Advances in Botanical Research, Vol. 12

Advances in

BOTANICAL RESEARCH VOLUME 12

Advances in

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

Department of Plant Biology, University of Birmingham, Birminghum, 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 Depurtmrnt of Plant Biology University of Birmingham Birmingham, Englund

VOLUME 12

I986

ACADEMIC PRESS Harcourl Brace Jovanovich, Publishers

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PRESS INC. (LONDON) LTD COPYRIGHT 0 1986 BY ACADEMIC ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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86878889

9 8 1 6 5 4 3 2 1

CONTENTS CONTRIBUTORS TO VOLUME 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

ix

Light/Dark Modulation of Enzyme Activity in Plants LOUISE E . ANDERSON I . Occurrence of Light Modulation . . . I I . Proposed Mechanisms .............................. 111. Regulation of Light Modulation ...................... IV . Dark Modulation ................................... V . Changes in the Target Enzyme ....................... VI . The Function of Light Modulation . . . . . . . VII . Effect of Sulfur Dioxide on Light Mo VIII . Osmotic Stress and Light Modulation IX . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . ............................

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

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

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

3 6 24 28 30 36 38 38 39 39

Algal Toxins WAYNE W . CARMICHAEL .. . . . . . . . . . . . . . . . . . I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .................. I1 . Naming Algal Toxins ............................... 111. Occurrence. Growth. and Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Isolation Characterization and Toxinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Environmental Role of Algal Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

.

47

52 52 67 91

93

Plant Transposable Elements PATRICIA NEVERS. NANCY S. SHEPHERD. AND H E I N Z SAEDLER 1. The Phenomenon of Variegation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 . Recognizing Mutations due to Transposable Elements by Classical Genetic

104

Means . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

109

111. Transposable Elements at a Molecular Level ............................. IV . A Model of the Mechanism of Transposition .............................

151

V . Interaction between Transposable Elements .............................. V

170 177

vi

CONTENTS

v1. Induction of Cenomic Instabilities. ...................................... VII. Addendum ............................................................ References ...........................................................

188

193 194

The Dinoflagellate Chromosome D.C. SlGEE I. II.

Introduction ....................................

..................... 208 IV . Dinoflagellate Chromosome Fine Structure . . . . . . . . . . . . . V. . . . . . . . . . . . . . . . 222 VI. Chromosome Changes during the Cell Cycle . . VII. Macromolecular Composition of Dinoflagellate Chromatin . . . . . . . . VIII. IX. References ................. ............................ 260 111. DNA Levels and Chromosome Numbers

AUTHOR I N D E X . . . . SUBJECT INDEX.. ..

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

265 281

CONTRIBUTORS TO VOLUME 12 Numbers in parentheses indicate the pages on which the authors' contributions begin.

LOUISE E. ANDERSON ( I ) , Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60680, U.S.A. WAYNE W. CARMICHAEL (47), Department of Biological Sciences, Wright State University, Dayton, Ohio 45435, U.S.A. PATRICIA NEVERS (103), Max-Planck-Institut fur Zuchtungsforschung, Egelspfad, D-5000 Koln 30, Federal Republic of Germany HEINZ SAEDLER (103), Max-Planck-Institut fur Zuchtungsforschung, Egelspfad, D-5000 Koln 30, Federal Republic of Germany NANCY S. SHEPHERD' (103), Max-Planck-Institut fur Zuchtungsforschung, Egelspfad, D-5000 Koln 30, Federal Republic of Germany D. C. SIGEE (203, Departments of Botany and Zoology, University of Manchester, Manchester MI3 9PL, England

I Present address: E. I . DuPont de Nemours & Company, Inc., Central Research and Development Department, Wilmington. Delaware 19898, U.S.A.

vii

This Page Intentionally Left Blank

PREFACE The changes in enzyme activity in green plants caused by the transition from light to dark are now regarded as important regulatory processes directing metabolism towards synthesis of sugars and storage compounds in the light, and their breakdown in the dark. Light affects chloroplast enzyme activity in a number of diverse ways, through alteration of stroma1 pH, ion and metabolite levels. However, there are also changes in activity in some enzymes that involve post-translation (probably covalent) modification of the enzyme protein, and these are generally referred to as ‘light modulation’. In her article, Anderson reviews such plant enzyme systems, the biochemical mechanisms involved (probably by reduction of a disulphide bond), their potential molecular basis and the function of modulation in photosynthetic carbon metabolism. One of the most important developments in plant molecular genetics is the rapid improvement of our understanding of the nature and mechanisms of mutation induced by transposable elements. It is interesting to reflect that the origins of this lie in our fascination for variegated plants as horticultural curiosities! Because of our increasing interest in transposable elements for exploring the genetic origins of variation, or as systems for molecular biology and genetic engineering, the review of Plant Transposable Elements by the group at the Max-Planck Institute, Koln (Nevers, Shepherd and Saedler) is particularly welcome. How transposons will be used to isolate genes known only for their phenotypic effects will be seen in the future. The unicellular dinoflagellates are major components of marine and freshwater ecosystems. Apart from their general ecological importance, there are a number of reasons why they are of interest to biologists. In this volume we consider two such aspects. The article by Sigee discusses the very high level of DNA possessed by these organisms, the particular configuration of their chromatin and their nuclear organisation. These are of phylogenetic significance, and to summarise the position as presented in Sigee’s article, it now seems that dinoflagellates are to be regarded as true eukaryotes with some prokaryote features, and that probably they are ‘primitive’ rather than degenerate forms of more advanced ancestors. Algae that can produce toxins effective against animals are found in three of the eight algal divisions, including the dinoflagellates. Carmichael ix

x

PREFACE

reviews various aspects of biology and chemistry of these chemically diverse toxins, some of which can exert potent effects on humans, and considers their potential natural role. Few ideas of such ecological roles appear to have been subject to critical experimentation, and this article should provide a framework for such future work. J. A. Callow

Light/Dark Modulation of Enzyme Activity in Plants' LOUISE E . ANDERSON Department of Biological Sciences University of Illinois at Chicago Chicago. Illinois. U.S.A.

I. Occurrence of Light Modulation .........................................

II. Proposed Mechanisms ..................................................

3 6 8 14 21 21 22 23 24 24 25 26 27 27 27 28 28 29 30 30 30

A . The LEM System ................................................... B. The Ferredoxin-Thioredoxin Reductase. Thioredoxin System ............ C Ferralterin .......................................................... D . Activation of Pyruvic.Pi Dikinase .................................... E . Other Mechanisms .................................................. F Light Modulation of the Activity of Cytosolic Enzymes ................. 111. Regulation of Light Modulation .......................................... A NADP-Linked Malic Dehydrogenase .................................. B. Fructose-Bisphosphatase ............................................. C Sedoheptulose-Bisphosphatase........................................ D. Ribulose-5-Phosphate Kinase ......................................... E Glucose-&Phosphate Dehydrogenase .................................. F. Glyceraldehyde-3-PhosphateDehydrogenase ........................... IV . Dark Modulation ....................................................... A Thioredoxin in Dark Reversal of Light Modulation...................... B . Other Possible Mediators ............................................ C Regulation of Dark Modulation ....................................... V . Changes in the Target Enzyme ........................................... A . Change in the Target Enzyme as a Result of Light Modulation ........... B Change in the Target Enzyme as a Result of Thioredoxin-DTT-Catalyzed Modulation ............................. 32

.

. . . . . .

.

