observations on the incorporation of [214C]mevalonateand 14C02into isoprenoid compounds therefore needs re-examination. The first consideration is the method of application of these compounds to the plant tissues. Carbon dioxide is a normal exogenous substrate for plants and it can be assumed that 14C02 would be treated by the plant in a normal physiological manner. However, mevalonate is not normally (never?) an exogenous substrate for plant cells. The cell’s supply of mevalonate is normally generated from within the cell, and the assumption that [214C]mevalonatewould enter the normal subcellular pools of mevalonate may not be justified. Another consideration is the identity of the cells in the plant tissues responsible for [214C]mevalonateand 14C02incorporation. In no study has it been established that the cells incorporating 14C02 into chloroplast isoprenoid compounds are also incorporating [214C]mevalonateinto sterols and ubiquinone. In most experiments the cut ends of shoots are placed in a solution of [214C]mevalonate,whereas the 14C02is supplied in the gas phase. It is therefore possible that [214C]mevalonateis metabolized by cells associated with the vascular tissue, and little reaches the photosynthetically active cells where 14C02is fixed and incorporated into chloroplast isoprenoids. This point could be examined by tissue fractionation into vascular and nonvascular material after incorporation of [214C]mevalonate and 14C02.However, this experiment would probably not detect differences in incorporation of labelled precursors by cells of different developmental ages. Some indication that this contributes to the differential incorporation of precursors in maize tissue is provided by Threlfall et al. (1967). Maize

68

JOHN C . GRAY

leaf cells are produced by a basal meristem and the cells at the tip of the leaf are older than those nearer the base. Threlfall et al. (1967) showed, by cutting into three sections leaves that had been incubated with [214C]mevalonate or 14C02, that the basal third of the leaf showed a greater relative incorporation of [214C]mevalonateinto sterols, whereas the upper sections showed greater relative incorporation of 14C02 into chloroplast isoprenoids. However, they concluded that this effect of developmental age of the tissue could not wholly account for the differences in incorporation of [214C]mevalonate and 14C02 into isoprenoid compounds. Incorporation of labelled precursors by different cell types, together with some influence of the developmental age of the cells, may explain the experimental observations. The ability of cells to incorporate both I4CO2 and exogenously applied [214C]mevalonatemay be examined with relatively homogeneous cell populations provided by protoplast isolation techniques.

111. CONTROL OF THE ISOPRENOID BIOSYNTHETIC PATHWAY Our knowledge of the mechanisms operating to regulate the flux through the isoprenoid biosynthetic pathway, with all its branch points, is very poor. A simple view would have the synthesis of individual isoprenoid compounds regulated at the branch points from the main biosynthetic pathway, perhaps by feedback inhibition of the enzyme by the final product, as in many bacterial biosynthetic pathways. However, this possibility has not yet been examined in detail because of the inadequate characterization of the branch point enzymes (see Section 1.E). Regulation of the isoprenoid biosynthetic pathway can be envisaged as occurring at two levels: coarse control by the amounts of the enzymes present, and fine control of the enzyme activities modulated by metabolite concentrations and possible reversible covalent modification of the enzymes. It is clear that many of the changes in isoprenoid biosynthesis during plant development result from changes in the amounts of enzymes present (see Section 1II.B). This is also the case for phytoalexin synthesis stimulated by infection with pathogenic organisms. The evidence for fine control of enzyme activity comes mainly from a few studies on the effects of exogenous metabolites on short sections of the biosynthetic pathway in vitro (Watts and Kekwick, 1974; Knotz et al., 1977; Steiger et al., 1985), together with information on the properties of purified enzymes (Section I). The possibility of modulation of enzyme activity by protein factors and by covalent modification has been suggested for HMG-CoA reductase (Sipat, 1982a; Isa and Sipat, 1982; Russell et al., 1985). However, it is clear that our knowledge of the fine control of the

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

69

isoprenoid biosynthetic pathway will be limited until comprehensive well-formulated studies are carried out. The information required, and the attempts to obtain it, are discussed below (Section 1II.A). A . FINE CONTROL

The information needed to understand the control of a biosynthetic pathway in a particular plant tissue under a given set of conditions is enormous, but can conveniently be divided into four sections. These are: (i) knowledge of the flux of metabolites through the pathway; (ii) knowledge of the maximum catalytic activities of all the enzymes of the pathway; (iii) knowledge of the substrate concentrations in vivo; and (iv) knowledge of the properties of the enzymes, particularly with respect to modulation of enzyme activity by metabolites and protein factors. With this information the contribution of each of the steps of the biosynthetic pathway to the overall control of the biosynthetic pathway can be assessed. 1. Flux Through the Path way The overall flux of metabolites through a pathway from substrate to product is often difficult to measure accurately. This is particularly the case for branched pathways, where the flux of metabolities through the early steps of the pathway before any branch point will be greater than the flux through the individual pathways after the branch points. Thus the rate of synthesis of the product of a branched pathway reflects only the flux through the pathway after the branch point, and will clearly underestimate the flux through the earlier part of the pathway. Even attempts to estimate the rate of synthesis of individual products are not without difficulties. The rate of accumulation of a product in plant tissue may be an underestimate of the rate of synthesis if there is any metabolism or degradation of the product. Attempts to measure rates of synthesis from the rates of incorporation of radioactively labelled substrates also present difficulties in the choice of appropriate substrate and the possibilities of poor uptake by the cells and of dilution by endogenous pools of substrate. These problems were clearly recognized by Goad (1983) in attempts to measure rates of synthesis of sterols in suspension cultures of Acer pseudoplatanus. The incorporation of radioactivity into sterols from a variety of substrates was examined. [U'4C]leucine and [ 1''C]octanoate were very poorly incorporated, although this is perhaps not surprising in view of the tortuous pathway for the incorporation of leucine (see Section 1.A) and the low activities of @-oxidationof fatty acids in plants (ap Rees, 1980). Incorporation of [U'4C]acetate and [methyl 14C]methionine into sterols gave higher rates of synthesis (13-14 nmol sterol synthesized per h per g dry weight of cells) which were exceeded only by the rate of synthesis calculated from the

70

JOHN C. GRAY

incorporation of [214C]mevalonate[31 nmol h-' (g dry weight)-']. This higher rate of synthesis measured with [214C]mevalonate was taken to indicate regulation of the flux from acetate to mevalonate in Acer cells (Goad, 1983). In a second experiment, the rate of sterol synthesis calculated from [214C]mevalonateincorporation was greater than the rate calculated from 3H20incorporation, again suggesting regulation of the flux at an early part of the pathway. Label from 3H20is expected to equilibrate with all the intracellular pools of water and to be incorporated into sterols at the reductive steps, either from protons in the medium or from NADPH (Goad, 1983). This method has been used for the measurement of rates of sterol synthesis in mammalian tissues (Lakshmanan and Veech, 1977). Other estimates of the rate of synthesis of isoprenoid compounds come from measurements of the rate of accumulation of these compounds during various stages of plant development, such as fruit ripening and greening of etiolated leaf tissue. Some selected rates of synthesis of individual isoprenoid compounds are shown in Table IV. None of these measurements allows a calculation of the true flux of metabolites through the early part of the isoprenoid biosynthetic pathway. However, values such as these, calculated for the plant tissues under investigation, may be extremely useful as a measure of the minimum rates of activity for enzymes expected to be associated with the pathway. Enzyme activities that do not exceed the rate of synthesis of isoprenoid compounds in the plant tissue must be viewed with suspicion. 2. Maximum Catalytic Activities of the Enzymes Measurement of the maximum catalytic activities of the enzymes of the pathway in plant extracts or organelle preparations, together with a knowledge of the flux through the pathway, may be extremely useful in helping to define the major rate-controlling steps in the pathway. However, only rarely has a number of enzymes of the isoprenoid biosynthetic pathway been assayed in the same plant tissue (see Table V). Unfortunately, in several of these studies enzyme assays were not carried out at saturating substrate concentration and therefore do not provide a measure of the full potential of the tissue for the enzyme activity. In the studies of enzyme activities in Hevea latex, only soluble enzymes were assayed and this is reflected in the low activity of HMG-CoA reductase (Lynen, 1969). This enzyme is located in membranes in the latex (Hepper and Audley, 1969; Sipat, 1982b) and was removed by centrifugation to produce the latex serum which was used for the enzyme assays (Lynen, 1969). Difficulties in accurate assessment of HMG-CoA reductase activity were also experienced in extracts of sweet potato root tissue infected with black rot fungus, where the measured enzyme activity (Suzuki and Uritani, 1976; Table V) is not great enough to account for the measured rate of terpene accumulation in this tissue (Suzuki etal., 1975; Oba et al., 1976;Table IV).

TABLE IV Rates of synthesis of isoprenoid compounds Isoprenoid compound Rubber Sterols Carotenoids Chlorophyll Ipomeamarone

Plant tissue

Hevea brasiliensis latex in vivo Acerpseudopfatanus cells in culture Pea shoots Phaseolus vulgaris leaves Sweet potato roots infected with black rot

Rate of synthesis

5 mg min-' (mI latex)-' 13 nmol h-' (g dry wt)-' 0.03 nmol min-' (g fresh w t - ' 5.8 pg min-' (g fresh wt)-' 14-19 mg day-' (g fresh wt)-'

"Calculatedon the basis that dry weight represents 10% of fresh weight.

Demand for IPP 500 nmo~min-' (mI latex)-' 2 nmol min-' (g fresh wt)-' a 0.24 nmol min-' (g fresh wt)-' 26 nmol min-' (g fresh

Reference Bealing (1975) Goad (1983) Kaethner and ap Rees (1985) Gray and Kekwick (1973a)

Wq-1

40-50 nmol min-' (g fresh wt)-'

Oba et al. (1977), Suzuki et al. (1975).

72

JOHN C. GRAY

TABLE V Activities of enzymes of the isoprenoid biosynthetic parhway

nmol min-' (g fresh wt)-' Infected Enzyme Acetoacetyl-CoA thiolase HMG-CoA synthetase HMG-CoA reductase Mevalonate kinase Mevalonate 5-phosphate kinase Mevalonate 5-diphosphate decarboxylase IPP isomerase Prenyl transferase Squalene synthetase

Germinating pea seeds'

-

37.8 12.0 2.6 3.4 0.4

0.7

Hevea brasiliensk latex

sweet potato

39' 232'

-

roots

0.078', 5.5" 149', 57Sd 44', 23d

0.15'

103', 216d

2.2f

8.9

-

-

"Green and Baisted (1972). 'Lynen (1969). "Sipat (1985). dCalculated from Skilleter and Kekwick (1971). assuming latex contains 5 mg serum protein m1-I. 'Suzuki and Uritani (1976). 'Oba et ul. (1976).

A similar discrepancy exists in the rate of terpene accumulation in suspension cultures of sweet potatoes and measurements of HMG-CoA reductase activity; the measured enzyme activity is approximately an order of magnitude lower than that required for terpene accumulation (Oba and Uritani, 1979). This is probably a consequence of carrying out enzyme assays on extracts of tissues in which only a small proportion of the cells are actively carrying out phytoalexin synthesis. However, without valid measurements of maximum catalytic activities in the cells carrying out isoprenoid biosynthesis it is not possible to assess the likely contribution of the different enzymes to the overall control of the pathway. The distribution of some of the enzymes between different subcellular compartments (Section 1I.A) is an added complication in the measurement of maximum catalytic activities. It is necessary to assess the activity in each of the subcellular compartments. Comparison of enzyme activity in the different compartments with estimates of the flux through the pathway may help in determining the subcellular location of sections of the biosynthetic pathway. This sort of comparison indicates that the mevalonate kinase activity associated with chloroplast preparations from pea and bean leaves is not great enough to account for the rate of accumulation of carotenoids or chlorophyll in these tissues (Table VI), even allowing for possible loss of

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TABLE VI Comparison of mevalonate kinase activity and demand for mevalonate in leaf tissue

r

Mevalonate kinase activit [nmol min-' (g fresh wt)- ] ~

Plant tissue "Bean (Phaseolus vulgaris) 24 h greening leaves

'Pea

~~

leaf Demand for mevalonate homogenate chloroplasts [nmol min-' (g fresh wt)-'] 90.5 2.4

16.7 0.02

26.2 0.24

(Pisum sativum)

7 day old leaves 'Calculated from Gray and Kekwick (1973a). The mevalonate kinase activity of the chloroplasts is the maximum possible allowing for a 10% recovery of ribulose bisphosphate carboxylase activity in the chloroplast preparation. The demand for mevalonate is that necessary for chlorophyll synthesis (see Table IV). 'Data for mevalonate kinase activities are obtained from Table 111. The demand for mevalonate is that necessary for carotenoid synthesis (Kaethner and ap Rees, 1985).

enzyme activity from isolated chloroplasts (assessed by the recovery of chloroplast marker enzymes in the chloroplast preparations). However, in both tissues the enzyme activity in the leaf homogenates was great enough for the measured rates of isoprenoid accumulation, and it must be concluded that cytoplasmic mevalonate kinase participates in the synthesis of chloroplast terpenoids. The activity of HMG-CoA reductase in chloroplast preparations from pea is also very low [0.11 nmol min-' (g fresh weight)-'], but the biosynthetic capacity of the pea shoots used for the chloroplast preparation was not reported (Brooker and Russell, 1975b). Comparison of the HMG-CoA reductase activity of chloroplasts with the rate of accumulation of chloroplast isoprenoid compounds may be helpful in determining the role of the chloroplast in the early stages of the isoprenoid biosynthetic pathway.

3. Substrate Concentrations in vivo There have been few attempts to measure the intracellular concentrations of the intermediates of the isoprenoid biosynthetic pathway. Although such measurements present many problems, it should be possible, using modern analytical methods, to establish the amounts of some intermediates in tissue extracts and subcellular fractions of tissues. Amounts of mevalonate in various fruits and vegetables have been measured by Modi and Patwa (1961) and Wills and Scurr (1975). Although mevalonate was below the limit of detection in some tissues, concentrations in the range 1-24 pmol (100 g fresh weight)-' were obtained (Wills and Scurr, 1975).

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Values for carrot root tissue [2-4 pmol (100 g fresh weight)-'; Modi and Patwa, 1961; Wills and Scurr, 19751 would represent a cytoplasmic concentration of 4-8x M if all the mevalonate were confined to the cytoplasm, and the cytoplasm accounted for 5% of the tissue mass. However, the subcellular location of mevalonate is not known, and it is not clear if mevalonate is present in the vacuoles of plant cells. Benz et al. (1983) have estimated the amounts of GGPP and phytyl diphosphate in etiolated oats leaves as 16 nmol (g fresh weight)-' and 2 nmol (g fresh weight)-' respectively. The value for phytyl diphosphate was approximately one-tenth of that previously reported for leaves of Phaseolus vulgaris [0.1-0.2 pmol (g dry weight)-'; Watts and Kekwick, 19741. The reason for the difference between the plants is not known, but may reflect the extraction procedures used. Benz et al. (1983) measured only cytosolic compounds, whereas the extracts of Watts and Kekwick (1974) would have included membrane-located phytyl diphosphate. Using estimates of etioplast volume from other plants, Steiger et al. (1985) have calculated that the stroma of oats etioplasts would contain GGPP at a concentration of 1.8X lov4M , if GGPP were confined to the etioplasts in oats leaves. Similar calculations would suggest concentrations of phytyl diphosphate of 2X lo-' M in oat etioplasts and 2x M in Phaseolus plastids. Using methods for the rapid separation of organelles from protoplasts (Robinson and Walker, 1979; Wirtz et al., 1980) it should be possible to obtain valid estimates of the metabolite concentrations in the organelles. With information on the concentrations of the intermediates in different subcellular compartments it should be possible to establish which of the enzyme reactions of the pathway are close to equilibrium and which are not. Enzymes catalysing reactions which are far from equilibrium are likely to play a major role in the regulation of the pathway. 4. Modulation of Enzyme Activity

Enzyme activity may be modulated by changes in metabolite concentrations or by interaction with protein factors, perhaps resulting in reversible covalent modifications such as phosphorylation. a. Modulation by metabolites. Five enzymes of the isoprenoid biosynthetic pathway must be considered as candidates for modulation of activity by metabolites, on the basis of the kinetic properties of the purified enzymes (Section I). These are HMG-CoA reductase, which is inhibited by coenzyme A and NADP+ (Bach and Lichtenthaler, 1984; Bach et al., 1986), mevalonate kinase, which is inhibited by prenyl diphosphates (Gray and Kekwick, 1972, 1973a; Shewry and Stobart, 1973), mevalonate Sdiphosphate decarboxylase, which is inhibited by IPP and ADP (Skilleter and Kekwick, 1971), IPP isomerase, which is inhibited by prenyl diphosphates (Ogura et al., 1969; Spurgeon et al., 1984) and prenyl transferase,

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which is also inhibited by prenyl diphosphates (Ogura et al., 1969; De La Funte et al., 1981). In all cases the metabolites appear to act as competitive inhibitors with respect to one of the substrates of the enzyme; there is no evidence for allosteric effects on any of the enzymes of isoprenoid biosynthesis. Unfortunately, without information on the intracellular concentrations of metabolites and substrates it is not possible to estimate the physiological relevance of these inhibitions. Several studies have used tissue homogenates or cell-free extracts to examine the regulation of sections of the isoprenoid biosynthetic pathway. Watts and Kekwick (1974) showed that the incorporation of [214C]mevalonate,but not of [414C]IPP, into phytol by homogenates of greening bean (Phaseolus vulgaris) leaves was inhibited by added phytyl diphosphate. In view of the potent inhibition of bean leaf mevalonate kinase by phytyl diphosphate (Gray and Kekwick, 1973a) it seems possible that the inhibitory effect observed by Watts and Kekwick (1974) acts M) of through this enzyme. However, the high concentration ( l x phytyl diphosphate needed to produce a modest inhibition (35%) of [214C]mevalonateincorporation does not accord closely with the Ki value of 3 . 5 lov6 ~ M phytyl diphosphate for the purified bean leaf enzyme (Gray and Kekwick, 1973a). A modest inhibition (40%) of [214C]mevalonate incorporation into GGPP by phytyl diphosphate in extracts of the endosperm of immature seeds of pumpkin (Cucurbita p e p ) has also been reported (Steiger et al., 1985). However, it seems unlikely that phytyl diphosphate has any physiological role in the regulation of mevalonate kinase activity in vivo, because of the separate subcellular locations of these two components (see Section II.A.3). The cytoplasmic location of mevalonate kinase would appear to preclude any feedback inhibition by intermediates such as GGPP and phytyl diphosphate, although inhibition by cytosolic FPP may be important. Steiger et al. (1985) have shown that added GGPP inhibits the incorporation of [214C]mevalonateand [1'4C]IPP into GGPP in pumpkin endosperm extracts. The incorporation of both [2'4C]mevalonate and [114C]IPPwas inhibited 8&90% by 2 . 2 ~ M GGPP. The inhibitory effect on IPP incorporation suggests that GGPP was not acting on mevalonate kinase, but on an enzyme utilizing IPP. Steiger et al. (1985) suggested an effect on prenyltransferase, which is possible in view of the inhibition of the pumpkin and orange enzymes by other prenyl diphosphates (Ogura et al., 1969; De La Funte et al., 1981) but effects on IPP isomerase cannot be discounted, in view of the potent inhibition of the pumpkin and tomato enzymes by GPP (Ogura et al., 1969; Spurgeon et al., 1984). Modulation of IPP isomerase activity by metabolites in vitro has not been sufficiently examined to draw any meaningful conclusions. However, the incorporation of [214C]mevalonateinto a-pinene in Pinus species is unequally distributed between the IPP-derived moiety and the DMAPP-

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JOHN C. GRAY

derived moiety (Banthorpe and Le Patourel, 1972). Approximately 90% of the radioactivity incorporated is located in the IPP-derived moiety, indicating a difference in the specific radioactivity of the DMAPP pool compared to the IPP pool. This suggests that IPP isomerase is not able to catalyse the isotopic equilibration of the two pools. Although this has been viewed as a consequence of the inequality of the flux through the pathway and the activity of IPP isomerase, such an inequality cannot exist in the steady state and is a consequence of the short period of incorporation of label. Inhibition of the activity of IPP isomerase by prenyl diphosphates may contribute to the control of the amounts of IPP and DMAPP available for isoprenoid biosynthesis. The synthesis of different isoprenoid compounds will require different proportions of IPP and DMAPP, and inhibition of IPP isomerase by DMAPP and GPP (Ogura et al., 1969; Spurgeon et al., 1984) may provide a means of ensuring an increased supply of IPP for elongation of these lower prenyl diphosphates. Knotz et al. (1977) examined the effects of energy charge on the synthesis of kaurene in extracts of the endosperm of immature seeds of Marah macrocarpa. They showed that the rate of kaurene synthesis from [214C]mevalonatewas influenced by the energy charge above 0.8. This modulation of activity was shown to be due to an inhibitory effect of ADP; AMP and inorganic phosphate had no effect on the rate. Energy charge or ADP did not affect the incorporation of [414C]IPPinto kaurene, indicating that the inhibitory effect was located at a step between mevalonate and IPP. It was shown that 2X lop3 M ADP increased the amounts of mevalonate 5-phosphate and mevalonate 5-diphosphate, and decreased the amounts of IPP and kaurene, when [214C]mevalonatewas incubated with the cell-free extract, suggesting that the inhibitory effect was on mevalonate 5-diphosphate decarboxylase. The overall incorporation of [214C]mevalonatewas the same in the presence and absence of ADP, indicating that ADP had no effect on mevalonate kinase. These observations are in accord with the known inhibition of mevalonate 5-diphosphate decarboxylase by ADP (Skilleter and Kekwick, 1971). There is no evidence from plant systems in vitro that modulation of HMG-CoA reductase activity by metabolite or coenzyme levels influences the flux through the pathway. However, the potent inhibition of the purified radish enzyme by NADP+ (Bach et al., 1986) suggests that in vivo HMG-CoA reductase activity may be regulated by the NADPWNADP+ ratio. The effect on HMG-CoA reductase activity of a number of compounds, such as sterols, abscisic acid, gibberellic acid and zeatin, which might conceivably act as feedback inhibitors, has been examined with pea microsomal preparations (Russell et al., 1985), but no inhibition was observed. There are unsubstantiated suggestions that HMG-CoA reductase activity may be modulated by covalent modification (see below).

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6 . Modulation by phospholipids. Modulation of isoprenoid biosynthesis in vitro by added phospholipids has been reported for extracts prepared from Pinus radiata seedlings (George-Nascimento et al., 1969) and from the endosperm of immature pumpkin seeds (Steiger et al., 1985). Phosphatidylethanolamine and phosphatidylcholine stimulated incorporation of [214C]mevalonateinto prenyl lipids by the Pinus extract approximately two-fold (George-Nascimento et al., 1969), whereas Steiger et al. (1985) reported a four-fold stimulation of the incorporation of [214C]mevalonate into GGPP by phosphatidylcholine. In both studies, membranes had been removed from the cell-free extracts by high-speed centrifugation, and it seems likely that the phospholipids were providing a lipid phase for the removal of hydrophobic reaction products which might otherwise inhibit the enzyme reactions in the aqueous phase. Higher prenyl diphosphates are potent inhibitors of IPP isomerase and prenyltransferase (Section LD), and accumulation of prenyl diphosphates in incubation mixtures in vitro would be expected to inhibit these enzymes. The stimulation of prenyltransferase activity in the presence of the detergent Tween 80 (Spurgeon et al., 1984) may be the result of the removal of prenyl diphosphates from aqueous solution. The removal of GGPP by membrane fractions is probably the explanation of the increased incorporation of [1'4C]IPP into prenyl lipids by stroma in the presence of membrane fractions from spinach chloroplasts (Block et al., 1980). Incorporation of [114C]IPPby the stroma fraction of spinach chloroplasts was principally into GGPP at low rates. The addition of chloroplast envelope or thylakoid membranes increased the incorporation of IPP approximately two-fold or eight-fold respectively. Neither envelope nor thylakoid membranes were able to incorporate IPP into prenyl lipids in the absence of stroma. It seems likely that the GGPP which accumulated in the stroma incubations inhibited incorporation of IPP, as described by Steiger et a f . (1985), and that partitioning into, and further metabolism of GGPP by, the membrane fractions relieved this inhibition. C. Modulation by protein factors. The possibility that HMG-CoA reductase activity may be modulated by protein factors has been suggested from several studies (Sipat, 1982a; Isa and Sipat, 1982; Russell et al., 1985). Isa and Sipat (1982) have described the presence of a trypsinsensitive heat-stable activator of HMG-CoA reductase in the latex serum of Hevea brasiliensis. This factor increases HMG-CoA reductase activity in vitro up to two-fold, but its likely physiological relevance is in doubt. Sipat (1985) has reported that its main effect is its ability to stabilize and prevent inactivation of the enzyme during preincubation. In mammalian tissues, the activity of HMG-CoA reductase is modulated by phosphorylation and dephosphorylation catalysed by protein kinases and phosphatases (Beg et a f . , 1979). The possibility that a similar regula-

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JOHN C. GRAY

tory mechanism operates in plants has been considered by Sipat (1982a) and Russell et al. (1985). Sipat (1982a) reported that the HMG-CoA reductase activity in membranes of Hevea brasiliensis latex was inhibited by 90% in the presence of 4 mM ATP and that the extent of the inhibition depended on the treatment of the membrane preparation. Washing the membranes with triethanolamine buffer or with 0.1% (wh) Triton X-100 removed any subsequent inhibition by ATP (Sipat, 1985). Russell et al. (1985) have also reported effects of Mg-ATP on the activity of HMG-CoA reductase in microsomal membranes and plastid preparations of pea leaves. Microsomal membranes preincubated with a high-speed supernatant fraction showed a 35% stimulation of HMG-CoA reductase activity compared to the activity in untreated membranes, and this stimulation was partly blocked by potassium fluoride. Preincubation of membranes with the supernatant fraction in the presence of Mg-ATP (concentration not stated) caused up to 80% decrease in HMG-CoA reductase activity which could be restored by incubation with bacterial alkaline phosphatase (Russell et al., 1985). These observations were suggested to be consistent with the inactivation of the enzyme by the action of a protein kinase, and the activation of the enzyme by the action of a protein phosphatase. The protein kinase was suggested to be a soluble activity because preincubation of microsomal membranes with Mg-ATP in the absence of the supernatant fraction did not affect the HMG-CoA reductase activity. Similar effects of Mg-ATP, KF and a stromal protein fraction on HMG-CoA reductase activity in pea plastid preparations have been interpreted in terms of a model for the regulation of HMG-CoA reductase activity by phosphorylatioddephosphorylation (Russell et al. , 1985). However, an alternative explanation has been suggested by Sipat (1985). He suggested that the inhibitory effect of ATP was due to the removal of mevalonate from the reaction mixtures by mevalonate kinase, and demonstrated the removal of mevalonate, previously accumulated in a 30 min incubation of Hevea latex membranes with HMG-CoA, on addition of 4 mM ATP (Sipat, 1985). This explanation would also partly account for the results of Russell et al. (1985) if mevalonate kinase activity were present in the high-speed supernatant fraction from pea leaves. Pea leaf extracts contain appreciable amounts of mevalonate kinase activity (see Table 111) and are also likely to contain phosphatase activity inhibited by KF as in other plants (Beytia et al., 1969; Shewry and Stobart, 1973). Incubation of membranes with Mg-ATP in the presence of the supernatant fraction would result in the further metabolism of the mevalonate produced by HMG-CoA reductase. Russell et d.(1985) assayed HMG-CoA reductase activity by the formation of mevalonate, and thus any removal of mevalonate by mevalonate kinase would appear to be an inhibition of HMG-CoA reductase activity. The presence of phosphatase activity in the

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79

supernatant fraction would relieve this apparent inhibition by hydrolysis of mevalonate 5-phosphate back to mevalonate. The inhibition of this phosphatase by K F would therefore be manifested as an increased inhibition of HMG-CoA reductase activity, as reported by Russell et al. (1985). The restoration of HMG-CoA reductase activity by treatment with bacterial alkaline phosphatase is also consistent with an effect on mevalonate 5-phosphate, regenerating mevalonate. Alkaline phosphatase is expected to hydrolyse preferentially small organic phosphates, rather than phosphorylated proteins. There is at present no unequivocal evidence in favour of a mechanism of protein phosphorylatioddephosphorylationfor the regulation of HMG-CoA reductase activity.

5 . Competitionfor Substrates The branched nature of the isoprenoid biosynthetic pathway (see Fig. 1) implies that there will be competition between enzymes using the same substrate. In the absence of full information on the activities and kinetic properties of the enzymes and the intracellular substrate concentrations, it is not possible to estimate the significance of competing activities on the fluxes through the biosynthetic pathways. However, some information is available to suggest that the fluxes through the pathways for the synthesis of chloroplast isoprenoid compounds are influenced by the availability of GGPP. Kreuz and Kleinig (1981) reported that in chromoplast preparations from daffodil flowers, the addition of chlorophyllide diverted GGPP away from carotenoid synthesis and resulted in the production of chlorophyll esterified with a geranylgeraniol side chain (Fig. 12). The amount of geranylgeraniol, presumably produced by phosphatase activity on GGPP, in the incubation mixtures was hardly affected by the addition of chlorophyllide. A similar effect on the channelling of prescursors away from carotenoid biosynthesis and towards chlorophyllide esterification has been observed with pepper chromoplasts (Dogbo et al., 1984). The addition of chlorophyllide to chromoplast preparations incubated with [ 1''C]IPP produced a 34-fold greater incorporation into chlorophyll than into carotenoids. However, the addition of protochlorophyllide, which is not readily esterified, gave a 33-fold greater incorporation into carotenoids than into protochlorophyll. Inhibition of chlorophyll synthetase with Triton X-100 resulted in greater incorporation into carotenoids than into chlorophyll in incubations with added chlorophyllide (Dogbo et al., 1984). These experiments serve to emphasize the importance of competition for substrates at the branch points of the isoprenoid biosynthetic pathway. However, a full understanding of the channelling of substrates towards different end-products will not be possible without further information on the activities and kinetic properties of the enzymes involved and on the concentrations of the substrates.

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JOHN C. GRAY

nmol chlorophyllide added

Fig. 12. Competition between the carotenoid and chlorophyll biosynthetic pathways for GGPP in chromoplasts of daffodil flowers. The addition of chlorophyllide redirects GGPP away from carotenoid synthesis. The incorporation of [1-'4C]IPP into p-carotene (O), geranylgeraniol ( A ) and chlorophyll (B) by chromoplast hornogenates is shown. Drawn from data in Kreuz and Kleinig (1981b).