The abbreviations used in this article include the following: CAM. Crassulacean acid metabolism; CMU. 3-(4-chlorophenyl)-l.I-dimethylurea; DCMU. 3-(3.4-dichlorophenyI)I. I-dimethylurea; DCPIP. dichlorophenolindophenol;DTT. dithiothreitol; FCCP. carbonylcyanide p-trifluoromethoxyphenylhydrazone;FeS. iron sulfur; GSH. glutathione; GSSG. oxidized glutathione; LEM. light effect mediator . ADVANCES IN BOTANICAL RESEARCH. VOL. 12

1 Copyright 8 1986 by Academic Press Inc. (London) Ltd . AU rights of reproduction in any form m e N e d .

2

LOUISE E. ANDERSON

VI.

VII. VIII. IX.

C. Change in the Target Enzyme as a Result of Dithiothreitol Treatment.. ... D. Change in Kinetic Parameters of the Target Enzyme.. .................. The Function of Light Modulation ....................................... A. Induction of Photosynthetic Carbon Dioxide Fixation ................... B. CFI-CFo Mg ATPase.. .............................................. C. Stornatal Opening.. ................................................. Effect of Sulfur Dioxide on Light Modulation ............................. Osmotic Stress and Light Modulation. .................................... Conclusions ........................................................... References ............................................................

32 34 36 36 31 31 38 38 39 39

Light activation of the reductive pentose phosphate cycle enzyme NADPlinked glyceraldehyde-3-P dehydrogenase was first observed by Irmgard and Hubert Ziegler while they were assaying for the activity of the enzyme in dehydrated specimens of the resurrection plant Myrothamnus flabellifolia. When they noticed rather large differences in activity levels, they realized that the enzyme was being activated in the plants on the sunflooded lab bench (I. Ziegler, personal communication). Subsequently they demonstrated that the phenomenon occurs in less exotic species such as spinach and maize (Steiger et al., 1971). It is now widely accepted that light/dark modulation of enzyme activity in green plants is an important regulatory process which directs metabolism toward synthesis of sugars and storage products in the light and toward the breakdown of these compounds in the dark. There is reason to suspect that many other processes are also controlled by the chloroplast photomodulation system. Here I will discuss first the extent and distribution of what is thought to be light-dependent posttranslational modification of chloroplastic and cytosolic enzymes, next the various mechanisms which have been proposed for this kind of light modulation, and finally the functions which have been suggested for this process. Because it has not yet been demonstrated that the light-activated and light-inactivated enzymes are actually covalently modified, I will refer to this process by the more general (and more ambiguous)term light modulation. For the purposes of this article light modulation will not be used to refer to those effects which clearly do not involve covalent modification, i.e., pH effects, Mg2+effects, metabolite effects. Light does, of course, also control enzyme activity through the alteration of stromal pH, and ion and metabolite levels. Light modulation of enzyme activity in higher plants has been the subject of several reviews (Anderson, 1979, 1983a,b; Anderson et al., 1980, 1981, 1982, 1983; Buchanan et al., 1979a,b; Buchanan, 1980a,b, 1981,

3

LIGHT/DARK MODULATION

1984a,b; Jacquot, 1984; Losada, 1976) and a workshop after the Sixth International Photosynthesis Congress (Scheibe, 1983a). I. OCCURRENCE OF LIGHT MODULATION

All Orevolving photosynthetic organisms probably have the ability to light activate or inactivate some enzymes. Light modulation has been observed in photosynthetic species ranging from cyanobacteria to C3,C4, and CAM plants (Tables I and 11). It has not yet been observed in any of the photosynthetic bacteria. The enzymes which are light modulated in chloroplasts of pea (Pisum satiuum) are shown in Fig. 1. Four Calvin cycle enzymes and NADP-linked malic dehydrogenase are light activated. Ribulose-bisphosphatecarboxylase is also “light activated” in that as pH and Mg2+levels in the irradiated chloroplast rise, the enzyme is starch

0Z-i

RuBP

6-P-gluconate F 6 P

I

I

4

Pyruvate

I

cytosol

NADP malate-oralacetate

4

chloroplast

Fig. 1. Effect of light on carbon metabolism in the pea leaf. Heavy arrows and sun symbols indicate activation by light; crossing out and sun symbols indicate inactivation by light. The abbreviations used are the following: DHAP, dihydroxyacetone-P; E4P, erythrose-4-P; F6P, fructose-6-P; FBP, fructose-I ,6-P2; GIP, glucose-I-P; G6P, glucose-6-P; G3P, glyceraldehyde-3-P; PGA, P-glyceric acid; R5P, ribose-5-P; RuSP, ribulose-5-P; RuBP, ribulose-l ,5-P2; S7P, sedoheptulose-7-P; SBP, sedoheptulose-l ,7-P2; XuSP, xylulose-5-P. Not shown in figure: transaldolase. From Anderson et a / . (1980) with permission.

4

LOUISE E. ANDERSON

converted to the active form (for a review see Miziorko and Lorimer, 1983). There is new evidence which suggests that an additional activator may be required to convert the enzyme to an activatable form in some plant species (Chastain and Ogren, 1985; Portis, 1985; Salvucci et al., 1985; Seeman and Berry, 1985; Vu et al., 1984). This process is currently not understood and is under intensive investigation. It will not be discussed further here. Two oxidative pentose phosphate pathway enzymes and four enzymes of starch metabolism and glycolysis are light inactivated. In addition, two cytosolic pea leaf enzymes involved in hexose phosphate metabolism are light inactivated. In C4 plants, the chloroplast enzyme pyruvate,Pi dikinase is light activated as is the cytosolic enzyme P-enolpyruvate carboxylase. In Crassulacean acid metabolism plants Penolpyruvate carboxylase is light inactivated (von Willert et al., 1979; Manetas, 1982). Four epidermal enzymes, which function in stomatal carbon metabolism in the pea plant, are also photomodulated. In addition to the enzymes shown in Fig. 1 and Tables I and 11, a lightactivated thylakoid-bound protein kinase controls the distribution of excitation energy between photosystem 1 and photosystem 2 (for a review see Bennett, 1983). There is also evidence that an enzyme in chloroplastic fatty acid biosynthesis, possibly acetyl-CoA carboxylase, is light activated (Roughan et al., 1980; Nakamura and Yamada, 1979). Probably many other enzymes which have not yet been tested are light activated as well, including some of those which are known to be DTT or DTTthioredoxin modulated, such as 6-aminolevulinic acid synthetase (Clement-Metral, 1979; Clement-Metral and Holmgren, 1983) and 6-aminolevulinic acid dehydratase (Balange and Lambert, 1983). To date the photogenes which have been studied appear to be phytochrome controlled (Jenkins et al., 1984), or blue light controlled (Steinmiiller and Zetche, 1985), but it seems possible that some gene expression may be regulated by the same systems involved in photomodulation of carbon enzyme activity. Activatiodinactivation of the enzymes listed in Tables I and I1 varies from about twofold to values approaching infinity. This, of course, reflects the method of calculation of activation (light activity divided by dark activity). Unfortunately there is no measure of absolute dark or light activity at present. For most of these enzymes, at least under optimal assay conditions, dark activity is significant. There is apparently species specificity for modulation. Whether this specificity is at the level of the target enzyme or at the level of the modulating system, or both, is not known. At physiological pH and fructose bisphosphate levels, activity of the dark form of fructose-bisphosphatase is much reduced relative to that of

LIGHT/DARK MODULATION

5

TABLE I Light Acfivafionof Enzyme Activity in Planfs and Cyanobacteria Reductive pentose phosphate cycle enzymes NADP-linked glyceraldehyde-3-P dehydrogenase Ulva, Enferomorpha, Bryopsis, Gracilaria, Polysiphonia (Ziegler and Ziegler, 1967); Ceramium (Ziegler et a/., 1968); Pisum (Anderson and Avron, 1976); Spinaciu (Steiger e f a/., 1971); Vicia, Nicoriana, Brassica, Valerianella, Befa, Myrothamnus, Trificum, Avena, Lemna (Ziegler and Ziegler, 1965);Zea, Saccharum (Steiger et al.. 1971); Tidesfromia (Bjorkman and Badger, 1977); Kalanchoe (Gupta and Anderson, 1978) Ribulose-5-P kinase Anacysfis (Duggan and Anderson, 1975); Pisum (Anderson and Avron, 1976); Spinacia, Zea (Steiger e f a/., 1971); Tidesfromia (Bjorkman and Badger, 1977); Kalanchoe (Gupta and Anderson, 1978) Fructose-bisphosphatase Pisum (Anderson e f a / . , 1979);Spinaciu (Steiger e f a/., 1971); Glycine (Alscher 1982b); Triticum (Leegood and Walker, 1983a) Sedoheptulose-bisphosphatase Pisum (Anderson and Avron, 1976); Spinacia (Champigny and Bismuth, 1976); Trificum (Woodrow and Walker, 1980); Kalanchoe (Gupta and Anderson, 1978) C4 pathway enzymes NADP-linked malic dehydrogenase Pisum chloroplastic (Anderson and Avron, 1976). epidermal (Rao and Anderson, 1983a); Spinacia (Wolosiuk et al., 1977);Zea (Johnson, 1971); Tidesfromia (Bjorkman and Badger, 1977); Kalanchoe (Gupta and Anderson, 1978) Pyruvic,P, dikinase Zea, Amaranfhus (Hatch and Slack, 1969) P-enolpyruvate carboxylase Pisum epidermal (Rao and Anderson, 1983a); Salsolu. Cyperus (Manetas e f al., 1983);Saccharum, Amaranrhus, Sefaria, Cynodon (Karabourniotis ef al., 1985) Other enzymes of carbon metabolism NADP-linked isocitric dehydrogenase Pisum epidermal (Rao and Anderson, 1983a) Glycerate kinase Maize (Kleczkowski and Randall, 1985) Enzymes of nitrate assimilation Nitrite reductase Anucystis (Tischner, 1983) Enzymes of amino acid metabolism Glutamine synthetase Anacystis (Rowell ef al., 1979); Chlorella (Tischner and Huttermann, 1980); Chlumydomonas (Cullimore, 1981) Glutamate synthetase Chlorella (Tischner and Huttermann, 1980) Phenylalanine ammonia lyase Spinacia (Nishizawa e f a/.. 1979) Other enzymes CFI-CFO Spinacia (Vallejos e f a/., 1983) NAD kinase Pisum, Trificum (Muto e f a/., 1981) Pyridine nucleotide-linked ascorbate peroxidase Spinacia, Sedum (Kow e f a/., 1982)

6

LOUISE E. ANDERSON

TABLE I1 Light Inactivation of Enzyme Acriviry in Planrs and Cyanobacteria Oxidative pentose phosphate cycle enzymes Glucose-6-P dehydrogenase Anacysris (Duggan and Anderson, 1975); Pisum chloroplastic and cytosolic (Anderson and Duggan, 1976); Spinacia (Lendzian and Ziegler, 1970) Transaldolase Pisum (Anderson, 1981) Glycolytic enzymes Phosphorylase Pisum chloroplastic (Heuer et a / . , 1982) P-glucomutase Pisum chloroplastic (Heuer et a / . , 1982) P-glucoisomerase Pisum chloroplastic (Heuer et a / . , 1982) P-fructokinase Pisum chloroplastic (Heuer et a / . , 1982). cytosolic (Kachru and Anderson, 1975) C4 pathway enzymes

P-enolpyruvate carboxylase Sedum (Manetas, 1982) Enzymes of amino acid metabolism Aspartate aminotransferase Pisum epidermal (Rao and Anderson, 1983a)

the light form (Charles and Halliwell, 1981b). Likewise greater activation of sedoheptulose-bisphosphatase is observed when physiological levels of sedoheptulose bisphosphate are used (Laing et al., 1981). Real activation of these and the other enzymes shown in Table I may then be considerably greater than is apparent since those values are for optimum assay activity. 11. PROPOSED MECHANISMS

The involvement of the photochemical apparatus in light activation of glyceraldehyde-3-P dehydrogenase was first indicated in experiments in Ziegler’s laboratory (Ziegler et al., 1968). It was shown that the action spectrum for activation of NADP-linked glyceraldehyde-3-P dehydrogenase follows the action spectrum for photosynthesis. Therefore the pigments of the photochemical apparatus, and not phytochrome or the cytoplasmic blue light receptor, are the photoreceptors. Inhibitor experiments and experiments with photosystem I particles demonstrate that the reducing side of photosystem I is involved in light modulation (Fig. 2). DCMU (Latzko et al., 1970; Leegood and Walker, 1980b; Avron and Gibbs, 1974; Anderson and Avron, 1976; Hatch, 1977; Gupta and Anderson, 1980) and CMU (Champigny and Bismuth, 1976),

7

LIGHT/DARK MODULATION

which block electron transport between the two photosystems, inhibit light modulation. This inhibition can be overcome by adding electrons from ascorbate or DCPIP into the system beyond the DCMU block (MvC Akamba and Anderson, 1981). Stimulation of electron transport with antimycin (Champigny and Bismuth, 1976; Leegood and Walker, 1980a,b) or tetramethylethylenediamine (Champigny and Bismuth, 1976) increases light activation, while electron acceptors such as nitrite (Leegood and 2 (Latzko et al., 1970; Leegood and Walker, 1980b), Walker, 1980b) or 0 or compounds which are reduced by photosynthetically generated NADPH, such as oxalacetate and CO2, inhibit or reverse light activation (Leegood and Walker, 1980a,b). Diquat (Avron and Gibbs, 1974; Anderson and Avron, 1976; Gupta and Anderson, 1980) and methylviologen (Wildner, 1975; Hatch, 1977; Mills, 1983), which shunt electrons from photosystem I to 02,are inhibitors, as is pyocyanin (Leegood and Walker, 1980b), which short-circuits electron transport by shunting electrons from the reducing to the oxidizing side of photosystem I. Clearly electron transport to photosystem I is required for light modulation of enzyme activity. In confirmation of the inhibitor experiments, photosystem I particles support light modulation (MvC Akamba and Anderson, 1981). Since FCCP inhibits light modulation of only one enzyme (glyceraldehyde-3-P dehydrogenase) (Anderson and Avron, 1976; Gupta and AnderMV Diquat I I

I

DCMU CMU

Ascorbate D T

I

p/hy

L P S II

\

\

NADP '700

Fig. 2. Effect of inhibitors on photosynthetic electron transport. DCMU and CMU block electron transport from photosystem 11 (Ps 11). Diquat and methylviologen (MV) shunt electrons from the system at the photosystem I level (Ps I). Ascorbate or DCPIP feeds electrons in between the two photosystems.

8

LOUISE E. ANDERSON

son, 1980), proton pumping and photophosphorylation appear not to be necessary for light modulation. Four mechanisms have been advanced to explain the light modulation of the activity of the carbon metabolism enzymes. The light effect mediator, or LEM system, has been developed in this laboratory (Anderson et al., 1981). Buchanan and co-workers have proposed a system which involves ferredoxin and thioredoxin (Buchanan, 1980a). The same group has decribed a ferredoxin-independent factor for light modulation, ferralterin (Buchanan, 1980a). In Australia, Hatch and co-workers (Sugiyama and Hatch, 1981; Ashton et al., 1984) have described an entirely different system involving phosphate and ADP and a single regulatory protein for the activation and inactivation of pyruvate,Pi dikinase in C4 plants. Properties of the proteins thought to be involved in light/dark modulation in the chloroplast are listed in Table 111.