B. COARSE CONTROL

The amounts and types of isoprenoid compounds present in plants change markedly during development and it seems probable that in most cases these changes are a consequence of altered activities of the isoprenoid metabolic pathways. However, only rarely have the enzyme activities of particular sections of isoprenoid metabolic pathways been assayed during plant development (Green and Baisted, 1971, 1972). These studies show changes in extractable enzyme activities, but in no case has a change in enzyme activity been correlated with a change in enzyme protein. The coarse control of metabolic pathways is mediated by changes in enzyme protein, either by synthesis or degradation, or a balance between the two. Unfortunately, few studies of the control of isoprenoid metabolism have examined the synthesis of enzymes during plant development. This section considers the changes in enzyme activity that have been monitored during plant development, or in response to factors such as light and elicitors of phytoalexin synthesis. 1. Seed Germination

Green and Baisted (1972) have measured the activities of all the enzymes

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involved in the conversion of mevalonate to squalene in pea seeds over the first 32 h of germination. The enzyme activities in seed extracts show rather different patterns of development. Mevalonate kinase activity remains more or less constant over the 32 h period, whereas most of the other enzymes, including mevalonate 5-phosphate kinase, mevalonate 5-diphosphate decarboxylase, IPP isomerase and squalene synthetase, show increased activity over the first 8-12 h and then remain constant. The increases in activities between 2 and 12 h germination are approximately two-fold for IPP isomerase and squalene synthetase, three-fold for mevalonate 5-diphosphate decarboxylase and five-fold for mevalonate 5phosphate kinase. The activity of prenyltransferase shows a more complicated pattern of development. Activity increases four-fold between 2 and 6 h, increases a further two-fold between 10 and 16 h, and then gradually declines to approximately half the peak activity over the next 16 h. This pattern of prenyltransferase activity is very similar to the pattern of squalene synthesis from [214C]mevalonatein pea seed extracts described previously (Green and Baisted, 1971). This suggests that prenyltransferase activity is the major rate-determining step in squalene synthesis in pea seed extracts. As shown in Table IV, prenyltransferase shows the lowest activity of enzymes converting mevalonate to squalene. Unfortunately, there has been no further work to investigate if the increases in enzyme activity are due to de novo synthesis of enzyme protein, or to activation of existing precursor forms. The location of the increased enzyme activity is also not clear. The increases may represent changes in particular tissues of the seed (e.g. root, shoot or cotyledons) or in different components of the cell.

2. Leaf Greening Changes in isoprenoid metabolism during illumination of dark-grown plants have been extensively studied. During leaf greening there are rapid increases in the amounts of chloroplast isoprenoid compounds, whereas the amounts of sterols and ubiquinone remain more or less constant (Goodwin, 1965). Although there are obvious changes in the patterns of isoprenoid biosynthesis during greening, there has been no systematic study of the activities of the individual enzymes. In pea and radish seedlings the activity of HMG-CoA reductase is higher in dark-grown plants than light-grown plants (Brooker and Russell, 1975a; Bach et al., 1980). A decrease in HMG-CoA reductase activity in microsomal membrane preparations is observed on illumination of darkgrown pea and radish seedlings (Brooker and Russell, 1979; Bach et al., 1980). Continuous white light results in a decrease in pea microsomal HMG-CoA reductase activity to approximately 50% of the activity in dark-grown tissue over a period of 12 h (Brooker and Russell, 1979), whereas 24 h white light treatment of dark-grown radish seedlings results

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JOHN C. GRAY

in a decrease to only 5 % of the activity in dark-grown seedlings (Bach et af., 1980). A short period (5 min) of red light was also effective in decreasing the activity of microsomal HMG-CoA reductase in dark-grown pea and radish seedlings, and this effect could be partially reversed if followed by a short period (2 or 5 min) of far-red light (Brooker and Russell, 1979; Bach et al., 1980). This suggests that the photoreceptor for the light response is phytochrome. HMG-CoA reductase activity in other membrane preparations from these illuminated pea and radish seedlings shows different responses. Bach et al. (1980) reported that HMG-CoA reductase activity in membranes sedimented at 16,OOOg for 4 h was not affected by red or far-red light treatments, but showed a decrease to approximately 30% of the activity in dark-grown seedlings on illumination with white light for 24 h. Wong et af. (1982) reported increased activity in plastid membrane preparations on illumination of dark-grown pea seedlings. Brief illumination with red light caused a transient increase in HMG-CoA reductase activity, and this increase could be abolished by subsequent treatment with far-red light. Red-light treatment gave increases of approximately 80% after 2 h , which then subsequently declined to activities about 30% higher than those in membranes from dark-grown tissue. These different responses of HMGCoA reductase activities in different membrane preparations have been used as evidence to support the existence of separately regulated enzymes in separate subcellular compartments (Bach et af., 1980; Russell et af., 1985). The increased activity of HMG-CoA reductase in pea plastid preparations on illumination is perceived to correlate with increased synthesis of chloroplast isoprenoid compounds (Wong et al., 1982). However, changes in enzyme activities measured in vitro do not necessarily correlate with the overall activity of biosynthetic pathways. The activity of protochlorophyllide reductase declines markedly on illumination of darkgrown plants, although the enzyme is essential for the massive synthesis of chlorophyll taking place (Mapleston and Griffiths, 1980; Ape1 e t a f . , 1983). Light effects on HMG-CoA reductase activity require further study, particularly to understand the effects of changes in activity on the flux through the pathway. However, it is essential that these studies take place only when the location of HMG-CoA reductase in plant membranes and the problems associated with assaying the enzyme in crude membrane preparations have been satisfactorily resolved. The only other enzymes of the main isoprenoid biosynthetic pathway whose activities have been shown to increase on illumination of darkgrown tissue are mevalonate kinase and mevalonate 5-phosphate kinase. Gray and Kekwick (1973a) measured an approximately three-fold increase in mevalonate kinase activity in the leaves on illumination of dark-grown bean plants for 24 h, and Thomas and Stobart (1970) reported a two-fold greater activity of mevalonate kinase and mevalonate 5-phosphate kinase

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83

in green callus cultures of Kulunchoe crenatu compared to dark-grown cultures.

3. Fruit Ripening The ripening of tomato and pepper fruits has provided well-studied systems of carotenoid synthesis. However, the enzyme activities present at different stages of ripening have not been determined. Genetic studies with tomato mutants with altered fruit pigmentation indicate that separate enzymes from those in the rest of the plant are responsible for the later stages of carotenoid biosynthesis in ripening fruits. Fruit pigmentation mutations do not affect the normal synthesis of carotenoids in leaves. This suggests that the genes for the enzymes of the later stages of fruit carotenoid biosynthesis are expressed, and the synthesis of the enzymes takes place, only during fruit ripening. Camara (1984) has shown that the increased activity of phytoene synthetase in pepper fruits artificially ripened by treatment with 2-(4-chlorophenylthio) triethylamine hydrochloride (CPTA) is due to synthesis de novo. The increased activity of phytoene synthetase was prevented by dipping the fruits in cycloheximide before dipping in CPTA. The increase in activity was not prevented by chloramphenicol or lincomycin, inhibitors of protein synthesis on plastid ribosomes, indicating that phytoene synthetase was synthesized on cytoplasmic ribosomes. 4. tnduction of Phytoulexin Synthesis Many plants produce isoprenoid phytoalexins in response to infection with bacteria or fungi (Brindle and Threlfall, 1983). Changes in the activities of enzymes involved in isoprenoid phytoalexin biosynthesis have been measured in sweet potato roots infected with black rot fungus, Cerutocystis Jimbriutu (Oba et ul., 1982), and in castor bean seedlings infected with Rhizopus stolonifer (West et al., 1979; Moesta and West, 1985). In each system, increased activity of enzymes of the biosynthetic pathway has been shown to be due to de novo synthesis of the enzymes. Infection of sweet potato roots with the fungus C. Jimbriutu results in the production of the phytoalexin ipomeamarone (Fig. 13). Increased activities of acetyl-CoA synthetase (Takeuchi et al., 1977), ATP-citrate lyase (Takeuchi et ul., 1981), HMG-CoA reductase (Suzuki et ul., 1975),

farnesyl diphosphate

ipomeomarone

Fig. 13. Scheme for the synthesis of ipomeamarone from FPP.

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JOHN C. GRAY

mevalonate kinase and mevalonate 5-diphosphate decarboxylase (Oba et al., 1976) have been measured within 24 h of inoculating the cut surfaces of sweet potato root slices with a spore suspension of C. firnbriata. Phytoalexin production is also induced in suspension cultures of sweet potato root cells infected with C. fimbriata (Oba and Uritani, 1979) and by treatment of cut sweet potato root surfaces with the abiotic elicitor, mercuric chloride (Uritani et al., 1960). This latter system has been used to examine the effect of protein synthesis inhibitors on the induction of enzyme activities of the ipomeamarone biosynthetic pathway. The other systems were not used because of possible inhibitory effects on the fungus itself. When sweet potato root discs were preincubated for 18 h and then treated with HgCI2, accumulation of ipomeamarone was detected within 3 h, reached a maximum at 6-9 h, and then gradually declined (Oba et al., 1982). Increases in HMG-CoA reductase and mevalonate 5-diphosphate decarboxylase activities were induced by this treatment (Oba et al., 1976; Jto et al., 1979). The increases in ipomeamarone, HMG-CoA reductase activity and mevalonate 5-diphosphate decarboxylase activity were inhibited by the application of cycloheximide immediately after cutting the discs, suggesting that the enzymes were synthesized de novo (Oba et al., 1976; Ito et al., 1979). However, when cycloheximide was added to the preincubated discs at the same time as the HgC12, the ipomeamarone content increased over a period of 24 h and showed a six-fold stimulation compared to discs treated with HgC&alone (Oba et a f . ,1982). Activities of HMG-CoA reductase and mevalonate 5-diphosphate decarboxylase were also greater in discs treated with cycloheximide and HgC12 than in discs treated with HgC12 alone (Oba et al., 1982; Ito et al., 1979). Cycloheximide appeared to be an effective inhibitor of protein synthesis in root discs incubated with cycloheximide and HgCl,; a 75% inhibition of [14C]leucine incorporation into protein was observed to 6 h after incubation with cycloheximide (Oba et al., 1982). These observations have been taken to suggest that the enzymes are synthesized de novo in inactive forms during the first 18 h of incubation in response to wounding and then the inactive forms of the enzymes are activated in response to treatment with HgC12 (Oba et al., 1982). This suggestion requires further consideration, but the discrepancy between the measured activity of HMG-CoA reductase and the rate of accumulation of ipomeamarone suggests that measurements of enzyme activity in vitro may not reflect the activities in vivo. Measurements of specific enzyme protein, obtained by the use of immunochemical techniques, are necessary for clarification of the processes taking place during phytoalexin induction. Infection of castor bean seedlings with the fungus Rhizopus stolonifer results in the production of phytoalexin casbene (Fig. 14). West et al. (1979) have shown increased activities of GGPP synthetase and casbene synthetase in extracts of castor bean seedlings prepared 16 h after exposure

CONTROL OF ISOPRENOID BIOSYNTHESIS IN HIGHER PLANTS

geranylgeranyl diphosphate

85

casbene

Fig. 14. Scheme for the synthesis of casbene from GGPP.

of the seedlings to fungal spores. The enzyme activities appeared to be localized predominantly in the proplastids of the castor bean seedlings (West et af., 1979). Casbene synthetase has been purified to homogeneity, and antibodies to the protein have been raised in rabbits (Moesta and West, 1985). These antibodies have been used to quantitate amounts of translatable mRNA for casbene synthetase after induction of phytoalexin synthesis with pectic fragments. Translatable mRNA increased from undetectable levels at the time of elicitation to reach a maximum at 6 h, after which casbene synthetase mRNA levels decreased. The increase in mRNA preceded the increase in casbene synthetase activity, which was initially detected at 2 h and increased to a maximum at about 10 h after elicitation. This suggests that the enzyme was synthesized de n o w as a result of increased levels of casbene synthetase mRNA. Further work is needed to determine if this increase in mRNA is due to increased transcription of the casbene synthetase gene, and how the transcription of this gene is regulated. C. CONCLUSIONS

Much work remains to be carried out before even a minimal level of understanding of the regulation of isoprenoid biosynthesis is attained. It is not even clear what constitutes the major rate-determining step in the isoprenoid biosynthetic pathway. Although, by analogy to animal systems, it is often assumed that the reaction catalysed by HMG-CoA reductase will be the main rate-determining step in plants, there is no unequivocal experimental evidence in support of this idea. This is partly due to the difficulties of obtaining realistic estimates of HMG-CoA reductase activity in plant extracts, but is also due to the absence of information on the flux of metabolites through the early stages of the isoprenoid biosynthetic pathway. The information needed to understand the control of the isoprenoid biosynthetic pathway is set out in Section III.A., and it is important that more work on isoprenoid biosynthesis is directed towards understanding regulation. The factors regulating the amounts of the enzyme proteins in plant

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tissues also require further investigation. The study of casbene synthetase and its synthesis during elicitation by pectic fragments (Moesta and West, 1985) is a model of the sorts of investigation that need to be carried out during changes in isoprenoid biosynthesis.

IV. GENERAL DISCUSSION An attempt has been made to assess all the relevant experimental evidence in order to produce a model for the regulation of isoprenoid biosynthesis in plants. Unfortunately, much of the published work on isoprenoid biosynthesis is of poor quality and does not allow unequivocal statements to be made. This is particularly the case with work on the subcellular location of enzymes, as discussed in Section 1I.A. The model of Kreuz and Kleinig (1984), as outlined in Fig. 11B, appears to represent the most realistic interpretation of the available experimental evidence on the location of the enzymes of isoprenoid biosynthesis. With this model as a framework it is important that the activities of all the enzymes and membrane carriers are estimated with a view to comparing these activities with measurements of the flux of metabolites through the pathway. As discussed in Section III.A, this information should lead to the identification of major rate-determining steps in the isoprenoid biosynthetic pathway. At present there is no definitive experimental evidence to indicate the identity of these major rate-determining steps. Further in the future, with the purification of the biosynthetic enzymes to homogeneity and the availability of monospecific antibodies, the mechanisms regulating the transcription of genes and the synthesis of the individual enzyme proteins can be explored. This will be essential if the changes in isoprenoid biosynthesis during plant development are to be understood.

ACKNOWLEDGEMENTS I wish to thank Dr T. ap Rees for helpful discussions, and Drs T. J. Bach, B. Camara, R. Douce, L. J. Goad, K. H. Grumbach, H. Kleinig, H. Lichtenthaler, W. D . Nes, W. Rudiger, H. Rudney, D. W. Russell, G . Schultz and C. A. West for providing preprints and reprints.

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Dunaliellu: a Green Alga Adapted to Salt

MARGARET GINZBURG

Botany Department, Institute of Life Sciences, The Hebrew University of Jerusalem, Israel

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. . . . . . . . .

. . . . . . . . .

126 126 128 128 130 130 131 133 133

. . . .

134 134

. . . . . . .

. . . . . . .

137 139 140 140 141 143 143

.

.

.

143 146 148 148

.

149

.

149

Regulation of Cell Volume . . . . . . . . . . . . A . Immediate Effect of Change in Osmotic Pressure of Medium . . . . . . . . . . . . . . . . . B . Recovery of Original Cell Volume After Osmotic Shock . C. Measurements of Ion Concentrations (K'. Na+. Cl-) . . D . Can Osmoregulation be Accounted for Entirely in Terms of GlycerolSynthesisandDegradation? . . . . . . . E . Two-Compartment Hypothesis for the Regulation of Cell Volume . . . . . . . . . . . . . . .

153

Composition of Dunaliella Cells . . . . . . . . A . Major Organic Compounds . . . . . . . . B . Protein . . . . . . . . . . . . . . C. Soluble Carbohydrates . . . . . . . . . D . Starch . . . . . . . . . . . . . . . E . Relation of Glycerol and Starch Contents to NaCl . F. Lipids and Carotenoids . . . . . . . . . G . Nucleic Acids . . . . . . . . . . . . H . Conclusions . . . . . . . . . . . . .

Enzymes . . . . . . . . . . . . . . . . A . Enzymes Concerned with Glycerol Metabolism . . B . Enzymes Concerned with Nitrate Reduction (Nitrate Reductase. Nitrite Reductase) . . . . . . . . C. Photosynthetic Enzymes . . . . . . . . . D . Enzymes Metabolizing Starch . . . . . . . . E . Phosphofructokinase . . . . . . . . . . . F . Effect of NaCl on Dunaliella Enzymes . . . . . G . Effects of Glycerol on Dunaliella Enzymes . . . . H . Special Features of Dunaliella Enzymes . . . . .

. . . . . . . . .

Photosynthesis . . . . . . . . . . . . . . . A . Products of Photosynthesis . . . . . . . . . . B . EffectofLight IntensityonPhotosynthesis . . . . . C. Photosynthetic Electron Flow . . . . . . . . . D . Rate of Photosynthesis After Long Period of Adjustment to a Given NaCl Concentration . . . . . . . . E . Effect of Change of NaCl Concentration on Photosynthesis . . . . . . . . . . . . . .

Conclusions . . . Acknowledgements . References . . .

.

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

153 160 167 173 174 175 177 178

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

95

I. INTRODUCTION The first mention of a small algal flagellate growing profusely in salt marshes was made by the French scientist Dunal in the 1830s. He was interested in the red coloration and the smell of violets of certain salt marshes along the French Mediterranean coast. Other French scientists collected similar organisms from the coasts of North Africa during the late 19th century and gave them names which have not persisted. The organism responsible for the red colour was described by Teodoresco and is now named Dunafieffasafina (Dunal) (Teodoresco, 1905). It is a unicellular, photosynthetic green alga which has been placed in the division Chlorophyta. It has two equal flagella, one cup-shaped chloroplast, an anterior nucleus, an eye-spot and a pyrenoid surrounded by starch grains at the posterior end of the cell. There is no rigid cell wall. The specific characteristics of the genus are its morphology and physiology. The cell morphology is Chlamydomonad-like. Physiologically, the genus is distinguished by the ability to grow in media with high concentrations of salt. Six freshwater species of Dunafieffahave been described; all appear to be extremely rare and their inclusion within the genus is uncertain (Melkonian and Preisig, 1984). The purpose of this chapter is to summarize and analyse the often conflicting data on Dunafielfa gathered during the past 30 years, to compare and contrast Dunafieffawith other green algae, and to further the understanding of the adaptation of Dunaliella to high salt concentrations.

11. TAXONOMY Dunafieffahas been placed in the class Chlorophyceae (Stewart, 1974; Mattox and Stewart, 1984). According to an alternative scheme, Dunaliella should be included in a special class, the Chlamydomonaceae [Ettl (1981) but see discussion by Deason (1984)]. It is generally agreed that Dunalielfa is more closely related to Chfamydomonas than to any other green alga (Mattox and Stewart, 1984). The anatomical basis for this view is given in Section V.D. Teodoresco (1905, 1906) divided the genus into two species: 1. D.safina-cells possess “haematochrome”; cysts are red. 2. D . viridis-cells and cysts are always green. A thorough taxonomic study was made by Lerche (1937), who distinguished between two types of D . safina,f. magna and f. obfonga.D . viridis was found to be very variable and was accordingly split into four new species, differing mainly in cell size (Table I).

TABLE I List of Dunaliella species

Species

Reference

Dimensions" ( m)

Cell volumeb (lo-'' m3)

Location S. Europe; N. African wast S. Europe; N. African coast Salt lake, California

salina

Teodoresw (1905)

12-16x2425

2670

viridis

Teodoresco (1906)

6-9x 13-15

400

peircei

7x15

385

panta

Nicolai and Baas Becking (1935) Lerche (1937)

7x12

308

media euchlora

Lerche (1937) Lerche (1937)

4x 13 5x9

109 118

minuta tertiolecta

Lerche (1937) Butcher (1959)

3x9 6 x 12

42 225

Romania; California; Australia Romania English Channel

bioculata

Butcher (1959)

4-8x8-10

170

Prague

quartolecta

Butcher (1959)

4-6 x 7-9

105

Off Plymouth?

polymorpha

Butcher (1959)

4-45x 7-9

105

Saline pool on Thames estuary

"Transverseand longitudinal cell axes, a and b. Cell volume = 4/34$]' $ .

Romania; Carinthia; saline pools in salt marshes

Characteristics Cells green or red; cysts red Cells and cysts always green Leaf-shaped (probably not a Dunaliella) Small green cells; no. 1919 in Cambridge Culture Collection

4-20 largish central granules; no. 81 in

Plymouth collection no. 1914 in Cambridge Culture Collection Refractive granules in anterior part of cell Density of granules irregular

DLJNALIELLA: A GREEN ALGA ADAPTED TO SALT

97

Teodoresco and Lerche limited their studies to algae isolated from salt ponds or lakes. In contrast, Butcher (1959) emphasized that Dunuliellu grows in seawater as well as in salt ponds and he identified several new species of the D . viridis type in British coastal waters (see Table 11). Butcher's criteria were morphological and it is hard to know whether to accept them: a major problem facing the taxonomist with regard to DunalieNa is that morphological criteria are hard to apply to green algae. Firstly, different genera can be distinguished only under the electron microscope. Secondly, cells of a given Dunaliellu strain vary morphologically with conditions of growth, both in appearance and cell size. Finally, strains that are morphologically indistinguishable from each other may differ in average cell size (Ginzburg and Ginzburg, 1985~). Thus, it appears that physiological and biochemical criteria might be more useful to the taxonomist than the usual morphological ones. The only Dunaliellu species to have been defined by these newer criteria is D. salina: any Dunaliella that has the capability of turning red, with a carotenoid-to-chlorophyll ratio greater than 6:1 (Loeblich, 1982).

TABLE I1 Distribution of Dunaliella SDecies

Location ~~

Reference

~

S. Europe and N. Africa N. Africa Salt evaporating ponds in Mexico Pink Lake, Victoria, Australia Solar salt pond, Australia

Teodoresco (1905) Lerche (1937) Loeblich (1972)

Romania Dead Sea Solar evaporation pond, San Francisco Bay Great Salt Lake

Teodoresco (1906) Elazari-Volcani (1940) Johnson et al. (1968)

parva

Dead Sea

Ginzburg (1969)

minuta parva

Lake Eyre, Australia

Baas-Becking and Kaplan (1956)

Great Salt Lake Salt lake and solar ponds, Bhavnagar, India Wadi Natrun, Egypt. (374g salts I-') Syowa Oasis, Antarctica Zabuye Lake, Tibet

Van Auken and McNulty (1973) Rao et al. (1982)

salina

viridis

euchlora SP. SP.

SP. SD.

Hammer (1981) Curtain et al. (1983)

Stephens and Gillespie (1976)

Imhoff el al. (1979) Tominaga and Fukui (1981) Zheng et al. (1985)

M. GINZBURG

98

Masyuk (1973) includes taxonomic data. Unfortunately, the book is not available in translation. Three Dunaliella species mentioned in the literature have not been formally described, as far as is known to the present author. These are D . marina (Kombrink and Woeber, 1980), D . maritimu (Riisgard, 1979) and D. bardawil (Ben-Amotz et al., 1982a). These three species have not been included in Table 1.

111. DISTRIBUTION AND ECOLOGY Members of the genus Dunaliella have been identified in seas and hypersaline lakes all over the world (Table 11). It should be remembered that almost as much of the world's inland waters are saline as fresh (Wetzel, 1964). However, there are many hypersaline lakes which do not appear to contain Dunaliella (Table III), and even in those lakes in which it occurs numbers fluctuate throughout the year. There are, therefore, limitations on the growth of the alga. TABLE 111 Hypersaline lakes in which Dunaliella has not been recorded Location Five lakes in Wadi Natrun Twenty-six alkaline, saline lakes in E. Africa Hypersaline lake in French Somaliland Lakes in Saskatchewan Saline lakes in Antarctic

Total salts (g I-')

Reference

92-394 47

Imhoff et al. (1979) Melack and Kilham (1974)

>350

Brisou et al. (1974)

41-47

Haynes and Hammer (1978) Hand and Burton (1981)

Conductivity = 15,000-30,000 ps.

For references on other saline lakes see Haynes and Hammer (1978).

The ecology of Dunaliellu has been studied in the Dead Sea (Kaplan and Friedman, 1970; Oren, 1981; Oren and Shiloh, 1982), in the Great Salt Lake (Brock, 1975; Stephens and Gillespie, 1976) and in Pink Lake, Victoria, S. Australia (Hammer, 1981b). Oren and Shiloh (1982) did not detect Dunaliella cells in the many water samples of the Dead Sea analysed between March and May 1980. There was then a rapid onset of growth and in June and July they found 7000-8000 cells ml-', restricted to the upper 5 m. There was only one species, D . parva. As the year progressed, the Dunaliella population declined rapidly and algae were found at greater depths (15-20 m), until in February 1981 few or none were found at any depth. Simultaneously with

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

99

cell numbers, Oren and Shiloh (1982) measured water density and temperature. Large amounts of fresh water flooded into the lake from December to March, probably bringing nutrients and certainly diluting the Dead Sea waters. These two conditions are essential for the growth of Dunaliella; rapid growth did not commence until the surface waters warmed up in the spring. The decline in numbers which occurred in August was attributed to the high temperature (36°C at the surface) and the re-establishment of high salinity in the surface water due to evaporation. Also, the waters were sufficiently turbulent to carry cells down to depths at which photosynthesis and hence growth was impossible because of the low light intensity. In this way, the surface waters steadily lost their population of growing cells. The conclusions were supported by laboratory experiments which showed that dilution of Dead Sea water and addition of phosphate were needed to bring about the growth of Dunaliella. Work on growth of Dunaliella in the Great Salt Lake is equally interesting. The body of water was divided into northern and southern basins by a causeway completed in 1957. The southern basin receives most of the surface inflow and is less saline than the northern basin [12.2-20% dissolved salts in the southern basin as compared with 29.6-34.9% in the northern, according to Brock (1975)]. The northern arm contains a reddish strain of Dunaliella and the southern a green one. Both are referred to as “ D . viridis” by Stephens and Gillespie (1976), who studied population dynamics in the southern arm and made measurements in January-July 1971 and April-November 1973. In the winter months the number of cells could not have been above lo00 cell ml-’; growth set in quickly in the spring and a maximum of 250,000 cell ml-’ was found in April-May. The onset of rapid growth was attributed to increases in light intensity and in temperature of the water; this latter was 1-9°C in winter and 10-17°C in April-May. There was a subsequent decline to levels barely above the limit of detection. A second flagellate appeared during the summer and was responsible for a minor peak in production during August and October. It is not clear whether the second species is a Chlamydomonm or a Dunaliella with smaller cells than the former species. Stephens and Gillespie (1976) were interested in the factors causing the decline in numbers in the early summer. They considered whether nutrients, in particular P and N, might be lacking, and they measured the concentrations of these substances in lake water, comparing them with optimum concentrations determined in the laboratory. P was found to be present in adequate amounts. Total N was very low indeed; when growth was measured in samples to which N or P had been added, stimulation of growth was found in those samples containing added N. Thus, the N content of the lake water must have been so low that growth was limited by lack of N. Addition of 10mgI-’ of C in the form of NaHC03 did not stimulate growth, probably because the amount added was too small

100

M. GINZBURG

compared with the usual lOOmgI-’ of NaHC03 needed to stimulate growth in the laboratory. The Great Salt Lake contains large populations of the brine shrimp Artemia salina, which feeds on Dunaliella. Stephens and Gillespie (1976) concluded that grazing by Artemia cannot account for the annually observed rapid decline in Dunaliella. They concluded that “light limitation imposed by the mass of algal cells may be the initial factor limiting production” and that lack of N is the next factor inhibiting growth and ultimately leading to decline in numbers. Hammer (1981b) studied four saline lakes in Victoria, S . Australia, in 1969-1970. A Dunaliella species was found in Pink Lake (salt content: - 3 NaCl), ~ the most saline of the four. There were two periods of growth, stretching from October to January and April to July. This latter is associated with reduction in salinity by rain. No explanation is given for the other flush of growth. The protozoan Paratemia zietziana Sayce preyed on Dunaliella and limited its numbers. However, the main limiting factor is the low nutrient concentration: nitrate could not be detected and P was present in trace amounts. The other lakes varied in salinity from 6 to 2 0 0 m ~and contained several diatoms, green and blue-green algae and Pyrrophyta, but no Dunaliella. Salinity may have been too low, though some strains do grow in 200 mM NaC1. The most likely explanation for the absence of Dunaliella from these less saline lakes is that it loses when in competition with other species; reasons for this are given in Section 1V.N. The lakes in which the growth of Dunaliella has been studied (Great Salt Lake, Dead Sea, Pink Lake) are all characterized by very low primary production (Hammer, 1981a). Rates vary as follows: Dead Sea Pink Lake Great Salt Lake

-4 g C m-2 year-’ 25 g C m-2 year-’ 200 g C m-2 year-’

Other saline lakes have primary production rates ranging up to 2000 g C m-2 year-’. The factors limiting production in the three lakes where Dunaliella is found are firstly, salinity (in each case dilution by rainwater was needed to induce growth), secondly, shortage of nutrients and especially P in the Dead Sea and N in the Great Salt Lake, and thirdly, temperature (low in the Great Salt Lake and Dead Sea in winter and too high in summer). Where Dunaliella is found, it occurs as a pure stand accompanied by halophilic bacteria but by no other alga. It appears that if there are enough nutrients and a low enough salinity to support other algae, it is these that develop by competing successfully against Dunaliella. The latter can maintain itself only in places where the conditions are too severe for other algae, and even here it develops only at certain times of year when the hypersaline waters are diluted by rainwater. Another essential condition is

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

101

suitability of temperature; this condition rules out the saline Antarctic and tropical lakes. The advantage that Dunaliella has over its algal competitors is its capacity to withstand long periods of drought in encysted form; during these times the cysts may be mixed with salt in solid crystalline form.

IV. GROWTH CONDITIONS The growth of Dunaliella has been studied by two groups of workers. Oceanographers have studied marine species, especially D. tertiolecta, as components of the phytoplankton and have made comparisons with other algae. On the whole, their media have consisted of seawater enriched with small amounts of N and P, and they have grown their algae in dilute suspensions (
Many workers have used buffered or unbuffered seawater to which trace elements are added (e.g. Guillard and Ryther, 1962). The compositions of several artificial media are shown in Table IV. The media contain Na+, K+, Mg2+ and Ca2+ as major cations and C1-, SO4*-, NOll- and phosphate as major anions. There are micromole amounts of Fe2+,Mn2+, Cu, Co, Zn and sometimes H3B03, Mo and V. A few workers have added vitamins. The table shows that the concentrations at which the elements have been used are very variable, even when the species of Dunaliella used has been the same. The pH of the medium has varied from 5.5 (Van Auken and McNulty, 1973) to 8 (Loeblich, 1972). It must be concluded that the Dunaliella strains studied do not have strict nutritional requirements. Some of the major variables which have been studied separately are discussed below. B. TOTAL SALINITY

Gibor (1956) concentrated seawater up to 10 times and compared effects of total salinity on relative growth, i.e. total cell counts after 16 days. He

TABLE IV Art$cial growth media for Dunaliella

(1) 410 1

44

44

10 1 0.1 24

10 1 0.1 24 65

460

100 1.5 185 7 0.8 0.02 0.0002

PH

Other substances Light (WEinstein mP2s-') S&cies

(2) 15 1

1.5 185 7 0.8 0.02 0.0002

(3) 50 1 4.75 1.25 1

0.1 2 52.5 1.5 185 7

0.8 0.02 0.0002

unstated

unstated

unstated

unstated

unstated

unstated

68 tertiolecta

68

(2) McLachlan (1960); medium B. (3) McLachlan (1960); medium C. (4) Loeblich (1972).