A. THE

LEM SYSTEM

Indications of the involvement of thiol compounds in light modulation first came from experiments of Latzko er al. (1970). They observed that arsenite, which reacts with vicinal dithiols (Fig. 3), and iodoacetamide, which reacts with thiol groups, inhibited light activation of ribulose-5-P kinase in intact chloroplasts, and that in broken chloroplast preparations, dithiothreitol activated the enzyme. They postulated that a vicinal dithiol was involved in light modulation. That the vicinal dithiol was membrane-bound and light generated was shown in experiments with crude broken chloroplast systems (Anderson and Avron, 1976). It was found that if the thylakoid membranes were illuminated and exposed to arsenite, photomodulation was subsequently inhibited in the recombined broken chloroplast system. Arsenite was not effective unless the thylakoids were irradiated during the treatment. The effect of arsenite was reversed by treatment with DTT. These experiments indicated that the vicinal dithiol group was generated when the thylakoids were irradiated. Sulfite, which reacts with disulfide bonds, giving rise to a free thiol group and a thiosulfonic acid (Fig. 3), also inhibited. Sulfite was effective even when the thylakoids were exposed to the inhibitor in the dark. These experiments indicate that a redox-active disulfide bond is reduced by transfer of electrons from the photosynthetic electron transport system giving rise to a vicinal dithiol. The membrane component containing these groups was named the “light effect mediator,” or LEM.

LIGHT/DARK MODULATION

9

Fig. 3. (A) Reaction of arsenite with vicinal dithiol; (B) reaction of sulfite with disulfide.

1. The LEM The LEM is removed from the chloroplast thylakoid membranes when they are treated with Zwittergent, a zwitterionic sulfobetaine detergent (Mohamed and Anderson, 198la). Light modulation is restored when the Zwittergent extract is dialyzed, concentrated, and recombined with the depleted thylakoids. The factor in the detergent extract is sulfite sensitive, but the depleted thylakoid membranes are not (Mohamed and Anderson, 1981b). Arsenite-treated thylakoids which have been stripped of the LEM are as effective as control thylakoids in a reconstituted light activation system. But the LEM activity in the Zwittergent extract of the arsenitetreated thylakoids is increased when the extract is treated with DTT and the DTT is removed by gel filtration (Anderson et al., 1985). Apparently the arsenite- and sulfite-sensitive LEM is the Zwittergent-stripped thylakoid membrane component necessary for light activation. Sonication enhances reconstitution (Anderson et al., 1985). This, and the requirement for a detergent for solubilization, indicate that the LEM is an integral membrane protein. Since it interacts with soluble stromal components, it is likely that the LEM is located on the surface of the thylakoid membrane. The coincidence of activity for activation of NADP-linked malic dehydrogenase, fructose-bisphosphatase, and NADP-linked glyceraldehyde dehydrogenase on different Procion dye-Sepharose columns indicates that there is only one LEM (Anderson et al., 1985). Specificity for the target enzyme must then be a property of the soluble component of the system. After partial purification and filtration through Sephadex G-75 or elution from Red H3BN Sepharose, the LEM has no apparent “thioredoxin activity,” as measured by DTT-dependent activation of NADP-linked malic dehydrogenase (Anderson et al., 1985). Neither thioredoxin nor plastocyanin can replace the LEM preparation in the light activation assay. EPR spectra of the stripped thylakoid membranes indicate the presence of iron sulfur centers A and B. The LEM then does not appear to be identical with any of these factors.

TABLE 111 Factors Proposed to be Involved in LightlDark Modulation Subunits Molecular weight

Number

Molecular weight

Visible spectra

),A(

Remarks

Thylakoid

Light effect mediator (LEM) E, Protein modulase

10,500

Femdoxin, tbioredoxin reductase

38,000

None

39,000

3

Source Pea

Anderson et al. (1985)

Pea

Anderson et al. (1985) de la Torre et al. (1979)

bound Requires ferredoxin None

2 = 12,500 1 = 14,000

408 nm

Spinach 3 Fe/mol

Spinach

Schiinnann (1981) Hutcheson et al. (1983) Yee et al. (1981) Hutcheson and Buchanan (1983a) Schiirrnann et al. (1981) Wolosiuk et al. (1979)

36,000

None

Maize

38,000 31,000

None

Nostoc Kalanchoe

Thioredoxin fa

11,200

Thioredoxin fb

16,000

Spinach.

None Heat labile

Reference

Spinach

16,000

Thioredoxin f

2

Spinach

Soulie e f al. (1981)

3 Isoenzymes

Nostoc Maize

Yee et al. (1981) Hutcheson et al.

Heat stable

Spinach

Wolosiuk et a/.

3 Isoenzymes

Nostoc Maize

Yee et al. (1981) Hutcheson e f al.

Monomeric in high ionic strength solutions

2 = 8,000

16,000 20,000

(1983)

Thioredoxin m

9,000

(1979) 9,000 10,000

(1983)

Thioredoxin mb

11,500

None

Spinach

Schiirmann et al.

Thioredoxin mc

12,300

None

Spinach

Schurmann e f a/.

Ferralterin

28,000

3

28,000

3

(1981)

-

(1981)

e

pYruvate,Pi dikinase activating factor

88,000

2= 1= 2= 1=

12,000 7,000 12,000 7,000

410 nm

3 Fe/mol

Spinach

de la Torre et a/.

410 nm

3 Fe/mol

Nostoc

de la Torre et al.

Maize

Nakamoto and Sugiyama (1982)

(1982) (1982)

12

LOUISE E. ANDERSON

The molecular weight of the LEM is about 10,500 as estimated by gel filtration (Anderson et al., 1985). The LEM is, in size and in having a redox active vicinal dithiol group, very much like thioredoxin (see Section I1,B) or glutaredoxin. But unlike these proteins, the LEM appears to be involved directly or indirectly in a 1 e- transfer reaction. There seems to be no reason to suspect that the LEM is a thioredoxin.

2. Protein Modulase Evidence for a soluble component in this system first came from experiments of Ashton and Anderson (1981) in which a stromal factor was shown to be required for light modulation. This factor was very unstable. Recently it has been resolved into two components (Fig. 4; Anderson et al., 1985). The first component is ferredoxin. The second has been extensively purified by adsorption onto ferredoxin-Sepharose and elution with a salt gradient. Soluble thioredoxin is not required, but involvement of thioredoxin has not been eliminated since some thioredoxin will be bound to the thylakoid membranes (Ashton et al., 1980) used in the light activation assay. It is possible that thioredoxin is a tightly bound modulase subunit. This system is not sensitive to fairly high levels of ferredoxin antibody (Ashton, Mohamed, and Anderson, unpublished). Therefore either this ferredoxin differs from the soluble ferredoxin involved with ferredoxin-NADP reductase in photosynthetic NADP reduction or the antigenic sites are shielded when it is complexed with the modulase protein.

Fig. 4. Separation of protein modulase (PM)activity into two components on Sephacryl S-200. The activity of the first component (0)is enhanced by the addition of the second component (0). which appears to be identical with ferredoxin.

LIGHT/DARK MODULATION

13

Fig. 5 . Hypothetical scheme for light activation catalyzed by the LEM system. In frame 1 all of the components are in the dark, oxidized state. In frame 2 the LEM is reductively activated by transfer of electrons from some component on the reducing side of photosystem 1 before ferredoxin. In frame 3 ferredoxin (Fd) is reduced and passes an electron to protein modulase (PM).In frame 4 protein modulase is reduced and passes its electrons to the target enzyme (TE).In frame 5 the target enzyme is reduced and undergoes a change in conformation to the light form.

If ferredoxin is involved in LEM system-catalyzed light modulation, what then is the role of the LEM? Originally it was thought to be a reductant, first for the target enzyme, then for protein modulase. Since the LEM becomes arsenite sensitive in the light in the absence of the soluble stromal factors ferredoxin and protein modulase, it seemed reasonable to assume that the LEM is reductively activated by the electron transport system. But if electron transport through ferredoxin is required for modulation, and if the LEM is reduced in the absence of ferredoxin (or under conditions which do not allow light modulation of target enzyme activity), then the LEM cannot be either the terminal target enzyme reductant or a 2 e- carrier. It is tempting to speculate that the LEM is a modifier or regulatory protein which must first be activated before light modulation can proceed. Such a possible scheme for light modulation involving these factors is shown in Fig. 5 . Alternatively, the LEM may be a 1 e- carrier which shifts orientation in the thylakoid membrane to expose vicinal dithiols in the light.

14

LOUISE E. ANDERSON

active

MDH<~ MDH2I inactive\

NADP

Fig. 6. Scheme for light modulation involving thioredoxin proposed by Buchanan and coworkers. Electrons are passed from ferredoxin (Fd) to ferredoxin-thioredoxin reductase (Fd,ThR) to thioredoxin (Th) and then to the target enzyme (MDH, NADP-linked malic dehydrogenase). From Anderson (1983a) with permission.

B. THE FERREDOXIN-THIOREDOXIN REDUCTASE, THIOREDOXIN SYSTEM

Buchanan et al. (1967) demonstrated a light- and ferredoxin-dependent activation of fructose-bisphosphatase in broken spinach chloroplast preparations. Light activation, according to the scheme later proposed by Buchanan and collaborators (Buchanan, 1980a), involves electron transport through ferredoxin to the soluble enzyme ferredoxin-thioredoxin reductase which, in turn, reduces thioredoxin. Reduced thioredoxin interacts with the target enzyme converting it to the light form. See Fig. 6. Ferredoxin-thioredoxin reductase may or may not be an iron sulfur protein (see the next section). Several thioredoxins have been implicated in light modulation. These are usually referred to as thioredoxin f (more active with fructose-bisphosphatase)and thioredoxin m (more active with malic dehydrogenase). Variants are designated thioredoxin fa, thioredoxin fb, etc. (see Table 111). 1 . Ferredoxin- Thioredoxin Reductase This enzyme is required for thioredoxin- and ferredoxin-dependent light modulation. It is thought to mediate the transfer of electrons from reduced ferredoxin to oxidized thioredoxin. An analogous enzyme, which is part of the ribonucleotide reductase system in bacterial and mammalian systems, uses NADPH as reductant. The ferredoxin-dependent enzyme does not appear to be structurally related to the NADPH reductases. There is disagreement as to the nature of the ferredoxin-dependent reductase, which was first separated from thioredoxin by Schurmann et al. (1976). Buchanan and co-workers have isolated a colorless protein (M, 38,000) from spinach (de la Torre et al., 1979), maize (Hutcheson et al.,

LIGHT/DARK MODULATION

15

1983), and Anabaena (Yee et al., 1981), which they claim has ferredoxinthioredoxin reductase activity. A smaller (3 1 kDa) reductase has been partially purified from Kulanchoe (Hutcheson and Buchanan, 1983a). This reductase cross-reacted with antibody to maize ferredoxin-thioredoxin reductase, but it was not sufficiently pure for spectral studies. Schiirmann (1981) has isolated a brownish protein (hm,,408nm) from spinach which consists of one 14 kDa and two 12.5 kDa subunits and has reductase activity. The spectrum of this protein is shown in Fig. 7. The highest ratio A408:A279 found is 0.37. The Orsay group (Droux et al., 1984; Jacquot et al., 1983) has isolated a similar brownish reductase from spinach. Antibody against this reductase completely inhibits light activation in broken spinach and pea leaf chloroplast preparations. Schiirmann (1981) has observed that a colorless protein, which is not involved in thioredoxin reduction, copurifies with the reductase. It remains to be determined whether there are two ferredoxin-thioredoxin reductases in spinach or whether one of these is an artifact. A similar protein, which reportedly functions independently of ferredoxin, was isolated earlier by the Berkeley group and named ferralterin (Section 11,C). In view of the similarities between the brownish ferredoxin-thioredoxin reductase and ferralterin, it seems quite possible that they are the same protein. Strangely, although ferralterin has a distinctive EPR spectrum (see Section I1,C) EPR studies of the brownish ferredoxinthioredoxin reductase appear not to have been done. The redox potential for oxidation and reduction of the iron sulfur group of the brownish ferredoxin-thioredoxin reductase has not yet been determined. If it is the same as that of ferralterin (Section I1,C) then some other as yet unidenti-

WAVELENGTH (nm)

Fig. 7. Absorption spectrum of the brownish ferredoxin-thioredoxin reductase. From Schiirrnann (1981) with permission.

16

L O U S E E. ANDERSON

fied group on the protein must be responsible for the electron transport properties of the molecule, since that FeS center gives the protein a very positive redox potential. If either the colorless reductase or the brownish reductase does transfer electrons from ferredoxin to thioredoxin (or to the target enzyme redox active disulfide) then the functional prosthetic group must be capable of a 1 e- to 2 e- transfer. Flavins and quinones usually fulfill this function. Ferredoxin-thioredoxin reductase is not restricted to photosynthetic organisms: An FAD-containing ferredoxin-thioredoxin reductase has been isolated from the obligate anaerobe Clostridium pasteurianum (Hammel et al., 1983). The analogous NADPH-requiring enzyme, a component of the system for ribonucleotide reduction in mammalian and bacterial systems (see above), is a flavoprotein. Ferredoxin-thioredoxin reductase activity has been found in darkgrown barley seedlings (Buchanan et al., 1978), but NADPH thioredoxin reductases have been isolated from wheat flour (Suske et ul., 1979) and dark grown carrot tissue culture cells (Cao et al., 1984). Droux et al. (1984) have shown that their brownish ferredoxin-thioredoxin reductase binds to ferredoxin-Sepharose, which indicates that the two proteins interact in uitro and hence might interact in uiuo. Kalunchoe ferredoxin-thioredoxin reductase and, according to Buchanan (1983), the reductases from Anabaena, maize, and spinach also bind to ferredoxin immobilized on Sepharose. It is not clear whether it is the colorless 38 kDa protein described by the Berkeley group or the iron sulfur protein of Schurmann which is bound in the later experiments. According to Droux et al. (1984) their reductase also binds to thioredoxin-Sepharose. According to Jacquot et al. (1983) the brownish reductase catalyzes the activation of NADP-linked malic dehydrogenase with either spinach or pea thylakoids. In the latter case, the system is thioredoxin dependent only after the thylakoids are treated with Triton X-100in the presence of high levels of salt. SouliC et ul. (1981) have shown that thioredoxin f can be reduced by ferredoxin-NADP reductase with NADPH as reductant. As has been pointed out by Lara et al. (1983), none of the ferredoxinthioredoxin reductases has been shown to reduce thioredoxin, either in catalytic or in substrate amounts. The enzyme may, then, be misnamed and might function in some way other than that proposed (see Fig. 6). For example, thioredoxin, reduced directly by the photosynthetic electron transport system, might interact with the reductase to form an active complex which is able to reduce the target enzyme. In such a complex the electrons for reduction might pass directly from the reductase to the target enzyme. Alternatively, thioredoxin might be a more effective reductant when complexed with the reductase.