5 0 42 r n ~ NaHC03 8

68

tertiolecta ~~

(1) McLachlan (1960); medium A.

(4) 860-4300 11.9 9.4 1.4 9 0.2 2 860-4300 350 9 45 9 0.15 0.17 0.16 0.10

tertiolecta ~

~~

(5) Wegmann and Metzner (1971). (6) Van Auken and McNulty (1973). (7) Abdullaev and Semenenko (1974).

(5) 1034 20

40 11.6 2.35 0.15 52 1054

2% coz

(6) 3000 2.3 2.3 2.8 2.8 2.3 2.3 3000

(7) 2000 54 202

100 46 9 0.8 0.2 0.3 1

30

W.4%

c02

5.5

-

45 9 202 2000

46 9 0.1 1 0.1 1.7% coz 7.5

420

6 soil extract 156

105

sunlight

salina

tertiolecta

SL?.

salina

103

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

found three different strains of D. viridis to have tolerances varying from 1/2 strength to x 3 seawater. One strain tolerated x10 seawater after training. D. safina grew best in X3 seawater and slowly in x10 seawater. Gibor also studied the growth of three other unicellular organisms, Stichococcus sp., Pfatymonas sp. and Stephanotera gracifis, which grew optimally in x 3 seawater and quite well in ~6 seawater. Dunafieffais not, therefore, unique in its capability to withstand saline solutions. McLachlan (1960) found that D. tertiofecta grew at a rate of 1 division per day in 1/4 to X 1 medium A; growth fell off outside this range. C. EFFECT OF NaCI CONCENTRATION ON GROWTH

McLachlan (1960) and Hellebust (1985) have found NaCI, in low concentrations, to be essential for growth. Effects of NaCl concentration may be expressed in terms of either growth rate (number of cell divisions per day) or yield (final number of cells per ml or weight of organic matter per day per area of exposed surface). The major findings are listed in Table V, which shows Dunafieffa species arranged roughly in order of tolerance to salt, with the most sensitive at the top. The first conclusion to be drawn is that there is an extraordinary range of variation in salt tolerance. This variation is also demonstrated in Fig. 1.

u.jn*slru -

14

A12

n I

1

1

1

1

1

1

1

1 2 3 4 1 2 3 4

I

1

I

1

I

1

1

1

1

1

1

1

I

I

I

I

I

-

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

[NaCI],,,

(MI

.,

Fig. 1. A: Yield (expressed in Klett Units) as function of [NaCI],,, at 26 and 36°C in six Dunaliella isolates. Measurements were made 5 days after inoculation of culture. 26°C; 0, 36°C. 100 Klett Units=2SOpg organic dry weight (ml suspension)-'. B: Doubling time, in hours, of six Dunaliella isolates as a function of [NaCI],,,. 0 ,26°C; 0,36°C. From Ginzburg and Ginzburg (198Sa). By permission of The British Phycological Society.

TABLE V Effects of NaCl concentration on growth of Dunaliella

Species tertiolecta

C source

Light (pEinsteins m-2 s-l)

C 0 2in air

68

3% c02

Temperature (“C) 18

150

Divisions day-’

NaCl (mM) 12-1400 2000 0-2600 1300 250-1250 2000

0.7 No growth 3

150

26

C 0 2in air

313

32-34

2000

2 Very Little growth 1

NaHC03

313 80

32 32

500-2000 2000

1 0.7

parva

2% c02 2% c02

333 333

26 36

2000 2000

1 2

parva

1.5% c02

18 25-33

750-3000 750 3000 300-750 1500-3000

29 29 29 29 2%39

24-42 30 30

Reference (1) (2)

maximum

3% c02

parva

Yield

0.4-0.6 2

2 2 2

(3)

5 g/m-2 day-’

(4)

NaHC03

salina

420

860

1

1700-2600 34004300

1.2 0.2

26

2000 4000

Some growth

25

Max.

(7)

(3x106

celldml)

viridis

1

(3)

salina

NaHC03

90

25-35

1000-2000 4000

2 Very little growth

(8)

bardawil

NaHC03

90

2>38 25-30

1000-2000 4000

2 Some growth

(8)

(1) (2) (3) (4) (5) (6) (7) (8)

McLachlan (1960). Frank and Wegmann (1974). Borowitzka and Brown (1974). Ginzburg and Ginzburg (1981). Ginzburg and Ginzburg (1985b). Gimmler et al. (1981). Loeblich (1972). Ben-Amotz n ol. (1982a).

106

M. GINZBURG

Each isolate appears to have its own range of tolerance which may be modified by “training” (Gibor, 1956; Borowitzka and Brown, 1974). The most sensitive species grow in NaCl concentrations ranging from 12 to 2000 mM (e.g. D . ferfiolecfu),while less sensitive strain.s may grow, albeit slowly, in 4000mM. It should be noted that this is some way below the saturation point of NaCl (5400 mM). A few Dunuliella strains have been found to have an increased tolerance to NaCl when their supply of C has been enriched. Thus, D . tertiolecfu grew in 2 6 0 0 m ~NaCl when air containing 3% COz (vh) was bubbled through the cultures. This degree of tolerance is not to be expected in natural habitats exposed to the atmosphere (COz content of air is 0.03%). Effects of temperature, light and NaCl on growth are interconnected; thus Fig. 2 shows that 40°C was tolerated by D . parva cells in 3 0 0 0 m ~

30

1” optimum

3~ NaCl

NaCl

-/

(N)

I 10

@ ,‘

.

.

I

20 Temperature (“C)

Growth of D.Darva cells adaDted to different salinities as influenced by growth temperature. Insects show dependency of optimal and maximal temperatures on salinity (upper inset) and temperature range at which growth can occur as function of salinity (lower inset). Redrawn from Gimmler et al. (1981).

NaC1, though not in 750 mM. However, strains may vary, since D. burduwil grew only between 25 and 30°C in 4000 mM NaCl, while it tolerated temperatures of up to 38°C in 1000-2000m~NaCl (Table V). High light intensities were tolerated by cells in 300 mM NaCl, though not in 750 mM (Fig. 3). In general, the higher the light intensity, the higher the NaCl concentration in which growth is possible.

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

107

Light intensity (klux) Fig. 3. Influence of light intensity on growth of synchronized D . parva cells adapted to salinities of 0.3 ( A ) , 0.75 (O), 1.5 ( 0 )and 3.0 (8)M NaCI. Insets show (A) optimal light intensity, in klux, and (B) maximal growth rate, in cell divisions day-', as functions of salinity. Redrawn from Gimmler etal. (1981).

The marked effects of COz and light intensity on the degree of tolerance to salt suggest a direct involvement of photosynthesis. In addition, the existence of a separate set of reactions is indicated by effects of temperature on salt tolerance. D. EFFECTOFK'

McLachlan (1960) found that the growth rate of D. fertiolecfu was increased by addition of 50 mM K+ to his three media (Table IV). Further increases to medium C caused inhibition, while it took the addition of 150 mM K+ to medium A to cause inhibition. In this latter medium, nearly maximal growth was obtained with 30 p~ K+. McLachlan concluded that the specificities for Na+ and K+ are very strict and that a definite ratio must be maintained between the two. E. EFFECTOFLi'

All concentrations are inhibitory to D. ferfiolecfu(McLachlan, 1960). Fujii et al. (1983) were unable to grow any one of seven Dunulieflu strains in 0.5 M LiCI.

108

M. GINZBURG F. EFFECT OF sof-

McLachlan (1960) found 150 p~ to be needed for D . ferfiolecfu;SO,"could replace C1- without inhibiting growth. Ginzburg (unpublished) found no growth of D . parvu below 5 mM S042-. G . EFFECT OFCaZ+

McLachlan (1960) found that D . tertiolecfu in medium A without Ca2+ grew at the rate of 0.7 divisions day-'; addition of 10 mM Ca2+ improved the rate to 1 division day-'. Effects were greater when Ca2' was added to the B and C media in which the Na+ concentrations were lower. Thus, Ca2+ and Na+ appear to replace each other. Ginzburg (unpublished) did not find addition of Ca2+ to have any appreciable effect on growth in Dunuliellu. It should be noted that the element is present as an impurity in the chemicals used to make up the medium, so that traces are always present even when no specific addition has been made. H. EFFECT OF Mg2+

Ginzburg (unpublished) has not managed to grow any strain of Dunuliellu in the absence of Mg2+. McLachlan (1960), by adding 40 mM MgCI2 to his medium C, increased the growth rate of D . ferfiolecfufrom 0.2 to 0.8 divisions day-'. Fujii ef al. (1983) found D . ferfiolectuto grow as well in 0.84 M MgS04 as in 0.5 M NaCl, provided that the algae were given 6% C 0 2 in air. In absence of this highly unphysiological concentration, the cells in MgS04 did not grow. D . primolecfu also grew in MgS04, while five other strains did not. 0.84 M MgS04 could not be replaced by 0.36 M MgC12. Incidentally, McLachlan (1960) concluded that Mg2+ was the most toxic of all the ions in the medium. I. EFFECTOFP

Gibor (1956) found concentrations of KH2P04 above 0.3 and 0.15 mM to be inhibitory to D . salina and to D . viridh respectively. This has not been the experience of Ginzburg, who found that yield, though not growth rate, was stimulated by progressive additions of phosphate up to 5 m ~ the , highest concentration tried. In general, the amount of phosphate that can be added to a liquid medium is limited by its solubility, especially when Mg2+ and Ca2+ concentrations are high. Thomas (1964) was unable to grow D. primolecfuin the laboratory if the medium contained less than 620 pg P I-' (0.3 p ~ ) .

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

109

J. TRACE ELEMENTS

Sandmann (1985) has determined that lop6 M CuS04 is helpful for growth, respiration and photosynthesis in D . parva Lerche. In the absence of Cu, plastocyanin becomes limiting and photosystem I is affected. This is because in Dunaliella, as in a few other algae, plastocyanin, which contains Cu, is essential; these algae cannot make use of cytochrome css3 for photosystem I.

K . EFFECTOFN

Gibor (1956) found that D . salina and D . vzridis utilized both NH4+ and NO3- as sources of inorganic N. In addition, D . salina was able to utilize uric acid, glutamic acid, urea and asparagine, but these sources of organic N were not used by D. viridis. A serious disadvantage of NH&l as source of N for Dunaliella is that its absorption is accompanied by output of H + ; the suspension becomes extremely acidic and the cells die unless buffer is added. Brewer and Goldman (1976) showed that there was a 1:l ratio between NH4+ uptake and H + output, while in cultures supplied with NO3- a 1:l ratio is maintained between NO3- taken up and O H - produced. This is true at all rates of growth. Bienfang (1975) compared the uptake of NO3- and NH4' in D. tertiolecta growing at high light intensity (0.097 calories cm-2 min-'=310 pEinstein mP2s-l). In most phytoplankton assimilating NO3- and NH4+ concomitantly, the assimilation of NO3- may be suppressed. In D . tertiolecta, on the other hand, there was no effect of NH4+ on NO3- uptake. The ratio of NO3- to NH4+ had no effect on growth rate. In contrast to NO3-, NH4+ was not stored, and the higher the proportion of NH4+ nitrogen, the lower the N content per cell, and also the lower the final cell density reached. Much of the above was confirmed and amplified by Goldman and Peavey (1979), who grew D . tertiolecta on limiting amounts of N supplied in the form of NO3-, NO2-, NH4+, or urea. They compared growth rates (measured as increase in cell number) in continuous culture as well as in batch culture. A maximum growth rate of 1.33 divisions day-' ( p , specific growth rate) was established for both types of culture. The source of N had no effect on growth rate. Residual N was virtually indetectable, representing extremely low half-saturation constants for growth. Thus there was a very strong and virtually identical dependence of growth rate on each of the N sources. The authors agreed that the situation may well be different when the supply of N is abundant. It is then that NH4+ is preferentially assimilated.

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It should be pointed out that in nature available N is rarely in abundant supply, and thus phytoplankton have adapted to exploit effectively different potentially available N sources in natural waters without appreciable differences in growth rate. These conclusions may need modifying when effects of light intensity are taken into account. Thus, Grant (1967,1968) and Grant and Turner (1969) found that the uptake of NO3- was 15 times faster in the light than in the dark. The uptake of NO3- and NO2- was stimulated by increasing the C02 content of the atmopshere or by providing NaHC03. In many algal species absorbing NO3- in the light, there is a stoichiometric relation between NOu3- absorbed and O2 evolved; this is not true of Dunaliella. Grant and Turner (1969) concluded that there may be two systems for nitrate reduction, one in the chloroplast and one outside. Grant (1970) and Leclaire and Grant (1972) have isolated a nitrate reductase and described its properties (see Section VI1.B). The effect of light on uptake of NO3- and NH4+ has also been studied by Sharfstein (1976). He found that the growth rate of D. tertiolecta was stimulated by NH4+ rather than by NO3-, the degree of stimulation depending on the light intensity to which the algae were subjected: the lower the intensity, the greater the difference between the growth in NO3and in NH4+. It was concluded that the light-dependent processes controlling NO3- uptake are saturated at half the light intensity of saturation of photosynthesis.

L. EFFECT OF C SOURCE

I . Dissolved Inorganic Carbon (DZC) This consists of C032-, H2C03, HC03- and C02. The relation between these compounds depends on the pH and salinity of the medium: C 0 2 + H 2 0e H + + H C 0 3 - =2H++C032- *H2C03 Above pH 8 most DIC is present in the form of carbonate. Bicarbonate predominates at pH values between 7 and 8. Below pH 7 dissolved C 0 2 is predominant. A physicochemical description can be found in Kerby and Raven (1985). There is still considerable controversy with respect to the chemical C species that crosses the plasma membrane of aquatic plants, as well as to the mechanisms used for uptake of C. The subject has been reviewed by Lucas and Berry (1985) and Kerby and Raven (1985). In Dunaliella, when a culture is transferred from high (5%) to low (0.03%) C 0 2 the rate of photosynthesis is at first strongly depressed, but adapts within some holm in such a way as to permit the cells to re-establish a high rate of photosy.1thesis in the presence of the low C 0 2 . The same

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

111

phenomenon is observed in some other algae. In the pioneering studies of Osterlind (1951) on low-C02-associated induction of Scenedesmus, it was concluded that “the factor to be activated is either connected to the adsorption of bicarbonate ions, or is carbonic anhydrase”. Both are now known to be applicable to Dunaliella. When Dunaliella salina cells grown at low C 0 2 are supplied with H14C03-, internal inorganic C accumulates to a level 10 times that outside (Zenvirth and Kaplan, 1981). Accumulation of inorganic C is reduced by metabolic inhibitors. The observed accumulation could be caused by facilitated diffusion. But if this were so, the membrane potential would have to be (inside) positive. As is well known, in plant cells all measured potentials across the cell membrane have been found t o be (inside) negative. It is reasonable to assume that such is the case in Dunaliella; if so, HC03- must be transported actively. Zenvirth and Kaplan (1981) found considerable amounts of carbonic anhydrase in crude cell extracts of D.salina grown at low concentrations of C02. They ascribed the uptake of C 0 2 to the activity of the enzyme. The DIC-accumulating system has enabled microalgae to develop a high photosynthetic affinity for COz, such that the C02-compensation point is very low. Lloyd et al. (1977) found compensation points of x0.15 mM for D. tertiolecta, Thallassiosira pseudonana and a Porphyridium species. In Dunaliella, carbonic anhydrase is produced in response not only to low C 0 2 concentrations in the medium, but also to increased salinity. It is evident, however, that an increase in salinity has the effect of reducing the concentration of C 0 2 . The importance of carbonic anhydrase to D . salina was demonstrated by Loeblich (1972) in experiments in which growth was measured with or without Diamox, an inhibitor of the enzyme, at p H values ranging from 5.8 to 9.3. In the presence of Diamox, growth was inhibited only at those pH values at which one expects carbonic anhydrase to be active, namely from pH 7.5 to 8.5. Latorella and Vadas (1973) found that the production of carbonic anhydrase by D.tertiolecta increased with the NaCl concentration of the growth medium. More than twice as much enzyme was produced by cells grown in 3.5 M as in those in 0.5 M NaCl (the solubility of C 0 2 in 3 M NaCl is one-half of its value at 0.5 M NaCI). These authors suggested that carbonic anhydrase encourages the formation of HC03-, which they postulated to be part of a Na+/I-I+ exchange pump. Aizawa and Miyachi (1984) measured 43.6k8.1 units carbonic anhydrase (mg chlorophyll)-’ in low-C02-grown cells. In contrast, cells grown with high C 0 2 had 5-7 units of enzyme (Aizawa et al., 1985). Both groups of authors found 90% of the enzyme to be active in intact cells, demonstrating that it is present on the cell surface. They supported this view by suppressing enzyme activity with 2% subtilisin, a protein molecule unlikely to cross the cell membrane.

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2. Organic Carbon Several attempts have been made to see whether Dunuliellu cells take up organic forms of C (Kwon and Grant, 1971; Ukeles and Rose, 1976; Couglan, 1977). Kwon and Grant (1971) tested for uptake of 5 mM glucose in D. tertiolectu in light and in dark. Cells in the dark supplied with glucose had not divided by the end of 3 days; the respiration of these cells fell to 10% of the contro1,value. The authors concluded that glucose is not taken up by whole cells of D . tertiolectu. The same conclusion was drawn for D. primolecta and D. euchforu by Ukeles and Rose (1976). However, suspensions of broken D. tertiolecta cells were found to metabolize glucose quite actively (Kwon and Grant, 1971); these broken cells were found to convert glucose to 3-phosphoglyceric acid, 3-phosphoglyceraldehyde and dihydroxyacetone phosphate when supplied with hexokinase and NADP. These results are consistent with the operation of glycolysis and the hexose-monophosphate shunt. Kwon and Grant (1971) were unable to stimulate the endogenous respiration of whole D. tertiolectu cells by treatment with a variety of organic substances. Ukeles and Rose (1976) tested D . primolecta and D. euchloru to see if they grew when supplied with pyruvate, succinate, sucrose, glycerol, alanine or a-ketoglutarate. Only acetate, pyruvate and glycerol penetrated the cells at rates that might affect growth; the yield of cells after 12 days was stimulated slightly by addition of glycerol to the medium. Coughlan (1977) was unable to detect any effect on growth of glycollate, acetate or glucose, though it is possible that the concentrations he used were too low to be effective. Coughlan is the only worker known to have studied the effect of long periods of dark on Dunalieflu: in the dark D . primolectu cells were found to become nonmotile, withdraw their flagella, and form a thick mucilaginous sheath. Cells remained viable for 6 weeks at 20°C and for 10 weeks at 5°C. These times were unchanged when organic substances were added to the medium. It is concluded that most organic C compounds are not absorbed either in the light or in the dark by Dunaliella cells, though acetate, pyruvate and glycerol may be taken up slowly. M. EFFECTS OF LIGHT

The effects of light intensity on doubling time of three Dunafiellu isolates are shown in Fig. 4. Units of illumination have been converted to photon flux density according to the formulae of Table VI. There is a clear effect of light intensity on rate of division: 100-200 pEinstein m-2s-1 were needed for maximum growth. This light intensity is higher than usual for microalgae (Fig. 5 ) . Very high light intensities have not been shown to inhibit growth of Dunufielfagrowing under otherwise optimal conditions. Effects of light-dark cycles on growth have been studied by Eppley and

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

I

0

I

200

1

I

113

I

400 600 800 Photon flux density (pEinsteins m-2 s-’)

Fig. 4. Effect of photon flux density on rate of division of Dunaliella. 0: D . salina in 2.56 M NaCl at 25°C; C source 0.48 mM NaHCO,; pH 8 (Loeblich, 1972). 0: isolate from Great Salt Lake in 3.2 M NaCl at 30°C; C source 2% CO,; pH 5.5; 20-4 L-D cycle (Van Auken and McNulty, 1973). U and 0: D. rerriolecta in seawater at 20-21°C. Redrawn from Eppley ei al. (1969) and Eppley and Sloan (1966).

Coatsworth (1966), Wegmann and Metzner (1971), Whitney (1973), Sharfstein (1976), Bruggemann et al. (1978), Nelson and Brand (1979) and Marano et al. (1978). There is an additive effect of light on growth. This was described by Eppley and Coatsworth (1966), who found daily cell production to be proportional to the product of intensity and time, when intensity was equal to or less than 224pEm-’s-’ (0.07 calories cm-2 min-’) with a total daily irradiance equal to or less than 70 calories cm-’ day-’. The product law holds at low daily irradiance provided either by changes in intensity or length of photoperiod. A linear relation between total light energy and number of divisions day-’ was also found by Sharfstein (1976). Whitney (1973) using two low light intensities (4 and 16 p E m-’s-’) and three day lengths found the relationship to be parabolic.

114

M.GINZBURG TABLE VI Equivalencies of units of illumination

Name

Value for full sunlight

Unit

Solar irradiance

1W m-’= 1 . 4 3 3 ~ calories cm-2 m in-’= 1oOO erg cm-’s-*

1000 W m-’ when using pyranometer and pyrheliometer sensors. 500 W m-2 when using photosynthetic irradiance sensor. 60 W m-*when using nearinfrared sensor.

Photosynthetic photon flux density

1 pEinstein m-’s-*= 1pmol m-2 s-I= 6 . 0 2 ~1017quanta m-2 s-’

2000 pE m-2 s-’ for 400700 nm waveband

Illumination

1 lux= 1lumen m-*= 0.0929 foot-candles

100 Klux

Conversion of units approx for 400-700 nm) 1 WEinstein = 51.2 lux = 0.218 W m-2 ~

~~

From Incoll er al. (1977); McCree (1972); Shibles (1976); and the Instruction Manual for the LiCor Integrating Photometer.

D. brighfwe/fii

C.huxleyi D.ferfiolecfo

I

I

I

0.15 lrradiance ( ly min-’1

Fig. 5. Specific growth rate as a function of irradiance for Coccoliihus huxleyi (Paasche, 1967). Skeletonerna costaturn (Jitts et al., 1964), Ditylum brightwelli (Paasche, 1966) and Dunaliellu terriolecta (Eppley and Coatsworth, 1966). All at 20-21°C. The langley (ly) is 1 calorie cm-*. From Eppley el al. (1969).

A second effect of light-dark cycles is that cell division becomes synchronized. While Sharfstein’s (1976) cells divided in the light, those of all the other groups divided in the dark. The difference may be due to the

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115

very low light intensity used by Sharfstein. Wegmann and Metzner (1971) imposed a temperature cycle in addition to the lightdark one, thus taking advantage of the fact that the optimum of photosynthesis was found to be 40°C while that of cell division was 20°C; 12 h of light and dark were accompanied by 12 h at 40°C and 12 h at 20°C. The cells divided throughout the dark phase. Bruggemann et al. (1978) grew D.parva which they synchronized by 14:lO 1ight:dark cycles at 30°C. A synchronization index of 0.82 was achieved, which was said to be as good as that of the other workers mentioned and comparable with that obtained for other flagellates. Marano et al. (1978) measured time of synthesis of the major macromolecules. They grew D.bioculata on a 8:lO 1ight:dark cycle. Cell division was restricted to the first part of the dark cycle. RNA and protein were synthesized in the light. Synthesis of DNA started 4 h before the start of the dark phase and continued for approximately 8 h . Their discussion includes a detailed comparison of division in Dunaliella and other Protista. There are small effects of quality of light on growth of Dunaliella (Vesk and Jeffrey, 1977; Jones and Galloway, 1979). In general, there is a very slight promotion of growth in blue as opposed to red light. Ruyters et al. (1984) have found respiration and carbohydrate breakdown to be faster in very dim light than in the dark. The effect is greatest at 450 nm. N. EFFECT OF TEMPERATURE

Temperature optima for the growth of several Dunaliella isolates are shown in Table VII. This shows that the optimum for division of small TABLE VII Temperature optima for cell division in Dunaliella

Species tertiolecta tertiolecta tertiolecta salina ’ ‘salina’’ bardawil SP.

(1) (2) (3) (4) (5)

Temperature optimum Reference [NaCl] (mM) Process measured (“C) lo00 divisions day-’ 21 loo0 photosynthesis 40 seawater cell counts in continuous 20 culture 28-32 doubling time 1000-2000 doubling time 25-30 25-38 1000-2000 doubling time doubline time 30-35 3600

Wegmann and Metzner (1971). Goldman (1977). Abdullaev and Semenenko (1974) Ben-Amotz et al. (1982a). Van Auken and McNulty (1973).

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M. GINZBURG

green-celled species is 20-25°C (Goldman, 1977; Wegmann and Metzner, 1971). Dunuliellu species with large red cells, e.g. D . sulinu and D . barduwil, have optima at 30°C or above. There may be a real difference in response to temperature between the two groups. In interpreting Table VII two factors should be borne in mind, firstly that the optima for cell division and photosynthesis are not the same in Dunuliellu, and secondly that the optimum is affected by the NaCl concentration of the medium and increases with it (see Section 1V.C). Goldman (1977) studied the effects of temperature on five marine planktonic species, including D . tertiolectu. He measured the rate of cell division together with particulate C and N in monocultures diluted at a constant rate of 0.6 day-' and kept under constant illumination (96 p E m-2 s-') on enriched media consisting of 50% secondarily treated waste water and 50% seawater. Four species including D . ferfiolectu, reached maximum cell numbers at 20"C, implying that the rate of cell division is fastest at 20°C. At 30°C only D . tertiolectu and Thulussiosiru pseudonunu still divided at a significant rate. Thus, the Dunuliellu species had a tolerance to high temperature unusual in these phytoplankton. The temperature of minimum P and N per cell was 20°C for D. tertiolectu and P. tricornufum. For a given growth rate, the production of biomass is fairly independent of temperature over a broad range. Thus, there is an uncoupling of cell division and biomass production in D. terfiolecfu and P. tricornufum. The same has been found for Chlorellu, a freshwater species (Williams, 1971). In the other two species studied by Goldman (1977) there was tight coupling between division and nutrient uptake. The minimum temperature of growth for D. tertiolectu is 9°C (Ukeles, 1961), rather higher than is usual for a marine alga. Effects of alterations of temperature have been studied by Lynch and Thompson (1982) on D. salinu. Division times of 20 and 80 h were found at 30" and 12°C respectively. When logarithmicallygrowing cells at 30°C were transferred to 12"C, growth stopped for 96h, after which it resumed with a division time of 80 h. While growth was arrested, changes occurred in the composition of the membranes. The cell volume was nearly three times larger at the lower temperature (976f89 pm3 and 2775+208 pm3 at 30" and 12°C respectively. (See also Section V1.F.) 0. EFFECTS OF INTERACTIONS BETWEEN PARAMETERS ON GROWTH

I . Light and Temperature Eppley and Sloan (1966) constructed a formula relating growth to light intensity and temperature: k=

(10'~036r-0~28)k~(l-10-100(ch'a))(12.6~OS0. 18) 0.015+10(1- 10-"0"h'a')(12.6Zo+0. 18)

DVNALIELLA: A GREEN ALGA ADAPTED TO SALT

117

where k = number of cell divisions per day t = temperature ("C) I, = incident light intensity in calories cm-2 min-' chla = pg chlorophyll a pmF3 cell volume This formula was found to apply to several phytoplankton, including D . tertiolectu. Chlorophyll a was introduced into the formula because the pigment is reduced in amount as light intensity rises. A slight modification enables effects of photoperiod to be included (Eppley and Coatsworth, 1966). The equation applied equally well to all the photoperiods tried. The formula indicates that for a given temperature, growth is proportional to amount of light received, whether as intensity or duration. Temperature and light are independent variables; thus, strictly speaking, there is no interaction between the two.

2. Temperature and Nutrient Supply Thomas and Dodson (1974) studied the effects of interactions between temperature and nitrate supply on the rate of cell division of Gymnodinium splendens and a Dunaliella species. Both were grown in enriched 75% seawater. The major results, summarized in Table VIII, were felt to show that cells growing at a higher temperature had higher requirements for nutrients. This may be due to the general increase of all enzymatic reactions with increasing temperature, rather than to a specific effect on growth rate. Here again, Dunaliella apparently behaves like other phytoplanktonic species. TABLE VIII Growth of two algal species on 75% enriched seawater Organism

Temperature ("C)

Duanaliella sp.

15 25

Gymnodinium svlendens

15 25

Nitrate K, (PM) 0.28 f0.68 0.95 f0.72 1.02 k 0.99 6.55 f 0.85

Growth rate (PlIlax)

1.93k 0.26 1.85 & 0.24 0.48 4 0.06 0.83 & 0.42

Source of N : N 0 3 - . From Thomas and Dodson (1974). K,: NO3- concentration at which growth rate is half-maximal; pmax: divisions per day when NO3- is unlimiting.

P. SUMMARY

In contrasting the conditions under which Dunaliella cells grow with those of other phytoflagellates, it has been seen that the most outstanding characteristic of Dunaliella is its tolerance to salt. A few other phytoflagellates are also salt tolerant (see Section 1V.B) but have been less studied.

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The nutritional requirements of Dunaliella cells do not appear to be different from those of other algae. Comparison is difficult, however, because data are lacking. It would, for instance, be interesting to know the requirements of Chlamydomonas for K+, Mg2+ and Ca2+ in order to compare them with those of Dunaliella. The N, P and DIC relations of many phytoplanktonic organisms, including Dunaliella, seem to be similar. Dunaliella is marked by its ability to produce large amounts of carbonic anhydrase, the amount increasing with the NaCl concentration of the medium. Dunaliella cells may be exceptional in their inability to utilize organic carbon and in their concomitant need for light, both for photosynthesis and for the reduction of NO3-. This genus has been seen to require higher light intensities than do other species with which comparisons have been made. It is therefore at a disadvantage in the open sea in comparison with its competitors. It can, however, fare better in rock pools, where the higher salinity and higher temperatures will act against most other unicellular algae, leaving Dunaliella as sole survivor (Eppley et al., 1969).

V. CELL ANATOMY A.

LIGHT MICROSCOPY

There are two distinct forms of Dunaliella, corresponding roughly to the two species originally described by Teodoresco. Cells of the D. salina type are rounded and reddish or orange in colour; their axes are 10-20 pm long. These cells move slowly and the two flagella are short, relative to the length of the cell. Most of the reddish forms lose their red colour when grown at low light intensities; it is then possible to see that the cell contains a green chloroplast which in general does not occupy more than half the cell volume. Cells of the D. viridis type are cylindrical to ovoid in shape and are green in colour. The two equal flagella emerge together from the front of the cell and point backwards; they are usually longer than the cell body. Such cells measure 3-5 pm by 6 8 pm and are indistinguishable from other green cells commonly referred to as Chlamydomonad, or of the Volvocalean type. These cells move comparatively quickly. The chloroplast occupies most of the cell body. The internal structure of both red and green cells is similar. The major organelle within the cell is the chloroplast, variously described as bell-, cup- or dish-shaped. The basal (posterior) portion is thickened and contains a single pyrenoid. The nucleus lies within the hollow of the cup or bell and is generally obscured in life by a collection of granules, the arrangement of which, according to Butcher (1959), has taxonomic importance.