LIGHT/DARK MODULATION

17

2. Thioredoxin Thioredoxins are small and rather unusual proteins. Most thioredoxins are heat stable; all contain redox-reactive vicinal dithiol groups. In the oxidized form the disulfide protrudes from the surface of the protein (Holmgren, 1981). Among enzymes studied to date, thioredoxin and the closely related protein glutaredoxin are unique in having protruding active sites. Thioredoxins are thought to be involved in the reduction of nucleotides to deoxynucleotides (see Section II,B,l), in sulfur metabolism, and in other reactions. For their role in dark modulation see Section IV,A. Levels of thioredoxin in chloroplasts are quite high (Scheibe, 1983b). A part of the “thioredoxin-like” activity of the pea leaf chloroplast is membrane bound (Ashton et al., 1980). The chloroplast thioredoxins are classified as thioredoxin f (active in the activation of fructose-bisphosphatase) and thioredoxin m (active in the activation of NADP-linked malic dehydrogenase). Two thioredoxin fs, thioredoxin fa and thioredoxin fb, and several thioredoxin m’s have been isolated (see Table 111). Buchanan’s group found two thioredoxins in spinach chloroplasts (Wolosiuk et al., 1979) and three in barley chloroplasts (Crawford et al., 1979). According to this group, thioredoxin f is heat labile and has a molecular weight of 16,000 when isolated from spinach. This thioredoxin, now named thioredoxin fb, has also been purified to homogeneity by SouliC et al. (1981). Apparently it is a dimer composed of two 8 kDa monomers which dissociate in high ionic strength solutions. The dimer seems to be the active form of the factor (or conditions which favor dimerization favor activation) (Soulik et al., 1981). A second thioredoxin f, now called thioredoxin fa, was first isolated by Schurmann and coworkers (1981) and later by Buc et al. (1983, 1984). The purified (active) protein exists only as a monomer with a pZ of 6.1 (Schurmann el al. 1981). The amino acid compositions of both thioredoxin f s have been determined (Schurmann et al., 1981; SouliC et al., 1981). The thioredoxin m isolated by Wolosiuk and co-workers (1979) from spinach is a 9 kDa heat-stable monomer. Schiirmann and co-workers (1981) find two m-type thioredoxins in spinach. They differ slightly in molecular weight (1 1,500 and 12,300) and in pZ values (5.2 and 5.0). Their amino acid compositions are similar and differ from that of thioredoxin fa. Antibodies to the two m-type thioredoxins cross-react with both of these thioredoxins, but not with thioredoxin fa. Likewise antibody to thioredoxin fa does not cross-react with the thioredoxin m’s. Three thioredoxins (two thioredoxin m’s and one thioredoxin f) have been separated from sorghum and from bean (Phaseolus vulgaris) leaves by Jacquot et al. (1978). Two thioredoxin f s and one thioredoxin m have been isolated from

18

LOUISE E. ANDERSON

Kalunchoe leaves (Hutcheson and Buchanan, 1983a). These appear to be similar in molecular weight and specificity to those from spinach and maize, but considerably less stable. A much larger protein (30 kDa) with thioredoxin activity has been isolated from Scenedesmus by Follmann and co-workers (Berstermann et al., 1983). Two smaller thioredoxins, apparently cytosolic, M, about 12,600, have also been isolated from this green alga. A multiplicity of proteins with thioredoxin activity have been isolated from maize by Jacquot and Gadal (1983). It is suggested that some of these proteins may have other functions in the plant. Buchanan's group (Hutcheson et al., 1983) has separated three thioredoxin f s and three thioredoxin m's from maize. The three forms, in both cases, have different PI'S. Tsugita et al. (1983) have shown that in absence of effectors, such as fructose bisphosphate, only thioredoxin f, and not thioredoxin m, is capable of catalyzing the DTT-dependent activation of fructose-bisphosphatase. Buc et al. (1983) found that both thioredoxin fa and thioredoxin fb, but not thioredoxin m, catalyze the activation of fructose-bisphosphatase, whereas only thioredoxin fb supports the DTT-dependent activation of sedoheptulose-bisphosphatase. NADP-linked malic dehydrogenase, on the other hand, is activated by all of the chloroplast thioredoxins (Tsugita et al., 1983; Buchanan, 1983) and by Escherichia coli thioredoxin (Tsugita et al., 1983). The same specificity for thioredoxins is seen with the ferredoxinthioredoxin reductase system (Schurmann, 1981; Wolosiuk et al., 1979). Spinach thioredoxin f is more efficient than thioredoxin m in catalyzing the activation of phosphoribulokinase and NADP-linked glyceraldehyde3-P dehydrogenase with DTT and in the ferredoxin-thioredoxin system (Wolosiuk et al., 1979). Crawford et al. (1979) report that there are two thioredoxins in spinach leaf cytosol similar to chloroplastic thioredoxin f and thioredoxin m, which they designate thioredoxin cf and cm.The function of these thioredoxins is not known, but it seems possible that they are involved in modulation of enzyme activity in the cytosol, either as light or dark modulators (see below). Berstermann et al. (1983) have purified multiple forms of thioredoxins from soybean and wheat seeds. According to Schmidt (1980, 1981), two thioredoxins which he has isolated from the cyanobacterium Synechococcus 6312 (Anacystis nidulans) are able to catalyze the DTT-dependent activation of fructose-bisphosphatase isolated from that organism, but not the activation of the spinach chloroplast enzyme. Two thioredoxins have been isolated from

LIGHT/DARK MODULATION

19

Anabaena sp. 7119 (Nostoc muscorum) by Crawford et al. (1983a,b). According to these workers (Crawford et al., 1984) both thioredoxins are active in DTT-dependent and ferredoxin-thioredoxin-dependent activation of Anabaena fructose-bisphosphatase, sedoheptulose-bisphosphatase, and ribulose-5-P kinase and also in the DTT-dependent and ferredoxin-thioredoxin light-dependent activation of spinach fructose-bisphosphatase and NADP-linked malic dehydrogenase. Some differences in target enzyme specificity were observed between higher plant and Anabaena thioredoxins. In contrast, Gleason (1985) finds only one thioredoxin in this organism. Reduction, according to Gleason, occurs not by a ferredoxin-thioredoxin reductase, but by a membrane-bound NADPH thioredoxin reductase. Reduced thioredoxin is an efficient reductant for ribonucleotide reduction with the ribonucleotide reductase from this organism. In the presence of DTT this thioredoxin activities spinach NADPlinked malic dehydrogenase and fructose-bisphosphatase, but not Anabaena fructose-bisphosphatase (Whitaker and Gleason, 1984). Fructose-bisphosphatase from the closely related A . cylindrica is activated by the single thioredoxin isolated from A . cylindrica (Ip et al., 1984). Clement-Metral and Holmgren (1983) have purified thioredoxin from Rhodopseudomonas sphaeroides to homogeneity and have partially purified an NADPH-dependent thioredoxin reductase from this photosynthetic bacterium. The thioredoxin is similar to the thioredoxin isolated from Anabaena by Gleason and Holmgren (1981) with respect to amino acid composition. The Rhodopseudomonas system catalyzes the activation of 6-aminolevulinic acid synthetase (Clement-Metral and Holmgren, 1983) and might be responsible for the regulation of bacteriochlorophyll synthesis by 02 in the photosynthetic bacteria. Spinach thioredoxin mc and an active site peptide from thioredoxin f have been sequenced by the Swiss group (Tsugita et al., 1983; Schiirmann et al., 1989, and the sequence of Anabaena 7119 thioredoxin has been determined by Gleason et al. (1985) (see Fig. 8). Like all thioredoxins, these thioredoxins and the thioredoxin isolated from Anabaena cylindrica (Ip et al., 1984)contain the invariant tetrapeptide -Cys-Gly-Pro-Cys-.The sequence around the active site of spinach thioredoxin m shows extensive homology with the same regions of the Anabaena, Escherichia coli (Fig. 8), and Corynebacterium nephridii thioredoxins. In contrast, the active site peptide of thioredoxin f shows little homology with the active site peptides of any of these thioredoxins (Tsugita et al., 1983; Schiirmann, 1983). Spinach fructose-bisphosphatase binds to thioredoxin-Sepharose (Pla and Lopez-Gorge, 1981). Elution can be accomplished by addition of

20

LOUISE E. ANDERSON

Spinach thioredoxin fa Val-Leu-~-Met-Phe-Thr-Gln-Trp-Cys-Gly-Pro-Cys-Lys-Ala-Asn-Gly-Asp-Lys-Glu-Ala-Thr-Gln-His

Spinach thioredoxin mc Met-Val-Asp-Phe-Trp-Ala-Pro-Trp-Cys-Gly-Pro-Cys-Lys-Leu-Ile-Ala-Pro-Val-Ile-Asp-Glu-Leu-~

Anabaena Leu-Val-Asp-Phe-Trp-Ala-Pro-Trp-Cys-Gly-Pro-Cys-Arg-Met-Val-Ala-Pro-Val-Val-Asp-Glu-Ile-~

E . coli

Leu-Val-Asp-Phe-Trp-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Lys-Met-Ile-Ala-Pro-Ile-Leu-Asp-Glu-Ile-~

30

24

40

Fig. 8. Amino acid sequence of spinach thioredoxin fa, thioredoxin mc, and E. coli thioredoxin. Sequences are aligned to show overlap. From Tsugita ef ul. (1983) and Gleason (1985).

MgC12. Binding is not specific for thioredoxin f. Thioredoxin m and the cytosolic thioredoxins are also effective. While the phosphatase does not bind to ferredoxin-Sepharose, it does bind to a number of amino acids and related compounds attached to Sepharose. Hence the binding may be relatively unspecific. Scheibe (1983b) estimates that the thioredoxin m concentration in pea chloroplasts is about 50 p M . Surprisingly, the levels of ferredoxin required by the ferredoxin-thioredoxin system have never been reported. In a broken chloroplast system (66 pg chlorophyll ~ m equivalent - ~ thylakoids and stroma) activity appears to be saturated by 2 p M concentrations (Buchanan et al., 1967). In a very preliminary experiment with purified components of the system (Jacquot et al., 1983) rather high levels of thioredoxin appear to be required for maximal light activation of NADP-linked malic dehydrogenase. On the other hand, the thioredoxin f concentration required for half-maximal activation of fructose-bisphosphatase can be calculated to be approximately 0.5 p M from data of Schurmann et al. (1981). This value is probably only valid for the conditions of the particular assay, but it seems likely that thioredoxin concentrations in the chloroplast (Scheibe, 1983b) could support activation by this system. The major problems with the ferredoxin-thioredoxin system lie in the sequence of events and the nature of the reductase. There is no direct evidence that thioredoxin is reduced by the reductase, nor is there direct evidence that reduced thioredoxin activates enzymes. It is not clear how ferredoxin-thioredoxin reductase, if its iron-sulfur group can only function as an oxidant under physiological conditions, can act as a one electron acceptor and transfer and two electrons to reduce a disulfide bond. Clearly additional biochemical characterization of this system is needed and its relationship to the LEM system defined.

LIGHT/DARK MODULATION

21

C. FERRALTERIN

Ferralterin is an iron-sulfur protein which is extraordinarily similar to Schiirmann’s and Jacquot’s ferredoxin-thioredoxin reductase (see above). This enzyme has been purified from Anabaena and from spinach (de la Torre et al., 1982). Some preparations of this enzyme support light activation of fructose-bisphosphatase without the addition of ferredoxin and thioredoxin. The activity of the enzyme in other preparations is stimulated by addition of ferredoxin and/or thioredoxin (Lara et al., 1983). The authors speculate that this occasional dependence on thioredoxin or ferredoxin could have been related to the state of the protein or of the thylakoid membrane. It may be that only very low levels of thioredoxin and ferredoxin are required for light activation and that these were sometimes present on the thylakoid membranes. The molecular weight of ferralterin is reported to be around 28,000 (de la Torre et al., 1982). Before the final steps of purification the enzyme appears to be somewhat larger, since it emerges from a Sephadex G-100 column ahead of ferredoxin-thioredoxin reductase (M,38,000) (Lara et al., 1980; Crawford et al., 1983b). As isolated, ferralterin consists of three subunits, two apparently identical 7 kDa monomers and a single 12 kDa monomer (de la Torre et al., 1982). It contains 3.3 nonheme irons per mole and a corresponding amount of acid-labile sulfur. Presumably there is one 4Fe4S center or two 2Fe2S centers per molecule. It is brownishyellow in color and has a peak in the visible spectrum at 410 nm. The highest ratio of A410:A280 found for the spinach enzyme is 0.35 and for the Anabaena enzyme, 0.39. The EPR signal from oxidized ferralterin is rhombic with g values of 2.10, 2.01, and 2.00. The midpoint redox potential is +410 mV, which is the most oxidizing potential ever reported for an FeS protein. Therefore it is unlikely that the FeS center of ferralterin is directly involved in electron transport. Exactly how ferralterin accomplishes activation of fructose-bisphosphatase is not known. No reports of activation of enzymes other than fructose-bisphosphatase by ferralterin have been published.

D. ACTIVATION OF

PYRUVIC,Pi DIKINASE

The system for activation of pyruvic,Pi dikinase is unique. There is no evidence for thiol involvement (Chapman and Hatch, 1981). Inorganic phosphate and pyruvicpi dikinase-regulatory protein are required for activation of the enzyme, which involves formation of PPi, with the phosphate moieties coming from the required Pi and dikinase threonine-bound phosphate (Ashton and Hatch, 1984; Ashton et al., 1984). Inactivation

LOUISE E. ANDERSON

22

.

E

Y

E

yip (active)

PVRUVATE

4

PPi

Fig. 9. Scheme for the activation and inactivation of pyruvate,Pi dikinase. PDRP is pyruvate,Pi dikinase regulatory protein. PEP is P-enolpyruvate. Phosphorylation of the histidine residue occurs during catalysis. Phosphorylation of the threonine residue occurs during modulation. From Burnell (1984) with permission.

involves the same regulatory protein (Burnell and Hatch, 1983),ADP, and very low levels of ATP (see Fig. 9). During inactivation the P-phosphate of ADP is transferred to the dikinase threonine (Ashton and Hatch,1983a, 1984; Ashton et al., 1984). The regulatory protein is then the only known protein kinase which utilizes ADP rather than ATP as the source of transferable phosphate. The function of the ATP is apparently to phosphorylate a His on the enzyme, rendering it susceptible to ADP-dependent phosphorylative inactivation (Burnell and Hatch, 1984a,b). ADP, AMP, and inorganic pyrophosphate inhibit activation; pyruvate and related compounds inhibit inactivation. It is possible that the light effect is mediated by energy charge and through changes in metabolite levels (Nakamot0 and Edwards, 1983b, 1984a). The enzyme in maize is activated in the dark under anaerobic conditions (Nakamoto and Edwards, 1983a).