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

119

The cell has no rigid cell wall of cellulose, chitin or other material. Instead, the plasmalemma is coated by a glycocalyx-like cell envelope. B. ELECTRON MICROSCOPY (Fig.

1.

6)

Chloroplast

a. D. salina type. Observations by Trezzi et al. (1964), Vladimirova (1978) and Melkonian and Preisig (1984) show that the chloroplast consists of thylakoids which have the appearance of long or short discs which bend and twist in every direction, sometimes combining to form slender sinuous or twisting threads or even bundles. The pyrenoid is penetrated by pairs of thylakoids which run towards the centre and end blindly there. Large grains of starch-like material surround the pyrenoid; there is little starch in the remainder of the cell. There are 10-20 small lipid globules around the periphery, just within the chloroplast.

Vacuole with electron- dense

Fig. 6. Diagram of Dundielh cell to show position of organelles. Pairs of thylakoids are represented by single lines.

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M. GINZBURG

b. D. viridis type. D. bioculutu has been studied by Marano and co-workers in many publications (e.g. Marano and hard, 1968; Marano, 1976, 1979; Marano et al., 1985). A very thorough study of D. primolecta was made by Eyden (1975). Hoshaw and Maluf (1981) have studied D . tertiolectu and have compared the ultrastructures of all the species studied up to now. In D. tertiolecta the bell-shaped chloroplast extends along the sides of the cell, almost completely enclosing the central region of the cytoplasmic matrix and nucleus. In living cells the chloroplast has a rugose texture, especially in older cells, and is yellowish-green. The thylakoids are arranged in dense stacks of 2 4 in log-phase cells and 8-10 in the stationary phase. As in D.sulina,the pyrenoid is penetrated by pairs of thylakoids and is surrounded by starch grains. Starch increases in stationary-phase cells; these latter also contain some oil droplets which are not as conspicuous as in D . sulina. 2. Eyespot This consists of one or two rows of lipid globules and is situated at the anterior tip of the chloroplast. Its conspicuousness differs in different Dunuliellu strains. In D . tertiolectu it is highly visible and orange-coloured; it lies just beneath the plasmalemma, free within the matrix of the chloroplast (Hoshaw and Maluf, 1981). According to Dodge’s (1969) terminology, the eyespot belongs to type A, seen in the Chlorophyceae and in two genera of the Cryptophyceae. Some hexagonal packing is seen, but the arrangement of the individual lipid droplets forming the eyespot is generally irregular and the units are irregular in size. Two layers of oil droplets are usually present. The eyespot appears to accumulate lipid with age, the number of droplets doubling between the log and stationary phase. 3. Nucleus The nucleus occupies most of the anterior part of the cell. It may or may not be enclosed within the flanges of the chloroplast. It contains a nucleolus and very evident chromatin strands. The membrane surrounding the nucleus is double, with an irregular space between the two layers. There are large pores crossing both layers of the membrane; the pores appear to be blocked by plugs. 4. Mitochondria Many mitochondrial profiles can be seen, mainly between the nucleus and the chloroplast, but also in other parts of the cell. Vladimirova (1978) says that the mitochondrial profiles are surrounded by extensions of the chloroplast membrane.

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5. Golgi apparatus This consists of two to four dictyosomes situated between the nucleus and the points of insertion of the flagella. The dictyosomes are particularly evident in cells from growing cultures. Acid phosphatase activity was demonstrated in D. prirnolecta (Eyden, 1975). The structure of the Golgi apparatus was studied by freeze-etching in an unnamed species of Dunaliella (Werz and Kellner, 1970). They found that the surfaces of the cisternal membranes contain many particles of different sizes. Small vesicles appear to become cut off from the tips of the dictyosomes and link the latter with endoplasmic reticulum. 6. Endoplasmic reticulum This was first noted by Berkaloff (1966), in an unnamed species of Dunaliella. There is some doubt, however, as to whether Berkaloff’s work really refers to a Dunaliellu, since the cells she described have an untypical structure: the thylakoids end in points at the posterior end of the cell and there are no evident thylakoids entering the pyrenoid. Endoplasmic reticulum has been mentioned by all subsequent workers in the field of ultrastructure of Dunaliellu. A branch is to be observed arching over the dictyosomes and leading into the body of the cell. 7. Vacuoles All cells contain vacuoles; these are especially evident in cells from stationary cultures. The vacuoles have an assortment of contents; some of the inclusions are apparently crystalline [possibly polyphosphate, according to Eyden (1975)] or consist of heterochromatin. Others contain tightly packed membranes arranged concentrically. Some vacuoles seem free of contents. Eyden classified the vacuoles according to whether they were lined by rough or smooth membrane. 8. Flagella Much attention has been given to plant flagella in recent years. The mode of attachment of flagella to the rest of the cell is uniform throughout the green algae (Moestrup, 1978; Melkonian, 1980). The flagella have the usual cross-sectional structure of nine peripheral microtubules and two central ones. in Dunaliella the two flagella continue down into the cell and each terminates in a flagellar or basal body. The two basal bodies are connected to each other by one distal and two proximal fibres. There are two additional basal bodies not connected to flagella. All four basal bodies are arranged in the form of a cross which is referred to as the “flagellar root system” and from which emerges a whole system of fibres and microtubules described in detail by Hyams and Chasey (1974). From each of the four basal bodies projects a branch or rootlet, two of which consist of two microtubules associated with fibres while the other two, also accompanied

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M.GINZBURG

by fibres, consist of 3+1 microtubules. It can be shown that the microtubules of opposite members of the root join at the posterior end of the cell. The fibres end about halfway down the cell. They are referred to as “System I” fibres by Melkonian (1980). A second set of fibres (System I1 fibres), this time in the form of a bundle, originating at one of the basal bodies and terminating at the nucleus, has been found in D.salina (Melkonian and Preisig, 1984). This was probably referred to as the “rhizoplast” by earlier microscopists. It was not found in D. bioculata (Marano et al., 1985). The precise course and arrangement of the fibres and microtubules are described in slightly different terms by different groups of workers; in particular, the accounts of Eyden (1975) and Melkonian and Preisig (1984) do not tally in every respect. Hyams and Chasey (1974) and Eyden (1975) found an intracellular axoneme lying between the plasmalemma and the chloroplast of D . primolecta. No structure of this sort has been found in other species. 9. Cell Envelope Dunaliella cells are commonly said to be naked forms of Chlamydomonas but this is an error (Oliviera et al., 1980; Melkonian and Preisig, 1984). The first group of workers studied D. tertiolecta and the second D. salina, so their conclusions may apply to the two major forms constituting the genus. Oliviera et al. (1980) used special techniques to show up layers of substance outside the plasmalemma; the cationic dyes ruthenium red and alcian blue made it possible to detect a discontinuous cell coat in which areas with a lumpy aspect alternate with others over which the covering is extremely thin. The covering is continuous over the flagella. They say that the organization of the coat reflects the functional activity of the cells and may be related to the production and subsequent release into the medium of material with a high affinity for alcian blue. The outer material of the coat could be digested and its thickness reduced by treatment with trypsin or pronase; neuraminidase made the cell coat look fuzzy. The observations led them to conclude that the surface coat consists largely of glycoproteins with a relatively high anionic charge density and possibly a few neuraminic acid residues. Melkonian and Preisig (1984) drew attention to the surface coat in their own pictures of D. salina and also in those of other workers where the coat can be seen, although no comment was made on it by the workers themselves (e.g. Peterfi and Manton, 1968; Trezzi et al., 1964; Marano, 1976). Eyden (1975) did mention that in some preparations of D . prirnolecta the external surface of the plasmalemma had a layer of amorphous material of variable thickness and distribution.

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123

C. INTERSPECIFIC ANATOMICAL DIFFERENCES

These have been studied by Hoshaw and Maluf (1981), who made a detailed comparison of the fine structure of D . salina, D . bioculata, D . primolecta, and D.tertiolecta. Differences were found in the flagellar region, the Golgi bodies and the eyespot. In the flagellar region D . primolecta was found to have four basal bodies and the other species only two; this is almost certainly an error, since Melkonian and Preisig (1984) subsequently found four basal bodies in D . salina, and the number is likely to be constant throughout the genus (Melkonian, 1980). An internal flagellum (axoneme) was found in D . primolecta, though in no other species. D . bioculata has a “microtubule-organizing centre”, D . primolecta a “reniform body” and D . salina a “rhizoplast-like structure”. The nature and function of these organelles is unknown. It is hard to see any definite differences in structure within the Golgi bodies or within the eyespots. It must be concluded that D . bioculata, D . primolecta and D . tertiolecta are hard to differentiate on anatomical grounds. Hoshaw and Maluf (1981) do not emphasize sufficiently the large differences in cell shape, size and chloroplast structure between D . salina and the other species they studied, all of which could loosely be classified as “ D . viridis”. In short, specific differences are almost impossible to define within the group of the marine species studied. D. INTERGENERICANATOMICAL DIFFERENCES

The general resemblance between electron micrographs of whole cells of Chlamydomonas and the marine species of Dunaliella is very striking. The resemblance is obscured in D . salina by the cell shape (round instead of ovoid) and by the arrangement of the thylakoids, which is disorderly in D . salina. At times, Dunaliella has been likened to Asteromonas (Peterfi and Manton, 1968) and Spermatozopsis (Melkonian and Preisig, 1984). Despite superficial resemblance, there is no evidence for a close relationship between the two genera. Dunaliella seems closest to Chlamydomonas. A comparison of major structural features is given in Table IX. Dunaliella differs from Chlamydomonas only in the absence of a nonelastic cell wall, the location of the Golgi body and the presence of vacuoles containing concentrically arranged membranes. E. EFFECT OF SALT ON CELL ANATOMY

Trezzi et al. (1965) are the only workers to have made electron micrographs of Dunaliella cells immediately after subjection of the cells to a change in tonicity. They found that when the tonicity was lowered, the cells swelled;

TABLE IX Morphological comparison of related genera of microalgae Character

Chlamydomonm

Spermatozopsis

Asteromonm Three to six longitudinal ridges run lengthwise down body

Dunaliella Ovoid to cylindrical

Cell shape

Ovoid

Spirally twisted; sickleshaped

Cell surface

Inelastic cell wall composed of glycoproteins

Absent

No cell wall; patches of glywproteins.

Dictyosomes

In perinuclear region

One at posterior end of nucleus

At flagellar end of cell

F'yrenoid

Embedded in chloroplast

Absent

Mitochondrion

Between chloroplast and cell membrane

One, compact, posterior to nucleus

Nuclear membranes

Regular; no pores

System I fibres (microtubules)

Overlie two-stranded microtubular roots; close to flagellar base

Overlie microtubular root

Underlie microtubular root; run all the way round cell under plasmalemma

Contractile vacuoles

Two

Two

Absent

Pressed against nucelus

As in Chlamydomonas Reticulum in anterior part of cell Spacesopen out between membranes; pores

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

125

the whole cell and the various cytoplasmic structures appeared equally swollen, showing that water had penetrated into every part of the cell and suggesting that all the various membranes were semipermeable. Their observations did suggest that the different membranes had somewhat different permeability properties; for instance, wide spaces appeared between the two nuclear membranes and also between pairs of thylakoids so that the blind ends swelled into saccules. Cells subjected to hypertonic stress lost water from every part of the cell. The two layers of the nuclear membranes became appressed together so that the space usually seen between them totally disappeared. Different thylakoids also became closely appressed. All the phenomena described are reversed fairly shortly after the osmotic shocks, so that the cells return to their usual appearance. This is probably why Hoshaw and Maluf (1981), who also made transfers of cells from media of one osmotic strength to another, were unable to detect any changes in structure. Their experiments differed from those of Trezzi et al. (1965), in that they took samples 90 min after the osmotic change took place, by which time recovery of the cells may be expected. Hoshaw and Maluf (1981) found that in cells grown at high salt concentration the stacking of thylakoids was more marked than in those at low concentration. A similar conclusion was reached by Pfeifhofer and Belton (1975), who found that the chloroplasts of intact cells grown at 6 0 0 m ~NaCl contained many thylakoid membranes, some of which extended over the entire length of the plastid. The thylakoids were usually seen as single membranes; stacking occurred only where the membranes made physical contact. In cells grown at 4 M NaCl the thylakoids appeared stacked over most of the membrane surface. They had a shrunken appearance except at the extreme tip of the thylakoid. These shrunken thylakoids were found to contain a higher density of B particles [thought to represent a photosynthetic unit, according to Park and Pon (1961)]. F. EFFECT OF LIGHT INTENSITY AND TEMPERATURE ON CELL ANATOMY

This has been studied by Vladimirova (1978), who grew D. salina under the following conditions: 1. 249 p E m-*s-'; 28°C 2. 1480 pE m-2 s-'; 28°C 3. 1480 pE m-2 s-'; 22°C

Cultures reached 50x106, 115X1O6 cells ml-' on the 6th day of culture under conditions 1,2 and 3 respectively. At the lower light intensity the chloroplasts had well-developed systems of thylakoids and looked more like those of a marine Dunaliella; the pyrenoid looked loose and distinct and there were very many small lipid globules scattered all over the

126

M. GINZBURG

chloroplast. At higher light intensity and 28°C the thylakoids were fewer and bended and twisted in every direction. At the higher light intensity and 22°C the thylakoid system was reduced and the intrathylakoid space shrunken so that the thylakoids looked like slender filaments. The pyrenoid was large but not dense. These cells contained more lipid than did the other variants; the drops were largest and were situated on the inner side of the chloroplast membrane. It is interesting to note that the chloroplasts shown by Trezzi etal. (1964) look like those of Vladimirova’s variant 1. Indeed, in Trezzi et al. (1964), cultures were grown at 22-24°C by natural light near a north-facing window; the light intensity cannot, therefore, have been high.

VI. COMPOSITION OF DUNALZELLA CELLS A. MAJOR ORGANIC COMPOUNDS

The relative proportions of the major compounds varies strongly with growth rate and NaCl concentration of the medium. Analyses have been made by Goldman and Peavey (1979), Ginzburg (unpublished) and Ben-Amotz et al. (1982a). Goldman and Peavey (1979) measured the N, P and C contents of D.tertiolecta grown in continuous culture in nitrogenlimited media. Cells growing at the maximum rate of 1.33 division day-’ were found to contain 8, 5.2-5.5 and 6.5 pg N, P and C respectively per cell. When N was limiting, protein synthesis was slow and C was channelled into the formation of starch. However, when N was not limiting, protein synthesis completely dominated the metabolic apparatus of the cell. At low growth rates P was stored in the form of polyphosphates; as the growth rate increased, P was diverted to the formation of organic compounds. Figure 7 shows the results of analyses of D. parva (19/9) grown in batch culture in Ginzburg’s laboratory. Thirty to 50% of the organic dry matter of the cells was found to consist of protein. There was little starch in the cells while the culture was actively growing (i.e. up to 3-5 days of culture). In older cultures starch increased very much. As expected, there was roughly three times as much glycerol in the cells in 1 . 5 ~ N a C l .The amount of lipid varied between 11 and 16% of total organic weight except for a single point on the first day of culture. In Fig. 7 the change in the ratio of protein to carbohydrate which occurred after the 3rd day of growth is unlikely to have been due to lack of N and is more probably a result of the reduced light intensity within the dense cell suspension. Ben-Amotz et al. (1982a) contrasted two species which they referred to as D. salina and D . bardawil. Their major results are shown in Table X. It

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

127

40

40 0

8

/’

20

0

Days

0

.,

Days

Fig. 7. Composition of D . parva (19/9) cells grown for 9 days in 0.5 M NaCl (A) or 1.5 M NaCl ( B ) . 0 ,protein; starch; 0, glycerol; 0, lipid. Unpublished results of B.Z. and M. Ginzburg.

should be noted that D.salina is defined as possessing cells capable of having a carotene:chlorophyll ratio of 6: 1 [Loeblich (1982), following Teodoresco (19091. Ben-Amotz’s D . salina is therefore improperly named, since the carotene:chlorophyll ratio is not more than 15. The protein measurements in Table X are similar to those in Fig. 7. BenAmotz et al. (1982a) found twice as much glycerol as is shown in Fig. 7; this is to be expected from the NaCl concentrations used by the two groups of workers. The organic material unaccounted for in the table is likely to be chiefly lipid, since the lipid content of the two species involved is 30% of total organic dry weight (see below).

TABLE X

Some major organic constituents of two Dunaliella species grown in 3 M NaCl ~~

Substance Chlorophyll Glycerol @-carotene Protein Unaccounted for Ben-Arnotz PI al. (1982a)

D . salina (YOdry weight)

1.5-3 24-40 0.25-0.3 30-40 20-46

~

D . barda wil (YOdry weight) 1-2 30-40 0.5-0.9 30-40 10-35

128

M. GINZBURG B. PROTEIN

Both Ginzburg (unpublished) and Ben-Amotz et al. (1982a) measured protein by means of the Lowry test. This test was later found to overestimate protein in Dunaliella when a comparison was made with N content measured by means of the Kjeldahl technique; in three of four strains tested, protein determined by the Lowry test was roughly twice that calculated from the Kjeldahl N content. Thus, the amounts shown in Fig. 7 and Table X are almost certainly overestimated. The amino acid compositions of two Dunaliella proteins are listed in Table XI, which also contains similar measurements made on four other algal species. The large excess of acidic over basic amino acids which occurs in Dunaliella should be noted. Acidity is also a characteristic of the proteins of halophilic bacteria: examples are malate and glutamate dehydrogenases, in which the excess of acidic over basic amino acids amounted to 11.5 and 9.6 per 100 residues (Eisenberg et al., 1977). In these acidic bacterial proteins, high salt concentrations have the effect of enabling adjacent protein molecules to come to within reacting distance of each other. The interactions between salts and proteins of Dunaliella have not been studied. C. SOLUBLE CARBOHYDRATES

Analyses of carbohydrates have been made by Craigie et al. (1966) and Ginzburg et al. (1983). The only soluble carbohydrate present in large amounts is glycerol. Glucose, fructose and ribose were each found to constitute 2-3% of dry weight. Sucrose was not detected by Ginzburg et al. (1983) in either of the two strains examined. Craigie et al. (1966) found weak traces of sucrose in D. tertiolecta and D . salina but none in D. parva or D.prirnolecta. However, Muller and Wegmann (1978a,b,c) found that sucrose was accumulated in algae maintained at temperatures above the optimum. At 0.3 M NaCl and up to 35"C, up to 30% of 14C incorporated during photosynthesis was recovered as sucrose. As the NaCl concentration of the medium rose, the temperature needed to bring about sucrose accumulation also rose. The authors labelled the membranes of whole cells with a spin-label compound and determined the temperature at which change in fluidity occurred. The temperature rose with NaCl concentration from 29.5"C at 0.3 M to 41°C at 2 M NaCI. It was hypothesized that the chloroplast membrane changed in fluidity at these temperatures and that a change in fluidity is related to a change in permeability. Sucrose was found to be synthesized from UDPG and fructose 6-phosphate by the action of sucrose phosphate synthetase. This enzyme was assumed to be in the cytoplasm. At low temperatures sucrose synthesis may be suppressed

TABLE XI Amino acid composition of algal proteins ~~

'70 molar ratio N in protein

Amino acid L-isoleucine L-leucine L-lysine L-phenylalanine L-tyrosine L-cysteine-cystine L-methionine L-threonine L-tryptophane L-valine L-cysteic acid L-aspartic acid L-serine L-glutarnic acid glycine L-alanine L-histidine L-arginine Proline

Dunaliella Protoplast

Plasmalemma

5.7 11.6 6.4 6.6 4.3 1.1 2.2 5.1

3.6 10.4 3.9 4.5 2.5

nm 5.85 4.0 12.2 5.1 12.6 6.0 8.5 1.4 6.6

nm

NH3

(Asp + Glu)-(Lys + Arg)

11.8

Chlorella vulgark

Anaboena cylindrica

Navicula pelliculosa

Tribonema aequale

3.5 6.1 10.2 2.8 2.8 0.2 1.4 2.9 2.1 5.5

3.9 6.2 36.6 2.9 21.6

3.5 7.2 38.3 3.4 31.9

4.1 6.4 39.0 2.8 3.0

nm

nm

nm

1.2 5.7 1.0 7.0

1.2 4.2 1.1 7.5

1.4 4.0 1.8 7.5

9 6.7 12.4 12.9 13.0 0.5 3.0 6.3 11.9

6.4 3.3 7.8 6.2 7.7 3.3 15.8 5.8

6.9 2.9 5.6 5.5 6.0 32.5 > 11.7 5.0

6.4 3.4 4.9 6.1 6.5 32.8 39.2 6.2

5.1 2.8 4.6 6.2 8.4 3.7 15.9 6.1

15

- 12

S -6

6 -6

- 15

0.6 5.7

nm 5

Dunaliefla protoplast: Gibbs and Duffus (1976). Dunaliella plasmalemma: Jokela (1969). Other algae: Fowden (1954). Analyses were made on the bulk proteins. nm: not measured.

130

M. GINZBURG

because fructose 6-phosphate is retained within the chloroplast and cannot move through the chloroplast membrane. At temperatures above the change in fluidity, fructose 6-phosphate is thought to cross the chloroplast membrane and come into contact with sucrose phosphate synthetase, thus bringing about synthesis of sucrose. D . STARCH

Eddy et al. (1958) identified starch in D.bioculata. The presence of a starch-type polysaccharide was indicated by its staining blue-black with iodine and by its degradation by a-amylase. The amylose content was found to be unusually low (12-14% as compared to 20-30%0 for most plant starches). The amylopectin component had a relatively high degree of branching, with average chain lengths of 15-16 glucose residues. Craigie et al. (1966) found that 60% of I4C fixed after 2 h of photosynthesis was present in the alcohol-insoluble residue, which they assumed to be starch. Starch accumulates in large amounts in stationary cultures of the green-celled strains (Fig. 7). The red-celled strains accumulate lipids instead. E. RELATION OF GLYCEROL A N D STARCH CONTENTS TO NaC1

A resume of published data for glycerol is given in Fig. 8, which shows that the cell glycerol content increases with salinity of the outside growth

SOL

h.parva

/

Fig. 8. Glycerol content of Dunuliellu cells grown under conditions of constant salinity.

0, pmol glycerol (mg chlorophyll)-' in D. parva (Gimmler and Moller, 1981). 0 , pmol glycerol (mg protein)-' in D. rertiolecru and D . viridis (Borowitzka and Brown, 1974).

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

131

medium. All measurements were made on cells grown under conditions of constant salinity. Under such conditions, the ratio of cell glycerol to starch rises with the NaCl concentration of the medium (Fig. 9).

contents of D.parva cells as influenced by salinity of Fig. 9. Glycerol (0)and starch (0) medium. Redrawn from Gimmler and Moiler (1981).

F. LIPIDS AND CAROTENOIDS

We have seen already that D. parva contains about 13% lipid per unit of organic dry weight (Fig. 7). In contrast, in D. bardawil grown under growth-limiting conditions (high NaCl concentration, low N), as much as 30% of total cell C was soluble in chloroform and assumed to be lipid (Ben-Amotz et al., 1982a). The large disparity in lipid content is due to the accumulation of carotenoids and p-carotene, which in D . bardawil amount to nearly half the total lipids (Table XII). It should be noted that the content of polar lipids, which are the principal constituents of cell membranes, is roughly similar in all three species (9% of organic dry weight in D . parva and D . tertiolecta; 10% in D . bardawil). Evans et al. (1982a,b) found that the major lipids of D . parva and D . tertiolecta were monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulphoquinovosyldiacylglycerol,phosphatidylglycerol and phosphatidylcholine. Fried et al. (1982) found the same compounds in D . bardawil These compounds are typical of those found in other photosynthetic organisms. An additional compound, diacylglycerol-O-N,N,N-

132

M. GINZBURG

TABLE XI1 Lipid composition Dunaliella

YO dry weight Polar lipids carotene Unidentified yellow pigments Free fatty acids Triacylglycerol Carotenoids

D. parva

D. tertiolecta

D . bardawil

9.1

8.8

10.2 4.3 2.7

1.7 1.9

0.9 2.0

-

-

-

0.5 10.2

D. bardawil was grown under growth-limitingconditions in 3.5 M NaCI. Data adapted from Fried er al. (1982) ( D . bardawil) and Evans el al. (1982a) ( D . parva, D. tertiolecta).

trimethylhomoserine was characterized by both groups of investigators. It had previously been reported in only four organisms, Chlamydomonas reinhardii, Volvox carteri, Ochromonus danica and Epidermophyfon floccosum. The fatty acid composition of D . parva and D . ferfiolecfawas similar and agreed with that of D. ferfiolecfu(Ackman ef al., 1968). The major acids found were linolenic, palmitic, linoleic and oleic. D . bardawil also contained stearic, 3-trans-hexadecenoic and hexadecetetraenoic acids. Evans et al. (1982a) comment that “notable features of the results are the high degree of unsaturation typical of photosynthetic organisms, the high levels of polyunsaturated 16-carbon acids, frequently observed in algae, and the absence of 20- and 22-carbon acids. Ackman ef al. (1968) also reported the virtual absence of 20- and 22-carbon acids in D . ferfiolecfa;in contrast, Chuecas and Riley (1969) measured high levels of 20- and 22-carbon acids in D . ferfiolecfa.The reason for the discrepancy is unclear. It should be noted that Chuecas and Riley’s are the only results on Dunaliella lipids to be quoted in Stewart (1974). The effect of temperature on the composition of chloroplast and microsomal membranes has been studied by Lynch and Thompson (1982, 1984a,b). Membrane fractions were analysed for chlorophyll, glycolipid and polar lipids, and a comparison, made of cells grown at 30°C with those grown at 12°C. The chloroplast increased dramatically in volume at 12”C, though membrane material increased by only 12%, the difference being due to starch. Microsomes increased three-fold at 12”C,concomitantly with polar lipid content. The fatty acid compositions of both chloroplast and microsoma1 membranes differed, with a trend towards the lengthening of the carbon chains at 12°C. There was also a marked increase in the degree of unsaturation at this temperature. Unsaturation is said to make membranes more fluid.

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

133

The synthesis of p-carotene has been studied by Loeblich (1982) in D . salina and by Ben-Amotz et al. (1982a) in D . bardawil. Synthesis was found to be a function of high light intensity and low growth rate brought about by manipulating the NaCl concentration of the medium. Thus, the ratio of carotenoids to chlorophyll was 12:l at 20,000 lux and 4.3 M NaCl. About 30% of the carotenoids are contained in globules situated in the interthylakoid space along the perimeter of the cell. Ben-Amotz and Avron (1983) found that carotene production could be further increased by lowering the sulphate concentration of the medium to less than S mM in cultures reaching the stationary state of growth. The role of carotene in Dunaliella has been much discussed and the hypothesis advanced that carotene protects against high light intensity. In fact, there is little or no evidence to support the hypothesis. Ben-Amotz and Avron (1983) have conducted an experiment which disproves it, though their interpretation differs from the one given here. One lowcarotene and one high-carotene strain of Dunaliella were grown at a series of light intensities over a 10 day period. The low-carotene strain grew better at all light intensities even though the carotene content of the cells remained low throughout. It seems that the major purpose of carotenoids is to mop up excess photosynthetic capacity when cells of the large-celled strains are exposed to high light intensities. G. NUCLEIC ACIDS

Rahmsdorf and Schweiger (1972a,b) studied the ribosomes and ribonucleic acids of a strain thought to be a D. salina. The main components were 80s and 70s ribosomes (characteristic of the cytosol and chloroplast respectively). There was a pronounced peak at SOS, thought to be a subunit derived from 70s ribosomes, which are very unstable even at high concentrations of Mg2+ and K + ; high requirements for these cations are characteristic of algal ribosomes. The 80s ribosomes produced types of RNA of 26S, 23s and 17.5s. The 50s ribosomes contained RNAs centrifuging down at 23s and 17.5s. These findings are in agreement with those from other green plants. H. CONCLUSIONS

Many of the organic constituents (insoluble carbohydrates, lipids, nucleic acids) of which Dunaliella cells are composed are similar to those of other green algae. Exceptions are as follows: 1. Acidic nature of proteins. There is an excess of acidic over basic amino acids as in halophilic bacteria, whereas in non-halophilic algae

134

M. GINZBURG

related to Dunaliella the number of acidic amino acids is balanced by basic residues (Table XI). 2. Production of an unusual photosynthetic product, glycerol, in very large amounts; rate of synthesis and final cell concentration is determined by the NaCl concentration of the growth medium. 3. Large content of polar lipids, amounting to -10% of organic dry weight.

VII. ENZYMES At least 25 enzymes derived from various Dunaliella species have been studied (Table XIII).Enzymes concerned with glycerol metabolism have received much attention, largely because of the involvement of glycerol in the regulation of the cell osmotic pressure. Photosynthetic enzymes have also been studied, in part by those whose interest is in phytoplankton and in part by students of photosynthesis, who find Dunaliella cells to be convenient experimental material. A.

ENZYMES CONCERNED WITH GLYCEROL METABOLISM

Of all aspects of Dunaliella biochemistry, the metabolism of glycerol has received the greatest attention. A concise review can be found in Brown et al. (1982). Both the formation and degradation of glycerol must be accounted for, since these processes occur as responses to increases and decreases respectively in the osmotic pressure of the ambient medium. In both cases the responses are rapid. The major features of the glycerol cycle are shown in Fig. 10. Photosynthesis

Pi Starch-

7T+ L

FBP 7

ATP

ADP

G

+ H+

i

Pi P

7 J

NADH NAD -I-

G-3-P

ADP

DHAP T

A

1

DHA

Fig. 10. Glycerol cycle. From Haus and Wegrnann (1984a).

W

ATP

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

TABLE XI11 Enzymes studied in Dunaliella I Enzyme Glucose 6-phosphate dehydrogenase Glycerol 3-phosphate deh ydrogenase

Km, mM

0.11 (glucose) 0.5 (DHAP) 0.026 (NADH) 0.15 (G3P) 1.3 (NAD')

Glycerol 3-phosphate phosphatase Glycerol de hydrogenase DHA reductase DHA kinase Nitrite reductase Nitrate reductase

2000-3000 (glycerol) 1-2 (DHA) 0.6 (DHA) 1500 (Glycerol) 0.01 (DHA) 0.08 (ATP) 0.1 (NO,-) 0.26 (N03-) 0.18 (NADH) 0.10 (NADPH) 0.06 (FADH2)

Carbonic anhydrase

PEP carboxylase Ribulose diphosphate carboxy lase

CFO-CFl ; CFl ATPase Starch-metabolizing enzymes Phosphofructokinase 2 (F-6-P) 0.6 (ATP) Phosphoriboisomerase Phosphoglucoseisomerase Plasmalemma ATPase Flagellar ATPase ER ATPase Lactate dehydrogenase Lactate enzvmes Succinate dehydrogenase

Reference Johnson et al. (1968) Ben-Amotz and Avron (1972) Borowitzka and Brown (1974) Gimmler et al. (1984) Haus and Wegmann (1984a) Gimmler et al. (1984) Marengo et al. (1985) Brown et al. (1982) Sussman (1982) Gimmler et al. (1984) Borowitzka and Brown 1974) Borowitzka et al. (1977: Ben-Amotz and Avron 1973,74) Gimmler et al. (1984) Lerner and Avron (1977) Lerner et al. (1980) Grant (1967,1968) LeClaire and Grant (1972) Heimer (1973, 1975, 1976) Latorella and Vadas (1973) Loeblich (1972) Ginzburg (1978) Aizawa and Miyachi (1984) Aizawa et al. (1985) Appleby et al. (1980) Hellebust and Terborgh (1967) Johnson et al. (1968) Paasche (1971) Glover and Morris (1979) Vladimirova et al. (1982) Fine1 et al. (1984) Kombrink and Woeber (1980) Brown et nl. (1982) Paradies and Vetterman (1979) Kombrink and Woeber (1982) Johnson et al. (1968) Johnson et al. (1968) Gimmler etal. (1984) Gimmler et al. (1984) Gimmler etal. (1984) Ben-Amotz and Avron (1972) Gruber et al. (1974) Cherep (1982j

135

136

M. GINZBURG

A key enzyme appears to be glycerol phosphate dehydrogenase (EC 1.1.1.8), which converts dihydroxyacetone phosphate to glycerol phosphate, with the help of NADH. This enzyme has long been known as a cytoplasmic glycerol phosphate dehydrogenase and is of importance in muscle (see, e.g., Klingenberg and Bucher, 1960). The enzyme is soluble and appears to be located in the Dunaliella chloroplast (Brown et al., 1982; Gimmler and Lotter, 1982). Its properties have been described in detail by Haus and Wegmann (1984a,b) and Marengo etal. (1985). Glycerol phosphate is converted to glycerol in a reaction catalysed by glycerol phosphate phosphatase (Brown et al., 1982). A 40-fold purification was obtained by Sussman (1982). The enzyme does not appear to have been completely characterized and its location within the cell is not established. Its action is irreversible. It thus prevents glycerol being degraded by the glycerol phosphate dehydrogenase pathway. Glycerol is degraded to dihydroxyacetone (DHA) by means of glycerol dehydrogenase, an enzyme that uses NADP+ as H-acceptor (Borowitzka and Brown, 1974; Borowitzka et al., 1977). This enzyme was described under its alternative name, dihydroxyacetone reductase, by Ben-Amotz and Avron (1973,1974), who for some years held it to be the major enzyme responsible for glycerol synthesis, but later agreed that its major function is in glycerol degradation (Ben-Amotz et al., 1982b). The peculiarity of this enzyme is that its K , for glycerol is 3W1000 times greater than that for DHA (K,=1.5 M for glycerol and 0.6 mM for DHA; see Table XI11 for complete figures). It is clear that unless the K , for glycerol were to be very high, the substance would not accumulate within the cell. Glycerol dehydrogenase is located largely in the cytoplasm (Brown et al., 1982; Gimmler and Lotter, 1982). The pH optima of the backward and forward reactions differ sharply, being 9 for the reducing reaction and 7-7.5 for the oxidizing reaction. DHA kinase converts DHA to DHA phosphate (Lerner and Avron, 1977; Lerner et al., 1980). The enzyme requires ATP and 1 mM Mg2+.The K , values are 1 0 p ~and 8 0 p ~ for DHA and ATP respectively. The enzyme is soluble; its location within the cell is uncertain. There is a sharp pH optimum at pH 7. Its action is irreversible. DHA phosphate is produced by photosynthesis and also by degradation of polysaccharides. This explains how glycerol may be formed in the dark in cells containing sufficient reserves of starch (Gimmler and Moller, 1981). The glycerol cycle demonstrates some interesting features: 1. Enzymes with irreversible actions: glycerol phosphate and DHA kinase. 2. An enzyme, glycerol dehydrogenase, for which the K , for the

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

137

substrates for the backward and forward reactions differ by a factor of up to 1OOO; the two reactions have sharply differing pH optima. It is these features which enable the principal product, glycerol, to remain at high concentrations within the cell and to be synthesized and degraded quickly. B. ENZYMES CONCERNED WITH NITRATE REDUCTION (NITRATE REDUCTASE, NITRITE REDUCTASE)

The physiology of nitrate uptake has been described in Section 1V.K. Attention will be given here to the two enzymes known to participate in nitrate assimilation. Both have been isolated from Dunaliellu cells and characterized (Grant, 1970; Leclaire and Grant, 1972). A brief scheme describing nitrate reduction is given in Fig. 11.

NADHl Nitrate reductase

NAD+J

Nitrite reductase

N H ~

Ferredoxin reduced

Ferredoxin oxidized

Electrons for nitrite reductase supplied by : - FMN ,FAD in the cell; ferredoxin acts as carrier. -methyl viologen dithionite in viffo.

+

Fig. 11.

Principal features of nitrate reduction in plants

Nitrite reductase (EC 1.6.6.4) was present in the soluble fraction of Dunuliellu homogenates (Grant, 1970). In the cell, ferredoxin is likely to act as electron carrier; in vitro, methyl viologen and dithionite served as electron donors. The enzyme was unaffected by NAD and NADP; FMN and FAD always restored significant activity. The amount of nitrite removed was always balanced by an equal gain of ammonia. The K, for nitrite was lop4M. The pH optimum of the enzyme was 7-7.5, depending on the buffer used. The enzyme was not stable. It was very active, as compared with nitrite reductases from other algae. The molecular weight of the enzyme was 70,000. It had the same molecular weight and K, as nitrite reductase from other sources (Beevers and Hageman, 1969). Nitrate reductase (EC 1.6.6.2) is also a soluble enzyme (Leclaire and

138

M. GINZBURG

Grant, 1972). In contrast to the enzyme from other sources, the Dunaliella enzyme was found to use NADPH, as well as the more usual NADH, as electron donor, even at the highest specific activity obtained (0.86 p~ NO3- reduced min- (mg protein)-’). FADH2 also acted as electron donor. The K , values for N03-, NADH, NADPH and FAD& are 0.26, 0.18, 0.10 and 0 . 0 6 m ~respectively. The properties of the enzyme are consistent with its being a rnolybdoflavoprotein, as is usual for nitrate reductases from plant sources. The molecular weight of the Dunaliella enzyme is 5~ lo5 (usual range 5 X lo5to 6 X lo6). The ability of nitrate reductase to use both NADH and NADPH is peculiar to Dunaliella; the suspicion remained that the NADPH was used only because it was first converted to NADH. However, this conversion does not occur in Dunaliella; Heimer (1976) coupled nitrate reduction to a NADPH-regenerating system made from glycerol and a NADP-glycerol dehydrogenase extracted from D . parva. Thus, the only source of electrons was from regenerated NADPH. Heimer’s (1976) K , values for NADH and NADPH were 0.01 and 0.02 mM respectively. These are lower than the values of Leclaire and grant (1972). An unusual feature of Dunaliella nitrate reductase mentioned by LeqClaire and Grant (1972) was its inability to use reduced methyl viologen as an electron donor. This is a commonly used cofactor. The apparent abnormality was resolved by Latorella et al. (1982), who pointed out that methyl viologen is also used as cofactor by nitrite reductase. Leclaire and Grant had tested their enzyme by measuring the appearance of nitrite in the medium; when none appeared, they deduced that nitrate had not been metabolized, whereas in reality the nitrite had been removed as fast as it had been produced, the methyl viologen acting as cofactor for both reductases. Latorella et al. (1982) inhibited nitrite reductase by adding sodium arsenite to the reaction mixture. They were then able to demonstrate the appearance of nitrite, reduced methyl viologen acting as cofactor for nitrate reductase. Latorella et al. (1982) have succeeded in isolating and partially characterizing a number of nitrate assimilation mutants of D.tertiolecta. Eight were found to grow well on nitrite but not on nitrate; two more grew poorly on nitrite but not on nitrate. Five others would not grow on either nitrate or nitrite. All grew well on ammonium salts. The characteristics were maintained for several generations. Most of the mutants were shown to be unable to synthesize either nitrate reductase or nitrite reductase, though two of them which did contain nitrite reductase could not grow on nitrite, possibly because of the lack of a cofactor. Genetic analysis was not possible because the D. tertiolecta strain used did not form zygotes. The effects of salt and glycerol on nitrate reductase from Dunaliella and two other plant sources were measured by Heimer (1973) and are summarized in Table XIV. It is interesting to see that the enzymes from

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

139

TABLE XIV Effectof NaCl or glycerol on nitrate reductase

Concentration causing 50% inhibition mM

Source Chlorella pyrenoidosa XD tobacco cells Dunaliella parva

NaCl

Glycerol

200 150

450

400

800 No inhibition below 1400 mM

From Heimer (1973).

C . pyrenoidosa and from X D tobacco cells were inhibited by glycerol as well as by NaCl. The D . parva enzyme showed greater resistance to both substances. It is unfortunate that both were not tested together. C. PHOTOSYNTHETIC ENZYMES

1. Carbonic Anhydrase The physiological aspects have been described earlier (Section 1V.L). The Dunaliella enzyme does not appear to have been characterized biochemically.

2. Ribulose 1,5-bisphosphate carboxylase (RuBPCase) This catalyses the entry of C 0 2into the reductive pentose phosphate cycle: C02+ribulose 1,5-bisphosphate+H20+ 2(3-phosphoglycerate) The reaction is possibly the only one leading to a net gain of carbon. The activity of the enzyme has been used in the past by oceanographers as an indication of photosynthesis in the sea. However, the results obtained are approximate (Glover and Morris, 1979), not surprisingly since the activity of the enzyme from different algae composing the phytoplankton is extremely variable, that from Dunaliella being one of the highest. Paasche (1971) compared the activity of RuBPCase in Dunaliella cells grown in media containing nitrate or ammonium as a source of nitrogen. When the activity was calculated per unit of total protein, there was no effect of N source. However, as cells grown on ammonium contained more protein per cell, the activity of these cells did appear to be greater when calculated per cell or per unit of chlorophyll. Vladimirova et al. (1982) used a cytoimmunofluorescent method to localize RuBPCase. They found the enzyme to be concentrated in the region of the pyrenoid, both in Chlamydomonas reinhardii and in Dunaliella salina. They suggested that the pyrenoid acted as a giant carboxysome: a

140

M. GINZBURG

pool of actively working enzyme and possibly a centre of biosynthesis. RuBPCase, and also PEPCase, have been found to be more active in low-C02-grown than in high-C02-grown cells (Aizawa et al. 1985).

3. Coupling Factors The CFO-CF1 and CF1-ATPase from D . bardawil was purified and characterized by Finel et al. (1984). The activity of these factors is very much lower in D . barduwil than in spinach [220 and 1700 /AM ATP formed (mg chlorophyll)-’ h-’ in D . bardawil and in spinach respectively]. The D. bardawil enzyme was unaffected by glycerol and in some cases even promoted by it, in contrast to the spinach enzyme, which was progressively inhibited by increasing glycerol concentration. Cyclic photophosphorylation of the thylakoids from both sources was inhibited by salt. Curiously, D. bardawil thylakoids were even more sensitive than those of spinach; 50% inhibition occurred with 100 and 300 mM NaCl for D . barduwil and spinach thylakoids, respectively. The authors concluded that D . bardawil thylakoid membranes, CFO-CFI complex and CF1 exhibit a high tolerance to glycerol when compared to the same systems for spinach. D. ENZYMES METABOLIZING STARCH

Evidence for two enzymes involved in starch synthesis has been obtained from D . marina cells (Kombrink and Woeber, 1980). The enzymes are ADP pyrophosphorylase and starch synthase. Both enzymes were identified from a fraction claimed by the authors to be isolated chloroplasts. The claim was disputed by Brown et af. (1982), who pointed out that the chloroplast fraction contained large amounts of non-chloroplast enzymes and that therefore the fraction should be described as “chloroplastenriched”. D . marina cells also contained an a-glucan phosphorylase and an amylase which, when working together, degraded soluble starch, shell fish glycogen or a maltodextrin mixture. E. PHOSPHOFRUCTOKINASE

This enzyme converts D-fructose 6-phosphate to D-fructose 1,6diphosphate. A study of molecular structure was made by means of X-ray scattering on enzyme isolated from D . salina (Paradies and Vettermann, 1979). These workers found general resemblances between the enzymes of D . salinu, rabbit muscle, chicken liver and erythrocytes, but not of E. coli. The active enzyme was considered to consist of four protomers each with a molecular weight of 82,000. The hydration, 0.64 g H 2 0 g-’ protein, was higher than that of rabbit muscle phosphofruc-

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

141

tokinase. The intrinsic viscosities were 6.5 and 6.8 ml g-' at pH 6.8 and 7.5 respectively. These figures are in the very upper range for spherical proteins. It is not known whether the peculiar nature of the Dunaliella enzyme is due to the fact that it is derived from a plant, or to the fact that the plant in question is halophilic. The biochemical properties of phosphofructokinase were studied by Kombrink and Woeber (1982) on a partially purified preparation from D. marina. They found the enzyme to be located in the chloroplast. The pH optimum of the reaction was 7.0. Maximum enzyme activity was at about 2 mM fructose 6-phosphate and 0.6 mM ATP. ATP was an allosteric inhibitor. The enzyme was also inhibited by glycerate and phosphoenolpyruvate, and was activated by inorganic phosphate at low concentrations of fructose 6-phosphate.

F. EFFECT OF NaCl ON DUNA L l E L L A ENZYMES

One of the questions which most puzzled the early workers on Dunaliella was whether the organism had evolved enzymes capable of tolerating high NaCl concentrations, or whether the cell interior was essentially like that of a freshwater organism, NaCl being excluded from the cell by means of a cell-membrane impermeable to it. Johnson el al. (1968) were the first to test the effect of NaCI on specific enzyme reactions. Soluble extracts were obtained by centrifuging broken cells, and the NaCl concentrations needed to inhibit specific enzymatic reactions by 50% were determined. It was found that the inhibiting NaCl concentrations were very low, as compared with those outside, and the conclusion was drawn that the cell interior must be virtually free of NaCI. This conclusion has been widely accepted even though work done subsequently has not supported the contention that all Dunaliellu enzymes are inhibited by NaCl. Thus, out of six enzyme systems tested by Ben-Amotz and Avron (1972), only two were 50% inhibited at NaCl concentrations below 400mM. A survey of 25 Dunuliellu enzymes was carried out by Gimmler et ul. (1984), who concluded that Dunaliellu enzymes tolerate the Na+ concentrations, which they had actually measured within the cells. However, internal CI- concentrations may be high enough to inhibit those enzymes which use anionic substrates. Figure 12 provides a summary of 17 Dunuliella enzymes and the NaCI concentrations at which activity is reduced by 50%, and demonstrates that there is a very wide range of variation. It is clear that no general statement can be made concerning the sensitivity of Dunuliellu enzymes to salt. Two of the enzymes studied by Johnson et al. (1968), glucose 6phosphate dehydrogenase (G6PD) and RuBPCase, were subsequently studied by Gimmler ef ul. (1984). In both cases NaCl was found to be less

M.GINZBURG

142

2400 2200 2000 1800 1600 I 1400 E

-B

-z

1200 1000 800 600

Fig. 12. NaCl concentration at which the activity of various Dunaliella enzymes is inhibited by 50%. From Ginzburg and Cinzburg (1986).

inhibitory than was initially claimed by Johnson et al. There was also a large difference in rates: for G6PD the rate of NADP reduction, in p~ pl-' h-', was 0.11 and 2-3 for Johnson et al. (1968) and Gimmler er al. (1984), respectively. The latter workers emphasize the importance of substrate and enzyme concentrations in determining the degree of inhibition caused by NaCl: the higher the concentrations, the less the inhibition caused by a given NaCl concentration. Though the data are hard to compare, it seems that the substrate concentrations used by Gimmler et al. (1984) were uniformly higher than those of Johnson et al. (1968); this may be the explanation of the difference between the two sets of data. Gimmler et al. (1984) tried to avoid using diluted enzyme extracts. Another argument presented by Gimmler et al. (1984) is that the in vitro sensitivity of a given enzyme to NaCl need not necessarily be the same as that in vivo. The argument is supported by Ahmad and Hellebust (1984) in a study of intracellular ion concentrations in a marine Chlorella species which grew with fair yield in X 3 seawater, despite high concentrations of Na' and C1- within the cells. The authors point out that in the halophytic angiosperm Suedu rnaritirna the chloroplasts have C1- concentrations of up to 210 mM (Harvey et al., 1981). However, when the activity of G6PD is measured in vitro, there is strong inhibition by 167 mM NaCl. It is clear that

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

143

within the chloroplasts the enzyme must be protected in some way from inhibition by salt. The data and argument combined make nonsense of the claim that NaCl sensitivity of enzymes in v i m is virtual proof of low salt concentrations within the cell. It is concluded that many Dunaliella enzymes are insensitive to Na+ . Others are sensitive to CI- and may be protected by cell compartmentation and by the presence of glycerol. G . EFFECTS OF GLYCEROL ON DUNALIELLA ENZYMES

These are summarized in Table XV. Three different effects have been noticed: 1. Inhibition of enzyme activity, e.g. G6PD. 2. Stabilization of enzyme structure, e.g. DHA kinase; reduction of ferredoxin. 3. Counteraction of inhibition caused by salt, e.g. glycerol 6-phosphate dehydrogenase, D H A reductase, PEP carboxylase. Physicochemical bases of these effects have not been studied. H. SPECIAL FEATURES OF DUNA LIELLA ENZYMES

Many of these have been modified in ways that render them insensitive to ions, particularly Na+, and to glycerol. It should be stressed that those enzymes from other sources that have been tested were inhibited by both these substances. There must therefore be modifications in the structure of the enzyme molecules in Dunaliella. We have already seen that Dunaliella proteins have a high ratio of acidic to basic residues; it is reasonable to assume that this feature applies to enzyme proteins as well as to the bulk proteins. It is on such grounds that modifications in enzyme structure may be suggested. The only other clue to some difference in enzyme structure emerges from the work of Paradies and Vetterman (1979) on phosphofructokinase (Section VI1.E): the Dunaliella enzyme was found to be unusually hydrated and to have a high intrinsic viscosity. It is not known how these features are related to tolerance to NaCI.

VIII.

PHOTOSYNTHESIS

Rates of photosynthesis in Dunaliella have been measured by several workers and are listed in Table XVI. The usual maximum rate for algae is around 200 PM O2 (mg chlorophyll)-' h-' (Raven, 1976). The table shows that Dunaliella cells have been found to exceed this rate, even at NaCl concentrations several times that of seawater.

TABLE XV Effect on Dunaliella enzymes of glycerol in the presence or absence of salt Enzyme Glucose 6phosphate dehydrogenase

Glycerol 3-phosphate dehydrogenase (reaction measured in oxidizing direction) (reaction measured in reducing direction) Nitrate reductase DHA kinase Reduction of Fd by photosystem I DHA reductase

Glycerol alone

Glycerol + NaCl

Reference

No effect below 4 M, then inhibitory No effect at 460 mM

Additive inhibition

Borowitzka and Brown (1974)

Partial relief of NaCl inhibition

Gimmler et af. (1984)

Stimulatory to 5.87 M,then inhibitory No effect at 900 mM

Progressive inhibition with increasing NaCl NaCl inhibition is partially relieved Not measured Enzyme remains stable NaCl inhibition is not relieved by glycerol NaCl inhibition is partially relieved

Borowitzka and Brown (1974)

No effect below 1400 m M Stabilizes enzyme Stabilizes system

No effect

Gimmler er al. (1984) Heimer (1973) Lerner et af. (1980) Sheffer and Avron (1982) Gimmler et af. (1984)

TABLE XVI Rate of photosynthesk in Dunaliella Standardized rate NaCl

Light intensity (pE m-2 s-')

D. tertiolecta Seawater Seawater

166 600

Seawater Seawater 1.1 M

280 200 1000

D. parva 1.5 M

200

2.1 M

460

D. salina 1.7 M 1.5 M

200-800 350

[PM 0 2

Rate quoted

(mg chlorophyll a)-' h-'1

0.86-1.5 mg C (mg chla)-' h-' 50 p g atom O2cell-' min-' ( x 108) 2 mg C (mg chla)-' h-' 2 p g C (pg chla)-' h-' 270 pnole O2(mg chla)-' h-'

39-68

14@190 pmole C 0 2 (mg chi)-' h-' 170-188 prnole C 0 2(mg chi)-' h-' 6-14 ml O2(mgchla)-' h-' 350 @moleC 0 2 (rng chla)-' h-'

90 90 270

Reference Eppley and Coatsworth (1966) Falkowski and Owens (1978) Bates and Platt (1984) Turpin (1983) Frank and Wegmann (1974)

146190"

Bruggeman et al. (1978)

17@ 188'

Gimmler and Moller (1981)

136300 350

Loeblich (1982) Zenvirth and Kaplan (1981)

Calculation of the standardized rate of photosynthesis is based on the assumptions that the photosynthetic quotient is 1 and that 1 (CH20)6 molecule is formed per 6 0 2 evolved. uRates in terms of total chlorophyll.

146

M. GINZBURG A. PRODUCTS OF PHOTOSYNTHESIS

Craigie and McLachlan (1964) were the first to identify glycerol as the major soluble product of photosynthesis in Dunafielfa.The rate of glycerol synthesis was found to be directly proportional to the NaCl concentration of the medium (Table XVII). Craigie and McLachlan (1964) and Craigie el al. (1966) made a thorough study of products of photosynthesis in two Dunaliella species in seawater. Most of the carbon fixed was recovered as starch, the major soluble product being glycerol (Table XVIII). Sucrose, fructose and glucose were present either in trace amounts or were absent altogether. In addition, I4C-uptake experiments lasting for 2-180 s were performed on D . tertiolecta by Beardall et al. (1976). Most of the labelled I4C was recovered as glycerol after as short a time as 30 s. TABLE XVII Formation of glycerol by photosynthesizing Dunaliella tertiolecta Molarity of NaCl in medium 0.025 Cell number ( x lo9) Total radioactivity fixed (pCi) Neutral fraction ( p a ) Glycerol (pCi) Glycerol ( p ~ ) Glycerol specific activity [(&I mmoIe C)-’]

2.22 29.99 0.18 0.10 3.91 8.56

0.5

2.22 52.9 4.13 3.15 74.8 14.05

2.5 1.14 31.14 8.45 6.84 267 8.53

Cells were offered 1.2 m M I4CO2 for 2 h at 22.5”C. Cells had been growing at the given NaCl for five transfers before the experiment. Craigie, J . S. and McLachlan, J. (1964). Glycerol as a photosynthetic product in Dunaliella tertiolecta Butcher. Canadian Journal of Botuny 42, 777-778.

Glycerol is the major soluble carbohydrate in Chlamydomonus as well as in Dunaliella (McLachlan and Craigie, 1967). The former genus is not generally halophilic; it is concluded that the development of halophilism in Dunaliella is secondary to the choice of glycerol as major soluble photosynthetic product. Winkenbach et al. (1972) studied the effects of NO3-, NO2- and NH4+ on carbon metabolism in Dunaliella (Table XVIII). The presence of NH4+ was found to decrease the flow of C to the insoluble material; the soluble products, and in particular amino acids, increased very much. When comparison of photosynthetic products was made in Dunaliella, spinach and Acetabularia chloroplasts, neither alga was affected by NO2-, in contrast to spinach chloroplasts, which were inhibited by the ion. The results were explained in terms of the permeability properties of the various chloroplasts to NO2- and to N H 4 + , the algal chloroplasts being

TABLE XVIII Product of photosynthesis in Dunaliella

Species tertiolecta primolecta tertiolectu

tertiolectu

salina salina parva

Exposure tihe to I4CO2 2 h seawater 2 h seawater 15 min seawater 15 min seawater 15 min seawater 3 min 1.1 MNaCl 3 rnin 1.1 MNaCI 3mIn1.1~ NaCl 3minl.l~ NaCl 15 min 1.5 M NaCl

% total amount fixed

N source

Insoluble

Glycerol

Sugar phosphates

Sucrose

Amino acids

0.1 0 1

2.2 7.5 5

2 2

NO; 5 mM

60

30 14 31

NO; 5 mM

62

28

1

4.6

NH? 5 mM

29

44

0.5

21.7

3.6

28.2

0

31.2

33.6

0.6

65

13

67 58

KNO3 20 mM

-6

Craigie et ul. (1966)

NH4C120m~

1

2.7

KNO320m~

7

32.6

0

20.6

30.8

NH4C120m~

3

20.6

0

40.3

25.3

14-20

80

Reference

Winkenbach et al. (1972)

Pristavu and Wegmann (1978)

Gimmler and Moller (1981)

148

M. GINZBURG

permeable to NH4+ though not to NO2- and the reverse being true for spinach. The increased synthesis of amino acids in the presence of NH4+ was interpreted as a means of detoxifying the ammonium ion. Pristavu and Wegmann (1978) confirmed the results of Winkenbach et al. (1972) showing that the synthesis of 14C-labelled amino acids was stimulated in presence of NH4+, probably due to an increase of the flow of C into compounds synthesized through the PGA-PEP pathway. The same phenomenon is seen in Chlorella and Acetabularia. The small amount of 14C fixed in the insoluble fractions was a result of high NaCl concentration in the outside medium. B. EFFECT OF LIGHT INTENSITY ON PHOTOSYNTHESIS

The light intensity found by Falkowski and Owens (1978) for maximum rate of photosynthesis in Dunaliella was within the range found for a group of six unicellular planktonic algae with half-saturation at 500 pE m-' s-l at 15°C. Bates and Platt (1984) determined the effect of light intensity on Dunaliella grown at 20°C at intensities from 9 to 280 pE m-2 s-'. All cultures showed the same shape of response curve with a half-saturation point at 75pEm-*s-'. It is possible that the higher temperature employed by Bates and Platt (1984) accounts for the lower light intensity needed to reach half-saturation. However, the workers concerned do not mention the cell density at which their experiments were carried out: since light penetration is inversely proportional to the cell density of a culture, a given light intensity has relatively less effect on a dense culture than on a more dilute one. At intensities above the maximum, both Falkowski and Owens (1978) and Bates and Platt (1984) found reversible photoinhibition. C.

PHOTOSYNTHETICELECTRON FLOW

Ben-Amotz and Avron (1972) showed that D.parva cell fragments were capable of photoreducing ferricyanide, PMS (phenazinemethosulphate) and diquat, indicating that photosystems I and I1 were both active in the system. The fragments were also able to photophosphorylate ADP to ATP. They were not able to photoreduce NADP until Sheffer and Avron (1982) found that thylakoids isolated under conditions from which salt was excluded could carry out the photoreduction of NADP, ferredoxin and cytochrome c+ferredoxin. The photoreduction of ferredoxin and PS I were found to be 50% inhibited by 2 mM NaCl, in contrast to PS 11, which was very much more resistant.

DUNA LIELLA: A GREEN ALGA ADAPTED TO SALT

149

D. RATE OF PHOTOSYNTHESISAFTER LONG PERIOD OF ADJUSTMENT TO A GIVEN NaCI CONCENTRATION

There are two studies of this type (Craigie and McLachlan, 1964; Frank and Wegmann, 1974). In the latter, cultures of D . tertiolectu were grown for two weeks at NaCl concentrations from 0 to 1.7 M NaCl, and the rate of photosynthesis measured from the fixation of I4CO2 per unit of chlorophyll. NaCl concentration was found to have only a minor effect on rate of photosynthesis; there was a slight upward trend with increasing NaCl concentration, demonstrating that salt is not an inhibitor. The same conclusion is drawn from the results of Craigie and McLachlan (1964), summarized in Table XVII. E. EFFECT OF CHANGE OF NaCl CONCENTRATION ON PHOTOSYNTHESIS

Balnokin and Medvedev (1980) isolated chloroplasts (more probably, a ~ chloroplast fraction) of D.sufinu and D . muritimu grown in 1 . 5 and 0.5 M NaCl respectively, resuspended them in 3 M and 1.25 M glycerol respectively, and compared them with isolated bean chloroplasts. In all three cases, optimum O2 evolution in the presence of ferricyanide was found with the addition of -400 mM NaCI. Thus, no special halophilic characters were exhibited by the Dunuliellu chloroplasts. The authors did not test the effect of glycerol on bean chloroplasts, nor the need of Dunulieflu chloroplasts for glycerol. The principal findings on whole Dunuliellu cells are summarized in Table XIX. At first sight, they seem confusing, but several conclusions emerge clearly: 1. Rates of C 0 2 fixation and O2 evolution change drastically with time after a change in NaCl concentration. For example, Frank and Wegmann (1974) and Kessly and Brown (1981) found that after the imposition of salt stress a new level of photosynthesis is reached within minutes; restoration to the control level takes several hours. Gilmour et ul. (1982), on the other hand, found that recovery was virtually complete in 100 min. Thus, the time needed for recovery is variable. 2. The range of NaCl concentration in which measurements are made determines whether the reaction studied will be inhibited or stimulated. The following are examples of hypertonic shocks which have been studied: (a) D. ferriolectu originally in 0.17 M NaCl shows stimulation of O2 evolution when transferred to solutions of 0.174.5 M NaCI; at higher concentrations there is inhibition (Kessly and Brown, 1981). (b) D . tertiolectu originally in 1 M NaCl shows stimulation of C 0 2 fixation in solutions up to 2.7 M NaCl and inhibition at higher concentrations (Frank and Wegmann, 1974).

TABLE XIX Effects of a sudden change in NaCl concentration on photosynthesis in whole Dunaliella cells Species

Range of change

tertiolecta

1.03-tl.03-3

tertiolecta tertiolecta

1.03- 0-1.03 0.1741.5

tertiolecta tertiolecta

1.6- 0.17 0 . 4 4 0.751.75

tertiolecta

0.4404.4

palVa palVa parva

1.542.5 1.5-0.38 1.5+3

patVa salina

1.5-below 1.5-2.1

viridis viridis

f.5+3 or 4 4- below 4

1.5

Effect Incorporation of 14C02increased up to 2.7 M NaCl, then fell. O2evolution increased to 2.1 M NaCI, then fell. Rates changed with time. Most measurements made 45-50 min after shock Both C 0 2 incorporation and O2evolution inhibited Glycerol synthesized; O2evolution increased up to 0.5 M NaCI, then fell. O2measured in first few min after shock Glycerol fell; O2evolution inhibited; recovery took 24 h Glycerol synthesized in light and in dark; O2evolution stopped after 5 min but recovered after 100 min O2evolution inhibited Photosynthesis increased four times No change in photosynthesis CO;?fixation rose to 2.5 M NaCI, then fell. Parabenzoquinone reduction rose to 2.1 M NaCl, then fell C 0 2fixation and parabenzoquinone reduction fell O2evolution increased whether C 0 2 was present or not; in presence of C 0 2 , slight reduction of C 0 2 fixed O2evolution increased up to 3.5 M NaCI, then fell O2evolution inhibited

Reference Frank and Wegmann (1974)

Frank and Wegrnann (1974) Kessly and Brown (1981)

Kessly and Brown (1981) Gilmour et al. (1982) Gilmour et al. (1982) Bruggemann et al. (1978) Bruggemann et al. (1978) Gimmler et al. (1981)

Gimmler et al. (1981) Kaplan et al. (1980) Kessly and Brown (1981) Kessly and Brown (1981)

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

151

(c) D. viridis originally in 1.5 M NaCl shows stimulation of O2 evolution up to 3.5 M NaCl and inhibition at higher concentrations (Kessly and Brown, 1981). (d) All hypotonic shocks (= dilution stresses) are followed by inhibition of photosynthesis, irrespective of the initial NaCl concentration. 3. The photosynthetic quotient is no longer 1 after a change in NaCl concentration. An example is provided by the work of Kaplan et ul. (1980), who measured O2 evolution in D. tertiolectu cells subjected to an increase in NaCl concentration from 1.5 to 2.1 M ; they found a marked increase which was not due to C fixation, since it occurred when the COz concentration was below 2 p ~The . effect was abolished by DCMU and by - ~ The fixation of COz was slightly high concentrations of KCN ( 5 ~ 1 O M). inhibited when the NaCl concentration was raised. The increase in O2 evolution was not, therefore, due to a higher rate of photosynthesis but to the degradation of starch to glycerol: (CHz0),+2HzO+ ATP+ 2NADPH+2Hf+ 2C3H803+02+ADP+Pi+2NADP+ The effect is, however, complicated, since O2 was not evolved in the dark even though starch is known to be degraded and converted to glycerol in the dark. Kaplan et al. (1980) did not demonstrate the appearance of glycerol or disappearance of starch in cells deprived of COz, so their scheme remains speculative. The effect of DCMU was thought to be due to inhibition of the photosynthetic electron transport chain and the consequent lack of ATP and NADPH needed for the breakdown of starch. A second example of this kind of effect is given by the work of Frank and Wegmann (1974), who found different optima for O2 evolution and C 0 2 fixation after hypertonic shocks (2.1 M and 2.7 M NaCl for cells originally in 1.5 M NaCI). Thirdly, Gimmler et al. (1981) found different optima for COz fixation and for the reduction of p-benzoquinone. 4. Uncharged solutes [e.g. deoxyglucose (Wegmann, 1971)] are generally less effective than salt solutions exerting the same osmotic pressure (or causing the same water activity, to use A. D . Brown’s terminology). Thus, photosynthesis is drastically affected by osmotic shocks; the stimulation noticed with relatively small increases in tonicity may be due to increases in concentration of the reacting substances as water is lost from the cell. Similarly, the inhibition noticed after decreased tonicity is explainable in terms of dilution of reactants as water is gained. The major effect of hypertonic shocks is a marked redirection of metabolism towards increased glycerol synthesis, largely from the breakdown of starch; C 0 2 fixation is inhibited and remains so for some hours. The work described so far does not explain how osmotic shocks act to

152

M. GINZBURG

inhibit photosynthesis, except that we may note that there are separate osmotic and ionic effects, the first demonstrated by nonpolar reagents and the second by ionic solutions. Recent work of Gilmour et af. (1982, 1984a,b, 1985) has shed considerable light on the mechanism of inhibition by osmotic shocks. These workers studied the following effects:

1. Fluorescence emission from 650 to 800 nm. 2. Electrochromic absorption change at 519 nm. This is referred to by Gilmour et af. (1984a) as the “flash-induced field-indicating absorption change at 519nm” or A519. It measures the absorption of light by chlorophyll. From it, the magnitude of the charge separation between photosystems I and I1 and ion fluxes across the thylakoid membrane can be deduced. 3. Fluorescence emission at strictly defined wavelengths, particularly at 695 nm. This reflects electron transport as well as the p H gradient across the thylakoid membrane and the degree of spillover of excitation energy between photosystems I and 11, and it therefore provides a sensitive probe of integrated photosynthetic function. It is possible to distinguish between the two photosystems by using actinic light (predominantly absorbed by photosystem I) or a quartz-iodine bulb, the light of which is filtered by a 718 nm interference filter (photosystem I1 light). Gilmour et al. (1982) found that with increase of the NaCl concentration from 0.43 to 0.75 M, and thereafter in steps of 0.25 to 1.75 M, the rate of oxygen evolution decreased progressively almost to 0 within 5 min. There was almost total recovery after 100 min. It should be stressed that the size of their smallest increase almost doubled the initial NaCl concentration; they therefore apparently missed the range of oxygen stimulation. The effects of O2 evolution were paralleled by changes in the A519. KCI, NaCl and to a much lesser degree, sucrose and ethylene glycol, decreased the initial A519, suggesting that the initial charge separations at the reaction centres of photosystems I and I1 were inhibited more by ionic stress than by osmotic stress alone. The initial charge separation due to photosystem I alone was stimulated by high concentrations of NaCI, KCI and sucrose. The inhibition of the charge separation driven by both photosystems at high external osmolarities must therefore be due to inhibition at photosystem 11. Fluorescence studies with actinic light confirmed that photosystem I1 was strongly inhibited when the osmotic agents were NaCl and KCI, but less so with sucrose and ethylene glycol. The major conclusion of Gilmour and co-workers is that osmotic stress alone inhibits photosynthesis to a certain extent but that there is an additional inhibition due to ions. It appears that osmotic stress renders the thylakoid membranes leaky to ions which inhibit photosystem I1 and then photosystem I. Thus, at the time of osmotic shock, ions must cross both the

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

153

plasmalemma and chloroplast membranes. Recovery of inhibition occurs in parallel with synthesis of intracellular glycerol. The question to solve was the nature of the ion or ions which inhibited photosynthesis. The most obvious ions to consider are Na+, CI- and H + . The last of these was eliminated rather indirectly, and it was concluded that photosynthesis is inhibited in salt-stressed cells by an influx of Na+ and C1into the thylakoids. The major inhibitory action is considered to be on photosystem 11. There is an unresolved discrepancy between the conclusions of Gilmour and those of Sheffer and Avron (1982), who found that in D.parva cell fragments photosystem I was highly sensitive to salt. One wonders whether whole cells are necessarily as sensitive as cell fragments.

IX. REGULATION OF CELL VOLUME Wall-less cells separated from their medium only by a plasma membrane have to expend energy in maintaining a constant cell volume. This is because the Donnan pressure exerted by the cell proteins tends to bring about a flow of water into the cell and has to be opposed by reactions leading to the loss of water. Thus, a supply of energy for the process of osmoregulation is necessary to cells living in media of constant salinity. The reactions whereby some wall-less cells, e.g. erythrocytes, maintain a constant cell volume are reasonably well understood. In Dunafiefla, however, they have been neglected up to the present. The cells of this genus respond to changes in external osmotic pressure by alterations in the volume of the cell, which shrinks or swells according to whether the change is in the hypotonic or hypertonic direction. After some time the cell volume returns to what it was before the change. Both phases will be described in detail.

A. IMMEDIATE EFFECT OF CHANGE IN OSMOTIC PRESSURE OF MEDIUM

Wall-less cells are said to behave as osmometers. If so, the cell volume should behave according to the equation which describes the behaviour of an osmometer (Dick, 1966). This is the Boyle-Van’t Hoff equation: r,,(V-b) = +RTni where .rr,= osmotic pressure of the outside solution, I/= cell volume, b = nonosmotic volume of cell, 4 = average osmotic coefficient of the intracellular solute, R = gas constant, T = absolute temperature, and ni = number of moles of solute in the cell. On differentiating the equation with respect to r we obtain:

154

M. GINZBURG

If the membrane of the cell is semipermeable, only two factors have to be taken into account. These are b, the nonosmotic volume, which in many cells is 20-30°/0 of the total cell volume, and d @ d r , the change in the average osmotic coefficient. This may be important for concentrated protein solutions, but in Dunafiella, in which the main solute is glycerol, it is to be expected that d4/dn=0. The osmotic behaviour of the cell should therefore -be governed solely by r / V .dV/dr, provided that interactions of macromolecules and water are unimportant. Teodoresco (1905) was the first to observe the effects of changing salt concentration on Dunaliefla safina cells. He found the shape of the cell to be very variable; cells taken directly from the brine were elongated and ellipsoidal in shape (Fig. 13, 1-8). Cells in brine which was allowed to evaporate became elongated and deformed (Fig. 13, 9 and 10); it is clear that the posterior portion of the cell shrinks while the anterior portion remains more or less the same size. When water was added to the brine, the cells became globular (Fig. 13, 30-31); again, it is evident that the posterior part of the cell was swollen and the anterior portion is unchanged. These observations demonstrate unequivocally that the membrane surrounding the cell is not as selectively permeable as are most cell membranes. If it were, the cell would change in size but not in shape when exposed to osmotic shocks. Ben-Amotz (1974) made quantitative measurements of alterations of cell volume as a function of osmotic pressure of the medium. In D. parva cells grown at 1.5 M NaCl and transferred to concentrations between 0.6 and 1.5 M, the amount of cell water increased linearly with the decrease in salt concentration (see Fig. 14). There was very little decrease in cell water when the NaCl concentration was increased to 2 M. The data do not allow a plot of pellet volume versus l / r to be drawn; it is not, therefore, possible to determine whether all the cell water participates in the osmotic process or not. A careful biophysical study on D. parva was made by Rabinowitch et al. (1975) by means of a particle-size analyser (PSA). Cultures were grown in 1.5 M NaCl. A straight-line relationship was found between cell volume and l / r (Fig. 15), though the resulting nonosmotic volumes were large and variable. It is noticeable that the higher the concentrations at which measurements were made, the higher the nonosmotic volumes (their figures 3 and 4 should be compared in this respect). Solutions were used in which Na+ was replaced by K + , Mg2+, Ca2+ or Cs+; the slight difference in slope of the resulting lines suggests that the effects are not purely osmotic. A major contribution of Rabinowitch et al. (1975) was to analyse the

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

15s

Fig. 13. Line drawings of various forms of Dunuliellu: 1-8. cells kept under isotonic conditions; 9-17, cells in hypertonic medium; 31-34, cells in hypotonic medium. From Teodoresco (1905).

M.GINZBURG

156

c

I 0

c

Glycerol concentration A

A

- 2.0

Glycerol content

c

I

.t

z

c

c

ss,

c

-1.0

8

-

E

c

0

3

water

-

[NaCI],,,

u > m -0

0

(MI

Fig. 14. Effect of [NaCI],,, on intracellular glycerol and water in D. parva. The reaction mixture contained 10 mM MgCI,, 25 mM sodium-potassium phosphate buffer at pH 7.5,s IIIM sorbitol, 3pCiTH0,2pCi’4C-sorbitol,and NaCl at theconcentration indicated. Approximately 400 pg chlorophyll ml-’ D. parva cells. After 120 s incubation, the cells were separated from the medium by centrifugation. From Ben-Amotz (1974).

0

0.5

1.o

1.5

1 / r (osrnoles)-’

Fig. 15. Volume of D. parva cells as a function of the reciprocal of osmotic pressure of the suspending medium. The equilibrium volume at each concentration was reached 3-5 min after resuspension in the appropriate medium. The salt solutions contained 50 mM Tris buffer at pH7.4. Redrawn from Rabinowitch et al. (1975).

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

157

kinetics of the change of volume that occurred during the first 200 s after the change in tonicity of the medium. They assumed that the only substance to cross the membrane was water. On this assumption, water enters or leaves the cell in two phases with kinetic coefficients (crL ) differing by an order of magnitude (4.5 and 0.23 cm3 s-l dyne-'xlO-'') corresponding to the passage of water across two membranes in series (Fig. 16).

Time ( s )

Fig. 16. Change in volume of D.parva cells as function of time after diluting the NaCl in the suspending medium from 1.5 to 0.7 M. The medium included 50 mM Tris at pH 7.4. Redrawn from Rabinowitch et af. (1975).

Gimmler et al. (1977) also made measurements with a PSA on D. parva, but with synchronously dividing cells. They confirmed the straight-line

relationship between cell volume and l l w (Fig. 17). In the intermediate concentration range, they found a nonosmotic volume b of about 10% of the cell volume. At salt concentrations above 2 M, b increased to -33% of the cell volume. They also found that the change in cell volume occurs in two steps; they attributed the first to water movements alone and the second to effects of ion leakage into the cell on the movement of water. They apparently did not consider the possibility of two membranes in series. Ginzburg (1981b) found that when D. parva (strain 19/9) was transferred from 0.5 M to 1 M NaCl the cells shrank and gained Na+ and C1-. The observations were subsequently extended to show that for small increases in tonicity the cells lose water, while for larger increases loss of water is accompanied by gain of Na+ and C1- (Fig. 18). Ehrenfeld and Cousin (1984) measured the Na+ content of D. rertiolecra cells in medium in which the NaCl concentration was increased suddenly from 15 to 410 mM. They found that the Na+ content increased several-fold 2-3 min after the hypertonic shock, while cell water fell by 70%. Thus there is ample evidence in support of Gimmler's hypothesis accounting for the biphasal nature of volume adjustment in terms of water and ion movements.

M.GINZBURG

158

I

I

I

0.02

0.01

I

0.03

I/H (bar)-' Fig. 17. Volume of D.parva cells as a function of the reciprocal of osmotic pressure produced by different concentrations of NaCl (equilibrium volumes, 3-4 min). Insets describe resDonses to very low (A) and very high (B) NaCl concentrations. Redrawn from Gimmler el al. 11977).

glycerol

700 1.2

500

1.0

's 400

0.8

CI,

c

g

300 200

0.6

-

-5f c

B

-

volume

100

[NaCI]( M

Fig. 18. Effect of change of tonicity on D.parva (1919) cells transferred from 0.5 M NaCl to higher concentrations. Measurements were made on cells centrifuged within 60 s of the change in [NaCI],u,. 0 , glycerol; 0, Na'; 0 , volume of cell water in p1 THO. Ginzburg (unpublished).

159

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

In the light of this later work, it is possible to reinterpret the results of Rabinowitch et al. (1975) by saying that the first kinetic coefficient, uL,,, did indeed measure rate of water movement, while the second, lower value was actually an artefact brought about by the effect of ion penetration on water movement. It is worth noting the good agreement between Rabinowitch's crLpof 4.5 cm3 s-l dyne-' x lo-'' (equivalent to 1.65 x cm s-') and Degani and Avron's (1982) measurement of diffusional water permeability of 1 . 5 ~ cm s-l in D.salina (measured by means of NMR). The figures quoted are not overly different from that of 2 x given by Dainty (1963) for erythrocytes and some macroalgal cells. Of which Dunaliella membrane has the permeability been measured'? Degani and Avron (1982) assume that the membrane in question is the plasmalemma, but we have seen already that there are serious reasons for doubting that the permeability of the plasmalemma is low. These reasons were reinforced by M. Ginzburg (1969), who found that D . parva cells were apparently partially permeable to small organic molecules, and by B. 2. Ginzburg (1978), who cast doubt on the ability of the PSA to measure the true volume of Dunaliella cells. The latter compared volume sizes obtained by different methods for different types of cell particles (see Table XX) and found that all methods used gave similar results for polystyrene particles and yeast cells, live and fixed, but that with Dunaliella cells the results obtained with the PSA were consistently lower than those TABLE XX Comparison of methods for determination of particle volunie Method of measurement A

Particle Polystyrene Polystyrene Yeast cells Fixed yeast D . parva (I) (2) (3) D . tertiolecta in 20 mM NaCl in 410 mM NaCl in 1640 mM NaCl

110 165 54 54

49 52 35

B C (volume, m3x lo-")

106-1 18 116-178 63-80 56-65

56

95 92 79 200 156 185

D

88 89 106 89 111

227 211 259

A: volume measured by means of particle-size analyser. B,C: centrifuge methods hy which pellets, corrected for medium trapped between particles, are divided by number of cells in pellets. B: trapped volume measured with dextran with molecular weight of 70,000 or more. C: trapped volume measured with sorbitol. D: volume measured from microphotographs. Results for D.rerriolecra are taken from Ehrenfeld and Cousin (1982); other results from Ginzhurg (1978) and Zmiri CI a/. (198.1).

160

M. GINZBURG

obtained by other methods. The observation, which was subsequently confirmed by Curtain et al. (1983) and Zmiri et al. (1984), showed how important it is to measure cell volumes by direct methods such as observation under the microscope. Ginzburg (1978), who was unaware at that time of Teodoresco's work, suggested that there are one or several organelles within the cell (e.g. chloroplast, mitochondria) which together form the compartment measured by the PSA and are surrounded by impermeable membranes; the outer cell membrane is more permeable and allows relatively free movement to Na', K+, Cl-, and small organic molecules. In the meantime, workers have continued to use the PSA. Riisgard (1979) transferred D.marifima from seawater to more dilute solutions; the cells swelled but a plot of volume versus l/n yielded a curved rather than a straight line, once more casting doubt on the perfect osmometric properties of the cells. Grizeau et al. (1982/3) with D.bioculata found that the initial volume change which occurs when the tonicity of the medium is changed took 1-2 min. This time interval, similar to those obtained by other workers, is far too long for the change in volume to be due solely to movements of water across a single semipermeable membrane. B . RECOVERY OF ORIGINAL CELL VOLUME AFTER OSMOTIC SHOCK

Dunaliella cells that have undergone swelling or shrinking as a result of some change in tonicity of the medium, return in time to the volume before the change (Table XXI). All experiments published on this subject have been done with cells grown in media with NaCl concentrations from 0.5 to 1.5 M NaCl [with the exception of Ehrenfeld and Cousin (1984) who started at 0 . 0 1 5 ~ N a C l J .Thus, there are no studies with cells in high NaCl concentrations. For recovery to occur, the cells must be in the light, or they must have reserves of starch (Gimmler and Moller, 1981; Fig. 19). A possible exception is D . bioculata, which did not recover in the dark (Grizeau et al., 198213). The effects of several inhibitors have been noted (Table XXI). It is not possible at the present time to explain the effects of Cu2+ or of high K + concentrations. The fact that photosynthetic inhibitors such as DCMU and DBMIB inhibit recovery, points to the importance of metabolism, in particular photosynthesis. The process involved in the recovery of cell volume are as follows: 1. Adjustment of cell glycerol content. 2. Loss of Na+ and C1- that may have leaked into the cell. 3. Adjustment of cell water content so as to equalize activities across the cell membranes.

The first and second of these processes require metabolic energy.

TABLE XXI Table to show recovery of Dunaliella cell volume after osmotic shocks Range of change (NaCl], M 1.540.6 1S 4 0 . 7 5 1.543 0 . 4 5 ~ 0 . 7 5 1.2 4 sw+1.2 sw SW-+fSW

0.5-

1

a) 0.75-1.5 b) 1.5-3 0.5- 1

0.5-0.25

160- 923 rnOsrn

Observations on recovery In L and D; 90 rnin Yes No Yes Not in starved cells Unaffected by FCCP, 2 p~ Inhibited by Cuz+ Inhibited by high [K+] Cuz+ effect reversed by low pH and when Na+ is replaced by choline-C1 Yes Delayed by low temperature Yes, L and D Yes, L Yes, Land D; inhibited by dark, O"C, PSic and respiratory inhibitors Yes, L and D; inhibited by high K+, KCN+SHAM, 0°C Yes

Method

Species

PSA

Ben Arnotz (1974) Rabinowitch et al. (1975)

D . parva D. parva

PSA

Girnrnler er al. (1977)

D. parva

PSA

Riisgard (1979)

D . marina

3H20

Ginzburg (1981b)

D . parva 1919

PSA

Girnmler et al. (1981)

D . parva

Grizeau er al. (198Y3)

D . bioculata

3

~

~

0

Grizeau et al. (198Y3) Cytocrit

3~z0 D 70,000

SW: seawater.

Reference

Ehrenfeld and Cousin (1984)

D. tertiolecta

162

M. GINZBURG

1.50

Y

1.5 kmol m-3 NaCl

“4

1.5

a,

/ 1.5

bX.L-m-m

*-*-*1.5+

1

2

3

Time ( h )

Fig. 19. “Volume recovery’.’ of D. parva cells adapted to 0.75 or 1.5MNaCl after hyperosmotic shocks in light ( 0 , O )and dark (m, 0).The arrows indicate the magnitude of the osmotic shock. Experiments were performed on young cells without starch reserves. From Gimmler and Moller, 1981.

1.

Glycerol Synthesis

a. Constant salinity. Under such conditions, glycerol is synthesized only in the light. This was demonstrated on synchronously growing cells of D. parva by Gimmler and Moller (1981) (Fig. 20). Glycerol contents of cells grown under conditions of constant salinity are shown in Fig. 8. The biochemical processes whereby glycerol is synthesized and degraded are described in Section VILA. Reference has already been made to the work of Craigie and McLachlan (1964), which shows that the rate of glycerol synthesis is directly proportional to outside NaCl concentration (Table XVII).

b. After hypertonic shock. Ben-Amotz and Avron (1973) were the first to demonstrate the disappearance and synthesis of glycerol which occurred after subjecting D.parva cells to hypotonic and hypertonic shocks (Figs 21 and 22). It is stated that the synthesis of glycerol after hypertonic shocks

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

163

/-

I

8

16

1

Time (h)

Fig. 20. Glycerol content of D.parva cells adapted to 1.5 M ( x ) or 0.75 M NaCl (0) throughout the life cycle. Results corrected f o r glycerol in medium. In (b) glycerol content is calculated per cell. From Girnmler and Moller, 1981.

occurred at the same rate in the light and in the dark, and that it took 90 min for a new equilibrium to be reached after the change in NaCl concentration. Synthesis of glycerol after an increase in outside NaCl concentration has been demonstrated in the light and in the dark in D . virzdis (Borowitzka et al., 1977) and in D.parva (Gimmler and Moller, 1981). Results for D. parva are shown in Fig. 23. In this case synthesis of glycerol in the dark was found to be dependent on the amount of starch stored within the cells; thus cultures grown at high concentrations of salt and containing little or no starch, synthesized little or no glycerol after a hypertonic shock. Similarly, Wegmann has shown that glycerol formation did not occur in starchdepleted cells of D . tertiolecra (Fig. 24).

164

M. GINZBURG

Time (minutes) Fig. 21. Kinetics of degradation and synthesis of glycerol in D. parva in response to changes in the salt concentration of the medium. A concentrated algal suspension in 1.5 M NaCI, 5 mM phosphate and 2 mM MgCI,, pH 7.5, was diluted to obtain a final concentration of 0.6 M NaCl with other components unchanged. Ninety minutes after dilution, solid NaCl was added to a final concentration of 1.3 M NaCI. From Ben-Amotz and Avron (1973). By permission of The American Society of Plant Physiologists.

2

I

I

I

0

Extracellular NaCl concentration(M) Fig. 22. Effect of [NaCI],,, on cell glycerol concentration in metabolizing cells of D. parva. The reaction mixture contained 25 mM sodium-potassium phosphate buffer at pH 7.5, 10 mM MgClz and NaCl as indicated. Measurements made after 90 min at 25°C in light or in dark. From Ben-Amotz (1974).

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

0

165

47< /* X

E

- x .

1.5 -0.75

I

I

I

4

2

0

I

0

I

4

2

Time (h)

Fig. 23. Glycerol and starch contents of D. parva cells as influenced by hetero-osmotic shocks. Glycerol was measured in the light. starch in the dark. From Gimmler and Moiler (1981).

0

I

0

0

I

x 0

x

0

0

I

-

-.

1

1

1

0

n

o

1

1

0 0

1

1

1

I"1111U153

Effect of transfer of D. lerriolecra cells from 0.35 M to 0.8 M NaCl in the light 0 , 0) or in the dark (W, 0). 0, 0 : cells were depleted of starch before the onset of the cells treated with 5x10^" M DCMU Unpublished results of K . Wegmann. experiment. 0: Fig. 24

(X,

166

M. GINZBURG

D.pama cells subjected to hypertonic shocks were found to synthesize glycerol at the expense of starch, even in the light (Fig. 25). Since photosynthesis is inhibited during the period of glycerol synthesis (Section VIII), it is concluded that glycerol is synthesized from products of starch degradation both in the dark and in the light. There is evidence to show that glycerol is synthesized in response to an increase in osmotic pressure of the medium, whether the osmotic agent is NaCl or sucrose (Borowitzka et al., 1977) or deoxyglucose (Wegmann, 1971). In view of the inability of ethylene glycol to elicit a response in D . tertiolecta cells (Gilmour et al., 1984b), and since the nonpolar agents are less effective than NaCl, care should be used in expressing osmotic pressure in terms of water activity, as is the custom of Brown and his co-workers. Starch loor

A

0

1

3

2

4

5

Hours Glycerol

I

0

I

1

I

I

3

2

I

4

I

5

Hours

I higher NaCl Fig. 25. Effect of transfer of D.parva 1919 cells from 0.5 M N ~ C to concentrations on cell glycerol and starch. Cells were incubated in the light at 25°C. Concentrations to which cells were transferred: 0 ,0.5 M;0, 0.75 M; W , 1 M; 0,1.5 M NaCl. Glycerol was measured in pmole mg-', chlorophyll and starch in pmole glucose (mg chlorophyll)-'. Unpublished results of M. Ginzburg.

DVNALIELLA: A GREEN ALGA ADAPTED TO SALT

167

2. Effect of Inhibitors on Glycerol Synthesis and Disappearance Inhibitors are listed in Table XXII. We note that inhibitors of protein synthesis, cycloheximide and chloramphenicol and RNA synthesis, rifampicin, had no effect on glycerol synthesis, which is not, therefore, dependent on the synthesis of new enzymes. The inhibitory action of FCCP suggests that a supply of ATP is required for glycerol synthesis and disappearance. The effects of DCMU in this system are complex and controversial (Gilmour et al., 1985). TABLE XXII Effect of inhibitors on glycerol synthesis in Dunaliella cells subjected to osmotic shocks in the light Species

[NaCl] jump (M)

Inhibitor

parva

1.5-2.5

FCCP, 8

parva

1.5-0.5

FCCP, 8 p M

tertiolecta

1.03-+2.6 Antimycin A , 10-dM

viridis

1.5j3.5

tertiolecta 0.4- 1.7

Cycloheximide, 2PM, chloramphenicol, 12.5 pg ml-' DCMU, 17 /.LM

Effect

Reference

Prevents glycerol synthesis Prevents loss No effect No effect

Ben-Amotz and Avron (1973) Ben-Amotz and Avron (1973) Frank and Wegmann ( 1974) Borowitzka et al. (1977)

35% inhibition Gilmour et al. (DCMU given (1982) after hypertonic shock)

FCCP, carbonyl cyanide p-triffuoromethoxyphenylhydrazone, an uncoupler, inhibits production of ATP. Antimycin A interrupts respiratory electron transport chain between coenzyme Q and cytochrome b .

C . MEASUREMENTS OF ION CONCENTRATIONS

( K + , Na+ , Cl-)

1. Under Conditions of Constant Salinity Table XXIII presents a summary of ion concentrations measured in several species of Dunuliellu and demonstrates that results may be classified according to method of measurement, rather than by species. All measurements agree in finding less Na+ and C1- and more K+ than in the medium. Thus, Na+ and CI- must be excluded from the cells, while K + is concentrated some 20-150 times.

M.GINZBURG

168

TABLE XXIII Cell ion concentrations in Dunaliella Ion concentrations in cell water (mM) “aC~lo,, (mM)

A.

Na+

K+

C1-

422 517 724 750 4.6

77 62 111 134 70.7

12.9

410 1600 1500

71.7 291.5 770

120.4 150.3

96.7 330

1500

390 180 (log) (log) 680-1100 260

1500

tertiolecta

Jokela (1969)

tertiolecta

Ehrenfeld and Cousin ( 1982)

parva, str.75

Zmiri and Ginzburg (1983) Ginzburg and Ginzburg (1985b)

350 parva, 1919 (log) 930-1100

Trapped volume measured with sorbitol or Lii

750 parva 340 76 1500 800 94 20 -7.6 108 tertiolecta 410 -19.4 178.5 1600 -38.9 265.5 500 -30 salina 1000 1500 -40 2000 0 3000 - 100 4000 -100 1000 20 bardawil 1500 20 2000 80 3000 100 4000 100 C. Cells washed in isotonic non-polar solute 20 410 1600 20 3500 500 2000

Reference

Trapped volume measured with dextrans (Mol.wt. 70,000-2X lo6)

259 1034 1724 2586 20

B.

Strain

2.2 6.6 7.2 0 0 250 400

66.9 106 118

4.4 14 13.7

tertiolecta

maritima salina

Gimmler and Schirling (1978) Ehrenfeld and Cousin ( 1982) Katz and Avron (1985)

Katz and Avron (1985)

Ehrenfeld and Cousin (1982) Latorella and Vadas (1973) Balnokin et al. (1979)

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

169

The cell Na+ concentration is found to vary around 0 when measured in cells washed in glycerol or when Li+ or sorbitol is used as marker of the medium in the intercellular space in centrifuged pellets. With markers of molecular weight of 70,000 or above, cell Na+ and CI- concentrations have been variable but well above 0; when outside NaCl concentration is 1500-1700 mM, cell Na+ concentration values of 291.5 to 1100 mM have been measured, depending on age of culture. The low values are typical of actively growing cells. Katz and Avron (1985) consider that the larger molecules used as probes are prevented from equilibrating fully within the intercellular coat which extends into the medium beyond the cytoplasmic membrane (Oliveira et a l . , 1980). Ginzburg and Richman (1985) calculated the volume occupied by the surface coat and found that it occupied 2-370 of the total cell volume, certainly not enough to account for the large difference in intercellular spaces measured by sorbitol and by high molecular weight dextrans, which according to the data of Ehrenfeld and Cousin (1982) amounts to over half the cell volume and is equivalent to a layer 0.9pm around the outside of the cell (see Table XX). The difference between the two sets of measurements can be related to the finding of B. Z. Ginzburg that PSA measurements gave low estimates of cell volume. Thus there are two separate sets of observations which suggest that the Dunaliella cell consists of two physiological compartments. The inner normally has a very low Na+ content and is surrounded by a membrane impermeable to Lif, Na+, sorbitol and other small molecules. This compartment consists of the chloroplast and other small cell organelles. The second, or outer, compartment is surrounded by a membrane impermeable to high molecular weight dextrans, though not to small ions and soluble sugars: 8&90% of the cell Na+ and C1- is postulated to be within the compartment and is washed out when the cells are resuspended in glycerol. An advantage of this hypothesis is that it can account for the similarity between cell Na+ concentration measured in washed cells and in pellets in which the intercellular volume is measured with sorbitol or Li+. It also accounts for the similar values obtained for cell volumes measured by microphotography and from pellet volumekell number when the intercellular volume is measured with a high molecular weight dextran (Table XX). Katz and Avron (1985) comment that the image measured by optical microscopic estimations may be larger than the space occupied by the cytoplasmic membrane. No reason is given for this assumption. 2. Regulation of Cell Ion Concentrations in Dunaliella There is considerable evidence to show that cell Na+ and CI- are maintained at their usual levels by the expenditure of metabolic energy and with the aid of other cations in the outside medium. Ehrenfeld and Cousin

170

M. GINZBURG

(1982) found that incubation of D. tertiolectu cells in medium without K+ brought about increases of cell Na+ and a fall in cell K+ (Fig. 26).

1

KCI

r

Fig. 26. Effect of [K'],,, on Na+ and K + contents of D. rerriolecra cells in 1 6 4 0 m ~ N a C I .At time 0 the cells were transferred to K+-free medium. 1 0 m ~ K C lwas added at the time indicated by the arrow. From Ehrenfeld and Cousin (1982).

Similarly, Ginzburg and Ginzburg have found increased cell Na+ in two Dunuliellu isolates incubated in the dark without K+ or phosphate (Tables XXIV and XXV). In one case (D.parva, 19/9), loss of cell Naf was brought about merely by returning the cell suspension to the light. The other isolate, C9AA, which is probably a D. sulina, lost Na+ only after incubation in the light and the addition of K+ and phosphate to the medium (Table XXV). TABLE X X I V Effect of incubation in the dark and of subsequent return to light on solute concentrations in Dunaliella parva 1919. There was no K+ or phosphate in the medium used for incubation in the dark. Results for the subsequent light treatment consist of a group of experiments, to some of which K + andlor phosphate had been added. Time in dark: 24 h; time in light: 2 h. NaCI concentration in medium: 1.5 M. Concentration (mM) Condition Log-phase cell? After dark After dark + light

Na+ 440f 20 1154f39 562 f 15

"260 976f 35 541 f 24

1950f50 1281+46 1520 f20

Number of Orthodeterphosphate minations 225 112' n.m.

aFrom Ginzburg and Ginzburg (1985b). borthophosphate measured in cells in 2 M NaCl and left for 48 h in the dark.

4 8

171

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

TABLE XXV Cell Na+ and CI- concentrations and water content in Dunaliella C9AA cells incubated in the dark for 24 h and then for an additional 2 h period in the light. The medium used for the dark incubation contained 3 ~ N a c land all the usual components of the growth medium except for K + and phosphate, which were absent. K + and phosphate were added in concentrations of 20 and 2 mM respectively in the light, where indicated in the table. Number of experiments is shown in brackets. Condition of incubation

Iodchlorophyll (n mol pg-l) Na'

c1-

Dark (5) Light (2) Light + K+ (3) Light+phosphate

207k 15 250 4 20 186 k 8 154425

2342 11 230 2 25 199 k 16 151226

(3) Light + K+ +phosphate (4)

153k12

165211

H20/chlor.

iodcell water (mM)

(nl CLg-') Na+

c1-

9944 125 f. 12 120 5 12 159f.12

21434150 2032 4 220 1668f.150 1119f220

2374rt 133 1911 4 260 1751+_ 134 11084210

160t10

1043+60

1112f90

Ehrenfeld and Cousin (1982) found that the level of K+ needed to counteract a given concentration of NaCl increases with [NaCI],,,. Thus, 0.1 mM KCI was sufficient when [NaCI],,, was 20 mM; 0.5 and 5 mM KCI were needed when [NaCl],,, values were 410 and 1640 mM respectively. The same workers found that Na+ was exchanged for K+ in the ratio of 1:l. The exchange was inhibited in the dark, by cold and by DCCD. They linked their results to a Na+/Kt ATPase isolated from Dunaliellu cell membranes by Jokela (1969). On the other hand, the very large reductions in cell Na+ and Cl+ shown in Table XXIV cannot be linked with K+ movements in the opposite direction; a Na+/H+ exchange mechanism must be invoked. Latorella and Vadas (1973) and Kaplan and Schreiber (1981) have found indirect evidence for Na+/H+ exchange. At the present time, the chief importance of these experiments lies in their demonstration of an active mechanism for the control of cell Na+ and C1-. While the concentrations of these ions in a large part of the Dunaliellu cell are clearly very low, the maintenance of the low value is due to the continuous supply of metabolic energy and to the existence of ion pumps. In some other major part of the cell, the Na+ concentration must be close to that in the external medium. It may be debated whether this part is extra- or intracellular; no extracellular layer has been identified that could account for it, and the possibility must be considered that the plasmalemma has permeability properties similar to those of the outer membrane of the chloroplast, the mitochondrion and the E. coli cell (Ginzburg and Richman, 1985).

3. Ion Exchanges After Hypertonic Shocks Ginzburg (1981b) measured the ion content of D. p a r v a cells in media in

172

M. GINZBURG

which the NaCl concentration had been raised from 0.5 to 1 M, and followed the change in ion concentrations for the next 3 h (Fig. 27). There was a net influx of Na+ which was complete 30 min after the hypertonic shock; as time progressed, the cell Na+ fell and eventually reached the concentration found beforehand. Mg2+ was found to be needed for efflux of Na+. The experiments were repeated and elaborated by Ehrenfeld and Cousin (1984) on D. tertiolecfu. These authors measured cell Na+ by the three methods indicated in Table XX, and found that when [NaCI],,, was increased from 15 to 410 mM, cell Na+ increased several-fold and then fell somewhat. The fall depended on the presence of 1 mM K + in the medium. There was a sudden short increase in cell K + which took place 10-30 min

1.51

u 0 40 80 120 Minutes Fig. 27. A: Effect of osmotic shock on water content of D.parvu (1919) cells. Results at time 0 represent cells in 0.5 M NaCI. The NaCl concentration was increased immediately afterwards; all points subsequent to the first refer to cells in 1 M NaCI. B: K + (0)and Na+ (0)concentrations of cells in Fig. 26A. Temperature, 25°C. Na' concentrations have been correction for Na+ in the extracellular medium. From Ginzburg (1981b).

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

173

after the loss of Na+ and only if the medium contained Ca2+ or Mg2+. It was inhibited by DCCD, FCCP and DCMU, though not by ouabain, cyanide or PCMBS. The authors concluded that the net loss of Na+ and net gain of K+ cannot be linked directly, because of the time delay between the two phenomena. They proposed independent pumps, one for the exchange of Na+ and K + , and another for K + uptake. The actions of FCCP and DCMU show that K + influx is dependent on the production of ATP and on photosystem I. 4. Mechanisms of ton Regulation Cell Na+ and CI- are maintained at low values under conditions of constant salinity by the operation of a Na+/K+ exchange mechanism which opposes the passive diffusion of Na+ into the cell. At high external NaCl concentrations an additional mechanism is needed, dependent on light energy and on the presence of K + , Mg2+ or Ca2+ and phosphate in the medium. After hypertonic shocks brought about by an increase of [NaCl],,,, there is a net influx of Na+ into the inner cell compartment. This is subsequently lost as long as the medium contains K + or MgL+,possibly by the same mechanisms that operate normally in the light.

D . CAN OSMOREGULATION BE ACCOUNTED FOR ENTIRELY IN TERMS OF GLYCEROL SYNTHESIS AND DEGRADATION?

This view is quoted in recent textbooks on algal physiology. It is based on several faulty assumptions, namely that the osmotic pressure exerted by the cell glycerol is equal and opposite to the osmotic pressure of the medium, that the cell membrane is impermeable to small molecules and to ions, and that cells respond to osmotic shocks solely by changing the rate of glycerol synthesis. Measurements of glycerol concentration in terms of moles of glycerol per litre of cell water have been summarized by Ginzburg and Ginzburg (1985) (Fig. 28). This is the only parameter from which osmotic pressure can be calculated. The measured glycerol concentrations are insufficient to account for the outside osmotic pressure. The permeability of Dunaliefla cells to glycerol and to water has been measured; permeability to glycerol was found to be < 5 x lo-'' cm s-' (Brown et al., 1982b), and to water 1 . 5 - 1 . 8 ~ 1 0 - ~ c m s - '(Degani and Avron, 1982). These are very low values which demonstrate the existence of highly impermeable membranes in Dunaliella cells. Nevertheless, the impermeable membranes have not been identified with the outer cell membranes.

M.GINZBURG

174

[NaCI] ( M )

Fig. 28. Glycerol concentrations in Dunaliella. V , Ben-Amotz and Avron (1973); W , Ben-Amotz and Avron (1980); A , Borowitzka and Brown (1974); A,Gimmler and Schirling Ginzburg and Ginzburg (1985~).The solid line represents molal concentrations of (1978); glycerol equivalent to NaCI, in M on the abscissa. From Ginzburg and Ginzburg (198%).

+,

In order to hold the view that the outer cell membrane (plasmalemma) is impermeable, it is necessary to ignore Teodoresco’s observations and the big discrepancy that exists between cell volumes calculated with the use of high molecular weight dextrans and with sorbitol (Table XX), as well as the references made to unusual reactions with sorbitol, glucose and inulin (e.g. Kaplan and Schreiber, 1981; Riisgard, 1979; Ginzburg and Richman, 1985). The holders of the view that osmoregulation can be accounted for solely in terms of glycerol synthesis or degradation are obliged to ignore all the work done on ion content as well as on the movements of ions after hypertonic shocks. They are also forced to ignore the detailed studies of change of volume after hypertonic shocks, which showed that shrinkage occurred in two stages as well as the work of Gilmour et ul. (1982, 1984a,b, 1985) on photosynthesis. They also ignore the different effects caused by high concentrations of nonpolar agents (sucrose, ethylene glycol) and salts. Thus the hypothesis that changes in glycerol concentration alone are responsible for osmoregulation in Dunaliella cells is partly incorrect and partly incomplete. E. TWO-COMPARTMENT HYPOTHESIS FOR THE REGULATION OF CELL VOLUME

According to this model, the Dunaliella cell consists of two physiological compartments. The inner, of which the major components are assumed to

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

175

be the chloroplast, mitochondria and nucleus, comprises 50-70% of the cell (Ginzburg and Ginzburg, 1 9 8 5 ~Ginzburg ; and Richman, 1985). In this view, it is the organelles comprising this compartment which are surrounded by membranes with very low permeabilities to glycerol and small ions. In this compartment, which is essentially identical to the whole cell as seen by Avron and co-workers, the osmotic pressure is maintained by glycerol, Kf and phosphate; the salt content is kept low by action of metabolically driven ion pumps which counteract passive diffusion into the cell. The outer compartment is assumed to lie between the inner compartment and the medium. It is bounded on the inner side by the impermeable membranes of the inner compartment and on the outer by a membrane permeable to small ions and to small, soluble carbohydrates. It resembles the outer membrane of the chloroplast and mitochondrion. The major solutes in this compartment are Na+ and C1+. Because of the permeability properties of the outer membrane, this compartment is always at equilibrium with the medium. Hypertonic osmotic shocks bring about movements of water across both sets of membranes, including the impermeable ones, which become leaky to salt. The ensuing inrush of salt has several effects, particularly on photosynthesis, which is inhibited. The metabolism of the cell is diverted to the synthesis of glycerol and the extrusion of salt; these reactions take about 90 min to bring the cell back to its former condition. At the same time other reactions, as yet unknown, take place for about 24 hours. After this time growth may resume at the same rate as before the osmotic shock.

X. CONCLUSIONS This chapter is an attempt to draw a coherent picture from the sometimes confused literature on Dunafiella. The confusion has been found to have two sources. There is wide variation in units of measurement adopted by different workers, who record light intensity and rates of metabolic activity in ways often hard to bring to a common denominator. A second and more interesting reason emerges from the nature of the Dunufieffucell, which possesses extraordinary capacities of adaptation to environmental conditions. Few other organisms are able to grow over as wide ranges of pH and temperature and to be as accommodating to changes in composition of the growth medium. It seems that tolerance to salt is but one aspect of a general indifference to environmental conditions. Furthermore, cells of the same strain exhibit large differences in cell composition without overt physiological effects. Differences between species are few and hard to define; in any case, most of the work on Dunafiella has been done on a limited number of strains. Thus the variations encountered in cell composition and in response to the environment cannot be ascribed to specific

176

M.GINZBURG

differences. The causes of the remarkable adaptability of Dunaliella cells are as yet unknown. Dunaliella is indistinguishable in many respects from other green unicellular algae. Thus, morphologically, the green-celled members of the genus are similar to Chlamydornonus, while the red-celled members differ only in size and colour. Growth conditions are the same, except for minor differences in optimum light intensity and temperature, which may be of ecological significance since the ability of Dunaliella cells to tolerate somewhat higher temperatures than other members of the phytoplankton may be a determining factor in selection of habitat. The physiological mechanisms for uptake of N and C have been shown to be the same in Dunaliella as in other unicellular algae. So far, the only physiological character which has been much studied is Dunaliella’s unusual tolerance to salt. In this respect the genus is not as singular among green plants as is sometimes supposed; other species, e.g. Stephanopteris gracilis and Stichococcus s p . , are as tolerant, though they are still relatively unstudied. Dunaliella has been shown to be limited to temperate and subtropical bodies of salt water, where temperatures are neither extremely cold nor hot (Tables I1 and 111). Where Dunuliella does occur in pure stand, as for instance in the Great Salt Lake, Dead Sea and Pink Lake, it grows only during short periods of time after the surface waters have been diluted by rainwater. The alga does not grow in nature at salt concentrations much above 20%. Thus there are distinct limitations to the salt tolerance of the genus. Extravagant claims should be avoided. Dunaliefla cells are able to survive for long periods of time in saturated salt solutions and even among damp salt crystals in the light. The nature of this type of survival remains as yet unstudied. It is curious that no attention has been given to what is undoubtedly an unusual property. Dunaliella cells exhibit several characters which distinguish it from related algae. In nearly every case, the character consists of a modification of some property common to other algae. These distinguishing characters are listed below: A . Morphological

1. Lack of rigid cell wall. 2. Vacuolar structures consisting of concentrically arranged membranes (D.salina).

B. Physiological 1 . Cell divided into two regions surrounded by membranes with widely differing permeability properties. 2. Ability to withstand higher temperatures and light intensities than is usual in green algae.

DUNALIELLA: A GREEN ALGA ADAPTED TO SALT

177

3. Ability to concentrate intracellularly large amounts of glycerol and salts which match the high osmotic pressure of the outside medium. 4. Ability to extrude Na+ and C1- against high concentration gradients.

C. Biochemical 1. Proteins with net negative charges. 2. Variable sensitivity of enzymes to salt. 3. Modification of K, of certain enzymes, e.g. glycerol dehydrogenase. The consequences of these modifications are that Dunaliella cells can grow in media containing more salt than does seawater, and can also withstand sudden large changes in osmotic pressure without bursting on the one hand or becoming coagulated on the other. The manner whereby the modifications listed above act together in harmony is virtually unknown; the only processes so far studied in any depth are the synthesis and degradation of glycerol. These are no doubt interesting and important reactions. The regulation of cell glycerol content is, however, only one feature of a whole spectrum of osmoregulatory characters. To equate glycerol synthesis with osmoregulation is totally false. What is the genetic basis of adaptation to salt? Are all the apparently unrelated modifications listed above controlled by the same gene? The answer to these questions is not known. When comparing Dunaliellu with other halophilic microorganisms, e.g. halophilic bacteria, blue-green algae, halophilic Chlamydomonus, and Ochromonas, one is struck by the way in which certain characters appear in unrelated genera. Among these may be mentioned the absence of a rigid cell wall, high concentrations of some intracellular solutes, highly acidic cell proteins, and regions of the cell acting as buffer zones between the medium and certain cell compartments. It is the possession of one or more of these characters which enables microorganisms to survive in concentrated salt solutions.

ACKNOWLEDGEMENTS I would like to thank Professors David Jennings, who suggested writing this review, and Ben-Zion Ginzburg for many ideas and constant encouragement. Practical help has been given by Dr Brian Loughman and several other members of the Department of Plant Sciences at Oxford, where much of this review was written. The Botany Department, University of Liverpool, very kindly allowed me to make use of its word processor. I am indebted to Professor K. Wegmann, who has allowed me to use unpublished material. Figure 6 was drawn by Rosemary Wise. I am grateful for financial help given jointly by The Royal Society and the Israel Academy of Humanities and Sciences.

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AUTHOR INDEX

A Abdullaev, A. A,, 102. 115, 178 Ackman, R. G., 128, 130, 132, 146, 147, 178 Adams, S. R., 40, 41, 86 Ades, I . Z., 8 22 Aedo, A. R., 77, 88 Ahmad, I., 142, 173 Aizawa, K., 1 1 I , 135, 140, 178 Akiyoshi, D. E., 43, 89 Alberts, A. W., 34, 86 Allen, B. E., 40, 42, 87 Amancio, S., 113, I 15, 182 Anastasis, P., 29, 30, 87 Antia, N. J., 122, I82 Apcl, K., 82, 87 Appleby, G., 135, 178 a p Rees, T., 57, 58, 62, 69, 72, 87, 89, 91 Arebalo, R. E., 30, 37, 59, 87 Ashmore, M. R., 114, 181 Audley, B. G., 30, 70, 89 Avron, M., 98, 115. 126, 127, 128, 131, 133, 135. 136, 141, 144, 148, 150, 151, 153, 159, 162, 164, 167, 168, 169, 173, 174, 178. 179. 181, 183

Beevers, L., 137, 178 Beg, Z. H., 77, 87 Bcllini, E., 119, 122, 123, 125, 126, 183 Bclton, J. C., 125, 182 Ben-Amotz, A,, 98, 105, 115, 126, 127, 128, 131, 132, 133, 135, 136, 141, 148, 154. 156, 161, 162, 164, 167, 174, 178. 179 Benson, S. A., 3, 22 Benz, J., 46, 47, 64, 74, 87. 90. 91 Berkaloff, C., 121, 178 Bernard-Dagan, C., 38, 63, 64,8R Berry, J. A,, 110, I81 Bcyer, P., 45, 63, 66. 87, 89 Bcytia, E., 36, 43, 77, 78, 87, 88, 90 Bickel, H., 55, 87 Bidwell, R. G. S., 146, 147, 148, I83 BicnTang, P. K., 109, 178 Bisalputra, T., 122, 182 Bitsch. A., 16, 22 Blobel, G., 2, 20, 22, 23 Block, M. A,, 46, 48, 63. 64. 77. 87, 91 Bohni. P. C.. 12, 23, 23 Boney, A. D., 149, 150, 152, 166, 167, 174, 180 Bonner, J., 28, 30, 89 Bonner, W. M., 4, 22 Borowitzka, L. J., 105, 106, 130, 135, 136, 144, 163, 166, 167, 174, 178 Boutry, M., 8, 22 Braithwaitc, G. D., 29, 87 Brake. A. J., 21, 23 Brand, L. E., 113, 182 Brangeon, J., 62, 87 Brcwcr, H . B., 77, 87 Brcwcr, P. G., 109, 178 Brieolis, A. V., 42, 44, 45, XY Brindle, P.A,, 83, 87 Brisou, J., 98, 178 Britton, G., 54, 61, 65, 87, 90 Brook, T. D.. 98, 99, 178

B Baas Bccking, L. G. M., 96, 97, 178, 183 Bach, T. J., 30, 32, 33, 34, 50, 51, 52, 54, 58. 59, 60, 61, 74, 76, 81, 82, 87, 88, 89, 90 Baisted. D. J., 43, 80. 81. 88 Baker, A,, 20. 21, 22 Baker, N. R., 152, 167, 174, 180 Balnokin, Y u V., 149, 168, 178 Banthorpe, D. V., 40, 42, 76, 87 Barclat, F., 46, 47, 64, 66, 79, 87, 88 Bates, S . S., 145, 148, 178 Batschauer, A,, 82, 87 Beardall. J., 146, 178 185

AUTHOR INDEX

186

Brodie, J. D., 29, 87 Bromberg, S. K., 138, 181 Brooker, J. D., 30, 31, 33, 34, 58, 59, 60, 72, 81, 82, 87 Brown, A. D., 105, 106, 122, 130, 135, 136, 144, 149, 150, 151, 163, 166, 167, 174, 178, 181, 182 Brownlee, C., 128, I80 Bruemmer, J. H., 36, 90 Bruff, B. S., 97, 135, 141, 142, 181 Bruggemann, M., 113, 115, 145, 150, I 78 Bucher, T., 136, 181 Bucholtz, M. L., 42, 44, 45, 89 Buggy, M. J., 61, 65, 87 Burton, H. R., 98, 180 Butcher, R. W., 96, 97, 118, 178 Butow, R. A., 8, 22 C Canvin, D. T., 11 1, 181 Camara, B., 45, 46, 47, 59, 62, 63, 64, 66, 79, 83, 87, 88 Cashmore, A., 2, 14, IS, 22 Charlton, J. M., 61, 64, 65, 87 Chasey, D., 121, 122, 181 Chauhun, V. D., 97, 182 Chayet, L., 40, 41, 75, 88 Chen, C. M., 43, 86, 8 7 Cherep, M. N., 135, 178, 179 Cheucas, L., 132, 179 Chua, N-H., 8, 14, 22 Cline, K., 15, 16, 22 Coatsworth, J. L., 113, 114, 117, 145, I79 Cockburn, B. J., 65, 87 Colbeck, J., 135, 178 Colman, A,, 2, 6, 22 Colonia, A., 62, 65, 88 Cooke, R. J., 62, 65, 88 Coolbaugh, R. C., 68, 76, 89 Cori, 0.,36, 38, 40, 41, 75, 77, 78, 87, 88.89

Coughlan, S., 112, I79 Courtois, D., 98, 178 Cousin, J.-L., 157, 159, 160, 161, 168, 169, 170, 171, 172, 179 Craigie, J. S., 128, 130, 146, 147, 149, 179 Croteau, R., 42, 88

Culver, D. A., 111, 181 Curtain, C. C., 97, 160, 179

D Dainty, J., 159, 179 Daleo, G. R., 50, 88 Daniel, A. F., 29, 88 Davidson, H., 60, 90 Deason, T. R., 95, 179 Degani, H., 159, 173, 178, 179 de Kruijff, B., 19, 24 De La Funte, M., 40, 41, 75, 88 Denis, F., 98, 178 Dennis, D. T., 40, 63, 64, 66, 88, 90 d’Harlingue, A,, 46, 88 Dick, D. A. T., 153, 179 Dingwall, C., 2, 4, 5, 22 Dirkse, W. G., 43, 88 Dobberstein, B., 2, 22 Dodge, J. D., 120, I 7 9 Dodson, A. N., 117, 183 Dogbo, O., 46, 47, 64, 66, 79, 88 Doll, M., 34, 60, 88 Doolittle, W. F., 20, 23 Dorsey, J. K., 36, 88 Douce, R., 46, 48, 63, 64, 77, 57, 91 Douglas, M. G., 2, 6, 11, 23 Dudley, M. W., 83, 84, 85, 91 Deuber, M. T., 83, 84, 85, 91 Duffus, C. M., 128, 179 Duncan, J. D., 47,88 Durrand, A. M., 1 13, 115, 182 E Earnshaw, W. C., 4, 23 Eddy, B. P., 130, 179 Edmond, J., 50, 88 Ehrenberg, L., 29, 88 Ehrenfeld, L., 157, 159, 160, 161, 168, 169, 170, 171, 172, 179 Eichenburger, W., 131, 132, 179 Eilcrs, M., 19, 23 Eisenbcrg, H., 128, 179 Elazari-Volcani, B., 97, 179 Elias, B. A., 57, 88 Ellis, R. J., 1-24, 2, 13, 14, 15, 17, 18, 20, 2 1, 23, 24 Epplcy, R. W., 112, 113, 114, 116, 117, 118, 145, 179 Ettl, H., 95, 179

AUTHOR INDEX

Evans, R. W., 131, 132, 179 Eyden, 120, 121, 122, 179

F Faini, F., 38, 41, 89 Falkowski, P.G., 145, 148, 179 Feldbruegge, D. H., 44, 64, YO Feldherr, C. M., 6, 23 Fellows, B., 64, 90 Finel, M., 135, 179, 183 Fischer, I., 74, 87 Flemming, 1. D., 130, 179 Flick, J. S., 43, 88 Flint, A. P. F., 36, 88 Flowers, T. J., 142, I80 Forn, B., 55, 57, 88 Fowden, L., 129, 179 Frank, G., 105, 145, 148, 150, 151, 167, 179

Frederick, S. E., 135, 180 Freer, I., 29, 30, 87 Fried, A,, 131, 132, 179 Friedman, A,, 98, 181 Frost, R. G., 48, 88 Fujii, S., 107, 108, 179 Fujita. M., 83, 84, 90 Fukui, F., 97, 183 G Galli, M. G., 119, 122, 123, 125, 126, I83 Galloway, R. A., 115, 181 Garcia-Peregrin, E., 36, 62, 65. 88, 91 Gasser, S . M., 13, 23 Gaudilliire, J.-P., 46, 88 George-Nascimento, C., 38, 77, 88 Gibbs, N., 129, 17Y Gibor, A,, 101, 106, 108, 109, 179 Gillespie, D. M., 97, 98, 99, 100, 183 Gillespie, L. L., 9, 23 Gilmour, D. J., 149, 150, 152, 166, 167, 174, I80 Gimmler, H., 105, 106, 107, 113, 115, 130, 131, 135, 136, 141, 142. 144, 145, 147, 150, 151, 157, 158, 160, 161, 162, 163, 165, 168, 174, 178, 179, I80 Ginzburg, B. Z., 97, 105, 131, 132, 135, 142, 154, 156, 157, 159, 160, 161, 168, 169, 170, 172, 173, 174, 175, 178, 179, 180, 182, 183

187

Ginzburg, M., 93-183, 97, 105, 128, 131, 132, 142, 157, 159, 168, 169, 170, 171, 172, 173. 174, 175, 179, 180 Givan, C . V., 55, 57, 88 Gleizes, M., 38, 42, 63, 64, 88 Glover, H. E., 135, 139, 146, 178, 180, 182 Goad, J., 29, 34, 69, 70, 88, YO Goldberg, S. B., 43, 88 Goldfarb, D. S., 5, 23 Goldman, J . C., 109, 115, 116, 126, 178, 180

Gollmer, I., 82, 87 Goodwin, T. W., 29, 53, 54, 55, 57, 62, 64, 65, 67, 68, 81, 87, 88, 90, 91 Graebe, J., 43, 88 Grant, B. R., 110, 112, 135, 137, 138, 146, 147, 148, 180, /&'I, I83 Gray, J. C., 25-91, 36, 37, 62, 72, 74, 75, 82. 813 Gray, M. W., 20, 23 Green, T. R., 37, 40, 42, 43, 63, 64, 66, 80, 81, 88 Griffiths, W. T., 54, 67, 68, 82, 88, 89, 91

Grizeau, D. N., 160, 161, 180 Grossman, A., 16, 23 Grover, N. B., 154, 156, 157, 159, 161, 182 Grunibach, K. H., 55, 57. 60, 88 Guiard, B., 13, 24 Guillard, R. R. L., 101, I80 Guthoff, C., 46, 47, 90

H Hageman, J., 18, 23 Hageman, R. H., 137, 178 Hall, J. L., 142, 180 Hammer, U. T., 97, 98, 100, 180 Hampp, R., 65, 90, Y I Hard, R. N., 98, 180 Harmey, M. A,, 13, 23 Hartmann, G., 29,89 Harvey, D. M. R., 142, I80 Hasc, T., 9. 23 Hashagen, U., 40, 41, 72, 88 Haus, M., 134, 135, 136, 180 Hay, R., 2, 6, 23 Haynes, R. C., 98, 180 Heidekamp, F., 43, 89

AUTHOR INDEX

188

Heimer, Y. A,, 135, 138, 139, 144, 180, 181

Heineke, D., 57, 90 Heinstein, P., 38, 39, 40, 41, 42, 86, 91 Heldt, H. W., 74, 91 Helenius, A., 32, 89 Hellebust, J. A., 103, 135, 142, 178, I81 Hepper, C. M., 30, 70,89 Higgins, M. J. P., 29, 89 Highfield, P. E., 2, 13, 14, 23 Hill, H. M., 35, 62, 89 Hille, J., 43, 89 Hipkins, M. F., 149, 150, 152, 166, 167, 174, 180

Hirata, T., 29, 9 1 Hirosawa, T., 115, 182 Holdsworth, E. S., 135, 178 Holland, R., 55, 57, 90 Hommes, N. G., 43,89 Horwich, A. L., 12, 23 Hoshaw, R. W., 120, 123, 125, 181 Houser, A. R., 44, 64, 90 Howe, J., 38, 39. 40. 41, 42, 91 Howton, M. M., 50, 91 Hurt, E. C., 2, 6, 8, 9, 10, 11, 17, 21, 23 Hyams, J., 121, 122, 181

Jones, T. W., 115, 181 Joyard, J.,, 46, 48, 63, 64, 77, 87, 91

K Kaaden, R., 135, 141, 142, 144, 180 Kaethner, T. M., 58, 62, 72,89 Kalderon, D., 5, 23 Kaplan, A., 98, 111, 145, 150, 151, 171, 174, 181, 183 Kaplan, 1. R., 97, 178, 181 Karlin-Neumann, G . A., 14, 15, 23 Kates, M., 131, 132, 179 Katiyar, S. S., 42, 44, 45, 89 Katz, A.,98, 115, 126, 127, 128, 131, 133, 168, 169, 178, 181 Kerwick, R. G . O., 29, 36, 37, 62, 68, 72, 74, 75, 76, 82, 88, 89, 91 Kellner, G., 121, 183 Kemmerling, M., 46, 48, 64, YO Kent, B., 142, 180 Kerby, N. W., 110, 181 Kessly, D. S., 135, 136, 149, 150, 151, 163, 166, 167, 178, 181

Kilham, P., 98, 182 Kirchner, U., 135, 141, 142, 180 Kleinig, H., 44, 46, 48, 50, 53, 59, 62, 63, 64, 66, 67, 79, 80, 86, 87, 89

I Iccho, K., 29, 91 Imhoff, J. F., 97, 98, 181 Incoll, L. D., 114, 181 Isa, R. B. M., 68, 77, 89 Islam, M. A., 42, 89 Ito, R., 84, 89 Ivancic, N. M., 97, 160, 179 Izdrd, c., 120, 181

J Jacob, G., 38,41,89 Jeanne, N., 160, 161, 180 Jedlicki, E., 38, 41, 89 Jeffrey, S. W., 115, 183 Jennings, D. H., 128, I80 Jitts, H. R., 114, 181 Johnson, E. J., 97, 135, 141, 142, 181 Johnson, M. A,, 42, 88 Johnson, M. K., 97, 135, 141, 142, 181 Johnston, J. A., 28, 30, 89 Jokela, A. C. C. T., 129, 168, 171, 181

Klingenberg, M., 136, 181 Kloppstech, K., 16, 22 Knauf, M., 57,89 Knight, J. S., 68, 76, 77, 78, 79, 82, 90 Knotz, J., 68, 76, 89 Kombrink, E., 98, 135, 140, 141, 181 Koyama, T., 38, 39, 40, 74, 75, 76, 90 Kreuz, K., 44, 46, 53, 59, 62, 63, 64, 66, 67, 79, 80, 86, 87, 89

Krishnawamy, S., 120, 182 Kuhn, D. M., 57,89 Kukaeva, E. A., 168, 178 Kuntz, M., 17, 23 Kwon, Y . M., 112, 181 L Lakshmanan, M. R.,70,89 Laskey, R. A., 2, 4, 5, 22, 23 Latorella, A. M., 111, 135, 138, 168, 171, 181

Leclaire, J. A,, 110, 135, 137, 138, 181 Leicht, W., 128, 179 Lempert, U., 46, 90

AUTHOR INDEX

Le Patourel, G. N. J., 76, 87 Lerche, W., 96, 97, 181 Lerner, H. R., 135, 136, 144, 181 Lichtenthaler, H. K., 30, 32, 33, 34, 54, 58, 59. 60, 61, 74, 81, 82, 87, 88, 89, YO

Leiber, K., 138, 181 Liedvogel, B., 50, 57, 58, 63, 64, 66, 89 Lilley, R. M., 122, 135, 136, 178, I82 Liu, W., 97, 183 Lloyd, N. D. M., 1 1 I , 181 Lodish, H. F., 2, 4, 24 Loeblich, L. A., 97, 101, 102, 105, 111, 127, 133, 135, 145, 181 Long, S. P., 114, 181 Looney, F. D., 97, 160, 179 Lotter, G., 136, 180 Lucas, W. J., 110, 181 Lutke-Brinkhaus, F.. 48, 50, 63, 64, 66, 89 Lynch, D. V., 116, 132, 181 Lynen, F., 29, 30, 37, 70, 89 Lyrene, S. A., 42, 89

M MacElroy, R. D., 97, 135, 141, 142, 181 Majak, W.. 128, 130, 146, 147, 181 Maluf, L. Y., 120, 123, 125, I81 Manners, D. I., 130, 17Y Manton, I., 122, 123, 182 Mapleston, R. E., 82, 89 Marano, F., 113, 115, 120, 122, 181, I82 Marengo, T., 135, 136, 178, 182 Markelova. A. G., 139, I83 Mason, T. L., 6, 24 Masyuk, N.. 98, IN2 Mattox, K. R., 95, 182 Maudinas, B.. 42, 44, 45, 89 Mayor, F., 36, 62, 65, 88, 91 Mayumi, T., 107, 108, 179 McAllisler, C. D., 114, 181 McCarthy, J. J., 114, 118, 179 McCormack, D. K., 33, 59, 60, 82, 91 McCree, K. J., 114, 182 McLachlan, J., 102. 103, 105, 107, 108, 128, 130, 132, 146, 147, 149, 162, 178, 179, 182 McNulty, I. B., 97, 101, 102, 115, I83 Medvedev, A. V., 149. 168, 178 Melack, J. M., 98, 182

189

Melkonian, M., 95, 119, 121, 122, 123, 182 Melitz, D. K., 43, 87 Mercer, E. I., 53, 54, 55, 65, 88, 91 Metzner, 102, 113, 1 15, 1 16, 183 Mevorech, M., 128, 179 Miflin, B., 57, 89 Miller, E. M., 42, 89 Milligan, R., 4, 24 Mishkind, M., 2, 14, 24 Mitchell, E. D., 30, 37, 59, 87 Mitzka-Schnabel, V., 68, 74, 75, 77, 91 Miura, S., 12, 23 Miyachi, S., 111, 115, 135, 140, 178, 182 Modi, V. V., 73, 74, 89 Moesta, P., 83, 85, 86, 89 Moestrup, O., 121, 182 Moller, E. M., 105, 106, 107, 130, 131, 136, 145, 147, 150, 151, 160, 162, 163, 165, 180 MonCger, R., 46, 66, 87, 88 Morris, I., 146, 178, 182 Morris, L., 135, 139, 180 Morris, R. O., 43, 89 Mukerji, D., 146, 178 Muller, M., 2, 23 Muller, W., 128, I82 Murphy, D. J., 57, 89 N Nagy, F., 14, 23 Nakamura, Y., 11 I , 135, 140, 178 Nandi, D. L., 41, 42, 89 Nelson, D. M., 113, 182 Nes, W. D., 50, 51, 8Y Neupert, W., 12, 19, 24 Newmeyer, D. D., 6, 24 Nicolai, E., 96, 182 Nishi, A., 60, 89 Nishino, T., 38, 39, 74, 75, 76, 90 Novick, P., 3, 4, 24

0 Oba, K., 30, 31, 37, 70, 71, 83, 84, 89, 90, 91 Ogura, K., 38, 39, 40, 41, 74, 75, 76, 90 Ohashi. A,, 13, 23, 24 Oliver, D., 3, 24 Oliviera, L., 122, 182 Oren, A,, 98, 99, 182

AUTHOR INDEX

190

Oshima, K., 29, 90 Oshima-Oba, K., 35, 90 Osterlind, S., 11 I, 182 Overton, K., 29, 30, 87 Owens, T. G., 145, 148, 179

P Paasche, E., 114, 135, 139, 179, 182 Palade, G., 2, 24 Papastephanou, C., 42, 44, 45, 89 Paradies, H. M., 135, 140, 143, 182 Park, R. B., 125, 182 Patwa, D. K., 73, 74,89 Pauly, G., 38, 63, 64,88 Peavey, D. G., 109, 126, 181 Pennock, J. F., 45, 90 Perez, L. M., 40, 41, 75, 88 Peterfi, L. S., 122, 123, 182 Pfaller, N., 12, 24 Pfeifhofer, A. D., 125, 182 Pfisterer, J., 15, 24 Pick, U., 135, 179, I83 Picken, D., 29, 87 Platt, T., 145, 148, 178 Pon, N. G., 125, 182 Pont Lezica, R., 50, 88 Popjack, G., 50, 88 Porter, J. W., 29, 36, 38, 39, 41, 42, 43, 44, 45, 64, 74, 75, 76, 77, 87, 88, 89, 90, 91 Portilla, G., 40, 41, 75, 88 Potty, V. M., 36, 90 Pratje, E., 13, 24 Preisig, H. R., 95, 119, 122, 123, 182 Pristavu, N., 147, 148, 182 Pugsley, A. P., 3, 24 Puiseux-Dao, S., 160, 161, 180

Q

Quveshi, A. A., 43, 90

R Rabinowitch, S., 154, 156, 157, 159, 161, 182

Racusen, D. W., 28, 30,89 Rahmsdorf, H. J., 133, 182 Railton, J. D., 48, 64, 90, 91 Randall, D. D., 57, 91 Rao, K. S., 97, 182 Rao, P. S. N., 97, I82

Rau, W., 68, 74, 75, 77, 91 Raven, J. A., 110, 143, 181, 182 Regan, D. L., 97, 160, 179 Reid, G . A., 13, 24 Rety, J., 30, 32, 58, 59, 60, 61, 81, 82, 87 Richman, L., 169, 171, 174, 175, 180 Reitveld, A., 10, 24 Riezman, H., 9, 24 Riisgard, H. U., 98, 160, 161, 174, 182 Riley, J. P., 132, 179 Robinson, C., 1-24, 2, 6, 14, 17, 18, 22, 23,24

Robinson, J., 138, 181 Robinson, S. P., 74, 90 Rogers, D.M., 32, 33, 34, 52, 74, 76, 87 Rogers, J. N., 114, 118, 179 Rogers, L. J., 35, 57, 61, 62, 65, 89, 90 Rogers, S. G., 43, 88 Roise, D., 9, 24 Rojas, M. C., 40, 41, 75, 88 Rose, W. E., 112, 183 Roughan, P. G., 55, 57, 90 Rudiger, W., 46, 47, 64, 68, 74, 75, 77, 87, 90, 91

Rudney, H., 29, 32, 33, 34, 52, 74, 76, 87, 91

Russell, D. W., 30, 31, 33, 34, 58, 59, 60, 68, 72, 76, 77, 78, 79, 81, 82, 87, 90, 91 Ruyters, G., 115, 182 Rycroft, D., 29, 30, 87 Ryder, N. S., 29, 34, 90 Ryther, J. H., 101, 180, 182

S Sadler, I., 29, 87 Sahl, H. G., 97, 98, 181 Sandmann, G., 109, 182 Santa-Maria, A., 120, 182 Sargeant, J. M., 54, 90 Sathyamoorthy, N., 38, 39, 41, 42, 45, 74, 75, 76, 77, 91

Scharf, H., 57, 90 Schatz, G., 6, 8, 19, 20, 21, 22, 23, 24 Schekman, R., 3, 24 Schell, J., 15, 16, 24 Schindler, S., 34, 60, 88, 90 Schirling, R., 157, 158, 161, 168, 174, 180

Schleyer, M., 12, 19, 24

AUTHOR INDEX

191

Schneider, H., 65, YO Schmidt, G. W., 2, 13, 14, 22, 24 Schoch, S., 46, 90 Schreiber, U., 150, 151, 171, 174, 181 Schreier, P. M., 3, 15, 16, 24 Schultz, G., 29, 46, 48, 55, 58, 64, 87,

Strogonov, B. P., 168, 178 Stumpf, P. K., 57, 58, 89 Suarez, D., 36, 88, YO Suga, T., 29, 91 Suissa, M., 8, 24 Sussman, I., 135,, 136, 144, 178, 179,

Schulze-Siebert, D., 29, 57, 58, 90 Schwartz, M., 3, 24 Schweiger, H. G., 133, 182 Scurr, E. V., 73, 74, 91 Selman, B. R., 135. 179, 183 Selman-Reiner. S... 135.. 179. 183 Semenenko, V. E., 102, 115: 135, 139,

Suzuki, H., 30, 31, 33, 58, 60, 70, 83, Y l

GG, 91

178. 183

Seto, S.,'38, 39, 41, 74, 75, 76, 90 Shah, D. V., 44, 57, 62, 64, 65, Y o Sharfstein, B. A., 110, 113, 114, 183 Sheffer, M., 144, 148, 153, 183 Shen-Miller, J., 48, 90, 91 Shewry, P. R.. 36, 74, 78, YO Shibuya, T., 38, 39, 74, 75, 76, 90 Shiloh, M., 98, 99, 182 Shinka, T., 41, YO Shiro, M., 107, 108, 179 Simcox, P. D., 64, YO Simmons, K., 32, 89 Singh, S. B., 29, 30, 87 Sipat, A. B., 30, 31, 32, 33, 34, 68, 70, 77, 78, 8Y, YO, 91 Siperstein, M. D., 50, Y l Skilleter, D. N., 37, 74, 76, 91 Slack, C. R., 55, 57, YO Sloan, P. R., I 13, 1 16, 179 Smcekens, S.. 14, 15. 18, 24 Smith, A. E., 5, 21, 24 Solinian, 0. H. S., 97, 98, 181 S o l J., 46, 47, 48, 64. 68, 74, 75, 77, Y I Speer, H. L., 97, 135, 141, 142, 181 Spurgeon, S. L., 38, 39, 41, 42, 45, 74, 75, 76, 77, 91 Steiger, A., 68, 74, 75, 77, Y l Stephens, D. W., 97, 98, 99, 100, IN3 Stcphens. K., 114, 181 Stewart, K. D., 95, 182 Stewart, W. D. P., 95, 132, 183 Stitt. M.. 57, 74, YI Stobart, A. K.. 36, 74, 78, 82, 90, 91 Stonik, J. A,, 77, 87 Strickland, J. H. D., 141, 181

181, 183

T Takeda, H., 107, 108, 179 Takeuchi, A,, 83, 91 Tanga, K., 29, 91 Tatematsu, H., 37, 70, 84, 89 Teodoresco, E. C., 95, 96, 97, 154, 155, 183

Terborgh, J., 135, I81 Thomas, D. R., 82, 91 Thomas, W. H., 108, 117, 179, 183 Thompson, G. A., 116, 132, 181 Threlfall, D. R., 54, 67, 68, 83, 87, 88, Yl

Tictz, A., 131, 132, 179 Tobin, E. M., 14, IS, 23 Tobler, U., 157, 158, 161, 180 Tocher, C. S., 128, 130, 132, 146, 147, I 78 Tolbert, N. E., 135, 180 Tominaga, H., 97, I83 Trcharne, K. J., 54, 61, 64, 65, 87, Y l Trezzi, F., I 19, 122, 123, 125, 126, IN3 Truper, H. G., 97, 98, 181 Tsuritani, I., 60 Turner, I. M., 110, I80 Turpin. D. H., 145, I83

u Ukeles, R.. 112, 116, 183 Unwin, P. N. T., 4, 24 Uritani, I., 29, 30, 31, 35, 37, 58, 60, 70, 71, 83, 84, 8Y, YO, 91 Urilani, M . 84, Y l V Vadas, R. L., I I I , 135, 168, 171, 181 Valcnzuela, P., 36, 78, 87 Van Auken, 0. W., 97, 101, 102, 113, I IS, I83 Van den Broeck, G . , 3, 15. 16, 24

I92

AUTHOR INDEX

Van Loon, A. P. G. M., 2, 6, 8, 9, 10, 11, 24, 24 Van Ormondt, H., 43,89 Veech, R. L., 70, 89 Vesk, M., 115, 183 Vettermann, W., 135, 140, 143, I82 Vladimirovq, M. C., 119, 120, 125, 135, 139, I83 W Walker, D. A., 74, 90 Wasson, G., 29, 87 Wasmann, C. C., 17, 24 Watts, R. B., 68, 74, 75, 91 Wax, Y., 159, 160, 183 Webber, A. N . , 152, 167, 174, I80 Wegmann,K., 102, 105, 113, 115, 116, 128, 134, 135, 136, 145, 147, 148, 149, 150, 151, 165, 166, 167, 179, 180, 182, 183 Weiger, C., 1 13, 1 15, 145, 150, 178 Wciss, G., 48, 89 Wellburn, A. R., 54, 65, 87, 89, 91 Werber, M. M., 128, 179 Werz, G., 121, 183 West, C. A., 40, 42, 47, 48, 63, 64, 66, 68, 76, 83, 84, 85, 86, 88, 89, 90, 91 Wetzel, R. G., 98, 183 Weyand, A., 135, 141, 144, 180 White, L. W., 29, 91 Whitney, D. E., 113, 183 Wickner, W. T., 2, 4, 24

Widmaier, R., 38, 39, 40, 41, 42, 91 Wiedermann, C., 105, 106, 107, 150, 151, 161, 180

Wiley, M. H., 50, 91 Williams, F. M . , 116, I83 Williams, M . , 57, 91 Williamson, I. P., 36, 91 Wills, R. B. H., 73, 74, 91 Wilson, T. M., 68, 76, 77, 78, 79, 82, 90 Winkenboch, F., 146, 147, 148, I83 Wirtz, W., 74, 91 Woeber, G., 98, 135, 140, 141, 181 Wong, R. J., 33, 59, 60, 82, 91 Wood, G . W., 131, 179

X Xiang, J . , 97, 183

Y Yamada, H., 84, 91 Yamaguchi, M., 83, 91 Yamashita, K., 37, 70, 84, 89 Yu, R., 83, 84, 90

Z Zenvirth, D., 111, 145, 183 Zheng, M . , 97, 183 Ziegier, H . , 65, 90 Zimmerman, R., 9, 24 Zmiri, A., 159, 160, 168, 183 Zsebo, K. M., 21, 24

SUBJECT INDEX

A Acetyl-CoA synthesis, 55-57 and chloroplasts, 55-56 Algae, see Dunaliella Amino acids in Dunaliellu proteins, 129 Ammonium us. nitrate for Dunuliellu, 109-1 10 Amylose in Dunaliella, 130 Artemiu salina and Dunaliella, 100 ATP and HMG-CoA reductase inhibition, 78 and nucleoplasmin targeting, 6

Casbene synthetase, 84-85 and mRNA levels, 85 Castor bean, Rhizopus sto1onifL.r infection, 84-85 CPrcitoc,~,.stis.fimbriritci infection and phytoalexin synthesis, 83-84 Chlorophyll synthesis, 4 6 4 7 P.S. carotenoid, and precursor channelling, 79 and compartmentation, subcellular, 64 Chloroplasts in acetyl-CoA synthesis, 55-56 of Dunaliella composition of, and temperature, 132 under electron microscope, 119-120 under light microscope, 1 18 and HMG-CoA reductase activity, 58 isopentenyl diphosphates, permeability to, 66 isoprenoid formation in, 64 mcvalonate kinase activity in, 62 prenyl diphosphate synthesis in, 63-64 and protein targeting, 13-19 binding of precursors, 15-1 6 processing of precursors, 17-19 synthesis of proteins, 14-15 transport and energy, 16-1 7 in spinach and prenylation catalysis, 46 Chromoplasts, prenyl diphosphate synthesis in, 63-64 Compactin and sterol synthesis inhibition, 34 Compartmentation in Dirnrrliella, 169 and cell volume regulation, 174-1 75

B Black rot fungus and phytoalexin synthesis, 83-84 Boyle-Van’t Hoff equation, 153-154 Brine shimp and Dunaliella, 100 C

Calcium ions.and Dunaliella growth, 108 Carbohydrates in Dutiuliella from photosynthesis, 146, 147 soluble, 128, 130 Carbon dioxide as carbon source, 106, 107 Carbon source and Dunaliella growth carbon dioxide, and sodium chloride, 106, 107 inorganic, I I & 1 1 1 organic, 112 Carbonic anhydrase in Dunuliella, 1 1 1 Carotenoids in Dundidla, I3 1, I33 synthcsis, 44 4 5 L’S. chlorophyll, and precursor channelling, 64 and compartmentation, subcellular, 64 in ripening fruit, 83 193

I94

SUBJECT INDEX

Compartmentation-Confd. and isoprenoid biosynthesis, 52-68 and acetyl CoA formation, 55-57 hypotheses, 52-55 and isopentenyl diphosphate formation, 61-63 and isoprenoid formation, 64-65 and membrane permeability, 65-66 and mevalonate formation, 57-60 and prenyl diphosphate formation, 63-64 Copper ions and Dunaliella growth, 109 Coupling factors for photosynthetic enzymes, 140 Cytokinins, 43

D Dead Sea, Dunaliella in, 98-99 Dihydroxyacetone kinase, 136 Donnan pressure of cell proteins, 153 Dunaliella, 93-183 anatomy, 118-126 differences, intergeneric, 123, 124 differences, interspecific, 123 and light and temperature, 125-126 microscopy, 1 18-1 22 and salt, 123, 125 cell volume regulation, 153-175 and compartmentation, 174-175 and glycerol metabolism, 173-174 and ion concentrations, 167-173 and osmotic pressure change, 153-160 recovery after osmotic shock, 160-167 composition, 126-134 carbohydrates. soluble, 128, 130 lipids and carotenoids, 131-133 nucleic acids, 133 organic compounds, 126-1 27 proteins, 128, 129 starch, 130 distribution and ecology, 98-101 enzymes, 134-143 glycerol, effects of, 143, 144 glycerol metabolism, 134-1 37 nitrate reduction, 137-139 phosphofructokinase, 140-141 photosynthetic, 1 39 - 140

Dunaliella-contd. enzymes-contd. and sodium chloride, 141-143 starch metabolism, 140 structural modifications, 143 growth, 101-1 18 and artificial medium composition, 101, 102 and carbon source, 1 1 0-1 12 and cations, divalent, 108 and cations, monovalent, 106-107 and copper, 109 and interactions of parametcrs, 116-117 and light, 112-115 and nitrogen source, 109-1 10 and phosphorous, 108 and salinity, total, 101, 103 and sodium chloride, 103-107 and sulphate, 108 and temperature, I 15-1 16 photosynthesis, 143, 144-153 and electron flow, 148 and light intensity, 148 products, 146-148 rate, and sodium chloride concentration, 144 and sodium chloride, 149-153 taxonomy, 95-98

E Electron flow in Dunalirlla photosynthesis, 148 Endoplasmic reticulum in Dunaliellu, 121 Eukaryotic cell structure, 2 F Farnesyl diphosphate formation, 3 8 4 2 Fatty acids in Dunaliella, 132 long-chain, synthesis, mevalonate in, 50-52 Flagella of Dunaliella, 121-122 Fluorescence emission and photosynthesis in Dunaliella, 152 Fruit ripening and isoprenoid metabolism 83

SUBJECT INDEX

G Genetic engineering and protein targeting, 21-22 Geranyl diphosphate synthesis, 3 8 4 2 Geranylgeranyl diphosphate availability and isoprenoid synthesis, 79-80 Germination, isoprenoid metabolism in, 80-8 I Gibberellin synthesis, 4 7 4 8 Glucose as Dunaliella carbon source, 112 Glycerol in Dunaliella, 126, 127, 128 and enzymes, 143, 144 dehydrogenase, 136137 nitratc reductase inhibition, 138-139 metabolism enzymes for, 134-1 37 and osmoregulation, 173-175 as photosynthetic product, 146. 147 and sodium chloride, 130-131 synthesis. and osmotic shock recovery, 162- 166, 167 Golgi apparatus in Dunaliella, 121

I95

Isopentenyl diphosphate chloroplasts, permeability to, 66 enzymes utilizing, and compartmentation, 63-64 formation, 34-38 and compartmentation, 61-63 mevalonate kinase inhibition, 36 in prenyl diphosphate formation, 3842 isomerase modulation, 75-76 in isoprenoid metabolism, 27-28 phenyl lipids, incorporation into, 77 lsopentenyl diphosphate isomerase, 3840 Isoprenoid biosynthesis, 25-91 compartmentation, subcellular, 52-68 and acetyl CoA formation, '55--57 hypotheses, 52-55 and isopentenyl diphosphate formation, 60-63 and isoprenoid formation, 64-65 and membrane permeability, 65-66 and mevalonate formation, 58-61 and prenyl diphosphate formation, 63-64 control, 68-86 H competition for substrates, 79-80 Homogentisate prenylation and enzymc activity. 70, 72-73 plastoquinone, 48 enzymc activity modulation, 74-79 Hydroxy-methylglutaryl coenzyme A flux of metabolites, 69-70, 71 formation, 28-30 in fruit ripening, 83 mevalonate formation from, 30-34 in germination, 80-81 Hydroxy-methylglutaryl coenzyme A in leaf greening, 81-83 reductase activity phytoalexin synthesis induction, and cycloheximide/mercuric chloride 83-85 treatment, 84 substrate concentration, 73-74 and light exposure, 81-82 metabolic pathways, 27-52 and location, subccllular, 57-60 carotenoids, 4 4 4 5 and chloroplasts, 58 chlorophyll, 4 6 4 7 and mcvinolin inhibition, 59-60 cytokinins, 43 and microsoma1 membranes, 57 gibberellins, 4 7 4 8 and mitochondria1 membranes, HMG-CoA formation, 28-30 58-59 isopcntcnyl diphosphatc formation, modulation. 76 34-38 by protein factors, 77-79 mcvalonate formation, 30-34 nicvalonatc shunt, 50-52 I phylloquinone and tocopherols, Ion pumps, scc Osmosis and D u n ~ ~ l i e l l ~ i 4546 Iponieamaronc synthesis and black rot plastoquinone, 48, 49 fungus, 84 prcnols. long-chain, 50

SUBJECT INDEX

196

Isoprenoid biosynthesis-contd. metabolic pathways-contd. prenyl diphosphate formation, 3842 sterols, 4 3 4 4 ubiquinone, 48-50. structure, 26 K Kaurene synthesis, 47-48 and compartmentation, 64 and energy charge, 76

L Leaf greening, isoprenoid metabolism in, 81-83 Light-harvesting chlorophyll binding, 15 Light intensity and Dunaliella anatomy and temperature, 125-126 and carbon, organic, uptake, 112 and p-carotene synthesis, 133 growth, 112-115 and sodium chloride, 106-107 and temperature, 116-1 17 and nitrogen uptake, 110 and photosynthesis, 148 Lithium ions and Dunaliella growth, 107-108 Lipids in Dunaliellu, 131-133 M Magnesium ions and Dunalidlu growth, 108

Methyl viologen as nitrate/nitrile cofactor, 138 Mevalonate concentration in vivo, 73-74 formation and compartmentation, subcellular, 57-60 and HMG-CoA reductase, 30-34 and isopentenyl diphosphate formation, 34-38 incorporation inhibition, 75 shunt, 50-52 translocation and plastid permeability, 65-66 Mevalonate diphosphate dccarboxylase . . activity. 84

Mevalonate kinase activity, 78 and isopentenyl diphosphate formation, 36 and light exposure, 82-83 and location, 60-63 in chloroplasts, 61 in mitochondria, 63 Mevinolin inhibition of HMG-CoA reductase, 34, 59-60 Microscopy for Dunaiiella electron microscope, 1 19-122 light microscope, 118-1 19 Mitochondria of Dunaliella, 120 HMG-CoA reductase activity in, 58-59 isopentenyl diphosphate utilizing enzymes in, 63 mevalonate kinase activity in, 63 permeability to isoprenoid intermediates, 66 and protein targeting, 6-1 3 binding of precursors, 9-10 cleavage of precursors, 12-13 synthesis of proteins, 6, 8-9 translocation, 1&12 ubiquinone synthesis in, 64

N Nicotianm plunih(rginiji)lirrr mitochondria1 protein, 8 Nitrate for Dimdidla us. ammonium, 109-1 10 metabolism, 137-1 39 Nitrite reductase in Dunulic~llrr,137, 138 sodium chloride inhibition, 138-139 Nitrogen source and Dunuliellu, see also Amino acids in Dunaliellu proteins; Ammonium P S . nitrate for Dunaliella; Nitrate for Dunulit.llu; Nitrite reductase in Dunaliellr carbon metabolism, 146, 148 growth, 109-1 10 Nucleic acids in Dunuliellu, 133, w e cilso ATP Nucleoplasmin targeting, 4, 7 and ATP, 6 Nucleus of Dunulirllu, 120 and protein targeting, 4-6

SUBJECT INDEX

0 Osmosis and Dunaliellr Boyle-Van't Hoff equation, 153-1 54 and compartmentation, 174-1 75 and glycerol metabolism, 173- 174 ion concentrations, 167-1 73 constant salinity, 167-169 and hypertonic shock, 171-173 regulation, 169-171, 173 membrane permeability, 159-160 recovery after shock, 160-167 glycerol synthesis, 162-166 and thylakoid membranes, 152-153 water/ion movement, 157-1 59

P Pepper ripening and phytoene synthetase activity, 83 Phenyltransfcrases, 4 M 2 Phosphofructokinase in Dunuliellrr, 140-1 4 1 Phospholipid modulation o f enzyme activity, 77 Phosphorus' andl Dutiirlic~lltrgrowth, I08 Photosynthesis of Dutirrliolla, 143, 144-153 and electron flow, 148 enzymes in, 139-140 and light intensity, 148 osmotic stress and thylakoid membranes, 152-1 53 products, 146-148 rate, and sodium chloride concentration, 144 and sodium chloride, 149-153 fluorescence studies, 152 Phylloquinonc synthcsis. 45-46 and compartmentation, 64 Phytoalexin synthesis, 83H5 Phytoene in carotenoid synthesis. 44-45 Phytocnc synthctasc activity and pepper ripening, 83 Phytyl diphosphatc concentration in riro, 74 and mevalonate incorporation, 75 Pink Lake, Drrt~nlirllrin, 100 Plastid nicmbrane pernicability, 65-66 Plastocyanin precursor processing, 18 Plastoqiiinonc synthesis, 48, 49 and compartmcntation, 64

197

Potassium ions and Dunaliellu growth, 107 Prenols, long-chain, synthesis, 50 Prenyl diphosphate formation, 3 8 4 2 and compartmentation, 63-64 and isopentenyl diphosphate isomerase, 3 8 4 0 and prenyltransferases, 4 W 2 Prenyltransferases location, subcellular, 63 and prenyl diphosphate formation, 4042 Protein in Dunuliellu, 128 amino acid composition, 129 Factor, and enzyme modulation, 77-79 synthesis, mitochondrial, 6, 8-9 Protein targeting, 1-24 chloroplasts, 13-18 binding of prccursors, 15-1 6 processing of precursors, 17- I 8 synthesis of proteins. 14-15 transport and energy, 16-17 and conformation during transport, 19 and gcnctic engineering, 21-22 mitochondrial, 6 -13 binding of precursors, 9-10 cleavage of precursors, 12-1 3 synthcsis of proteins, 6. 8-9 translocation, 1 & 12 nuclcar, 4-6 presequcnce origin, 20-21 principles. 3 4 receptor origin, 20 rcvicw, historical, 2-3 structural setting, 2 Pyruvate dchydrogenase in acetyl-CoA synthesis. 55, 56

R Radish and isoprenoid mctabolisni, 34 R / ~ i x ~ / ~ ls/oloti;fir lu.s i n fect ion and phytoalexin, 83-85 Ribtilose hi.cphosphate carboxylase in D l l t ~ ~ / l i ( ~ lIl39t r , 140 and sodium chloride inhibition, 141 142

I98

SUBJECT INDEX

S Salt, see Dunaliella; Sodium chloride and Dunaliella Seeds germination, isoprenoid metabolism in, 80-81 Sodium chloride and Dunaliella concentration and cell anatomy, 123, 125 and growth, 103-107 and photosynthesis rate, 144 enzyme inhibition, 141-143 nitrate reductase, 138-139 and glycerol, 130-131 and growth and carbon dioxide as carbon source, 106, 107 and light, 106-107, 116-117 and temperature, 106 and photosynthesis, 149-153 fluorescence studies, 152 and starch us. glycerol synthesis, 131 Spinach chloroplasts and prenylation catalysis, 46 photosynthetic products and nitrogen source, 146, 148 Squalene synthesis, 4 3 4 4 Starch in Dunaliellu, 126, 130 us. glycerol and sodium chloride, 131 metabolism, enzymes for, 140 Sterol synthcsis, 4 3 4 4 inhibition and conpactin. 34 rate of, radiolabclling studies, 69-70

Sucrose in Dunaliella, 128, 130 Sulphate and Dunaliella growth, 108 SV40 large T antigen and nuclear targeting, 4-5 Sweet potato, black rot infection, 83-84

T Temperature and Dunuliella anatomy and light intensity, 125-126 chloroplast composition, 132 growth, 115-1 16 and light, 116-1 17 and nutrient supply, 117 and sodium chloride, 106 Terpenoids, see Isoprenoid biosynthesis Thylakoid membranes in Dunaliella chloroplasts, 119, 120 in cndoplasmic reticulum, 121 and osmotic stress and photosynthesis, 152-1 53 Tocopherol synthesis, 45-46 and compartmentation, 64 Tomato ripening and carotenoid synthesis, 83

U Ubiquinone synthesis, 48-50 in mitochondria, 64

V Vacuoles in Dunaliella, 121