E. OTHER MECHANISMS

Experiments of Scheibe and Beck (1979) and Alscher (1982b) suggest that there is a membrane-associated light activation system in spinach and soybean, respectively. This could represent a membrane-bound version of one of the systems described above. Rudd and Anderson (1983) report that light inactivation of a membrane-bound form of P-fructokinase from

LIGHT/DARK MODULATION

23

pea leaf chloroplasts is sensitive to DCMU and to Diquat, which indicates that photosystem I is involved in light modulation of this thylakoid-bound enzyme. A part of the glucose-6-P dehydrogenase, ribulose-5-P kinase, and glyceraldehyde-3-P dehydrogenase activities in the chloroplast are membrane bound and are released to the stroma when the chloroplast is irradiated (Ben-Bassat and Anderson, 1981; Anderson and Ben-Bassat, 1981). Release is DCMU sensitive. It seems possible that the light modulation system is involved in binding and/or release. Fructose-bisphosphatase from Anacystis nidulans is inactivated by glutathione and by DTT (Udvardy et al., 1983). According to these workers there is no detectable glutaredoxin activity in this photosynthetic prokaryote. It is suggested that GSH/GSSG levels may be important in regulation of the activity of this enzyme in uiuo. Halliwell and Foyer (1978) have clearly shown that GSWGSSG levels remain relatively constant during lightldark transitions, which would seem to rule out GSH as a mediator in higher plants. Schriek and Schwenn (1984) found that spinach chloroplast ferredoxinNADP reductase contains a protein capable of catalyzing NADPH-dependent activation of fructose-bisphosphatase. Although thioredoxin stimulates activation, there is substantial (50% of complete system) activation without the dithiol. Ferredoxin is not required. The activating protein has NADPH-ferredoxin and NADPH-DCPIP diaphorase activity. It seems possible that this enzyme is related to protein modulase or ferredoxinthioredoxin reductase. It has been suggested that NADP, NADPH, and ATP might be the light mediators (Lendzian, 1978, 1980; Lendzian and Bassham, 1975; Miiller, 1970; Miiller et al., 1969; Schmidt-Clausen et al., 1969; Ziegler, 1972). Although these compounds will act as effectors in uiuo, there is no indication that this type of modulation involves covalent modification. Activation through NADPH reduction cannot be ruled out (see above). Winter (1980) has suggested that the light inactivation of P-enolpyruvate carboxylase in the CAM plant is actually due to malate inhibition, but this possibility seems to have been eliminated by experiments of Manetas and Gavalas (1983) in which the extracts were desalted by passage through Sephadex G-25 before activity was measured. F. LIGHT MODULATION OF THE ACTIVITY OF CYTOSOLIC ENZYMES

The mechanism of this process has received little attention and is not understood. Since DCMU inhibits light inactivation of pea leaf cytosolic glucose-6-P dehydrogenase (Anderson and Nehrlich, 1977) and activation

24

LOUISE E. ANDERSON

of P-enolpyruvate carboxylase in Salsola soda (Karabourniotis et al., 1983), the photochemical apparatus must be involved.

111. REGULATION OF LIGHT MODULATION Although a number of enzymes are light modulated the only evidence at present for target enzyme specificity is in the difference in activity observed with thioredoxin f and thioredoxin m in the ferredoxin-thioredoxin system. Yet there clearly is differential modulation of target enzyme activity in the intact chloroplast. How is this differential modulation achieved and how is light modulation controlled? A unique solution to these problems seems to have evolved in the green plant. Apparently the target enzymes, rather than the modifying enzymes, interact with modifiers. In some cases these might be acting by blocking the redox-active disulfide bond. In other cases they may be altering the conformation of the target enzyme, either making the redox-active disulfide inaccessible to the modifying enzyme (be it protein modulase, ferralterin, or reduced thioredoxin) or changing the redox potential of the active disulfide so that it can no longer be reduced, or, in dark modulation, oxidized. The effect of modulators on activation has been the subject of a number of investigations.

A.

NADP-LINKED MALIC

DEHYDROGENASE

NADP inhibits the DTT-thioredoxin-dependent activation of NADPlinked malic dehydrogenase and the thioredoxin-dependent inactivation of the enzyme (Ashton and Hatch, 1983~).The effect of NADP is reversed by NADPH, but NADPH itself is without effect. A model has been constructed to show the effects of varying NADPINADPH ratios on the steady state activity levels of the target enzyme (see Fig. 10). Although many assumptions have to be made, it is significant that the modulation system itself must be subject to control.'That NADP does indeed inhibit light activation of this enzyme has been shown by Scheibe and Jacquot (1983). In experiments with intact chloroplasts these workers, as well as Leegood and Walker (1983b), demonstrated that electron acceptors, which would increase the NADP/NADPH ratio, inhibit the activation of this dehydrogenase. The enzyme is activated in the dark in intact maize leaves under anaerobic conditions (Nakamoto and Edwards, 1983a, 1984b).

25

LIGHT/DARK MODULATION

0

0.5

1.0

M D P H (RIM)

Fig. 10. Simulated rates of thioredoxin-dependentactivation and inactivation of NADPlinked malic dehydrogenase activity at different NADPWNADP ratios. From Ashton and Hatch (1983~)with permission.

B. FRUCTOSE-BISPHOSPHATASE

The DTT-dependent activation of fructose-bisphosphatase is enhanced by fructose bisphosphate and Mg2+(Rosa, 1981; Rosa and Whatley, 1984). The DTT-thioredoxin-dependent activation of fructose-bisphosphatase also appears to be enhanced by fructose bisphosphate (Wolosiuk er al., 1980; Nishizawa and Buchanan, 1981; Hutcheson and Buchanan, 1983b). Here it is possible that the substrate protects the enzyme from inactivation by overreduction. Hertig and Wolosiuk (1980) and Hutcheson and Buchanan (1983b) report that Ca2+ and fructose bisphosphate, or Mn2+ and fructose bisphosphate, stimulate thioredoxin f-DTT-dependent activation of fructose-bisphosphatase. The Ao.5 for fructose-bisphosphatase is 0.33 mM, and, for Ca2+,55 p M (Hertig and Wolosiuk, 1980). These concentrations are reasonably close to assumed concentrations in uiuo and it can be concluded that these compounds may modulate activation in the green leaf. Sedoheptulose bisphosphate, glucose I ,Qbisphosphate, or ribulose bisphosphate can replace fructose bisphosphate in the Ca2+-and thioredoxin f-dependent DTT activation (Hertig and Wolosiuk, 1983). Rosa (1981) suggests that Ca2+may be acting by inhibiting the breakdown of the enzyme protector (and substrate) fructose bisphosphate. Whatever the biochemical explanation for the effect, these experiments suggest that both substrate and metal cations might affect modulation in uiuo and that

26

LOUISE E. ANDERSON

the two might act together. Gontero et al. (1984), however, find little or no effect of the carbon substrates and Mg2+on thioredoxin-dependent activation. The strict specificity for thioredoxin fin DTT-thioredoxin activation of fructose-bisphosphatase is lost in the presence of fructose bisphosphate (Schurmann, 1983). Light activation of this enzyme in broken pea leaf chloroplast preparations is inhibited by sedoheptulose bisphosphate, 3-Pglycerate, ribulose bisphosphate, and NAD (Anderson et al., 1981). Fructose bisphosphate is without effect. In spinach chloroplasts light activation of fructose-bisphosphatase is inhibited by anaerobiosis (Leegood et al., 1982). Light activation of NADP-linked malic dehydrogenase was not affected by anaerobiosis in the same experiments. These data indicate that differential regulation of light activation can occur in the intact chloroplast. According to Charles and Halliwell (1981~)addition of dihydroxyacetone-P to the suspension medium enhances activation of fructose-bisphosphatase in spinach chloroplasts. In intact wheat leaves at low light intensities activation of fructose-bisphosphataseis sensitive to atmospheric levels of 0 2 (Leegood and Walker, 1983a). This could be a reflection of the effect of metabolite levels on modulation or a direct effect of O2on the reductant required for activation.

C. SEDOHEPTULOSE-BISPHOSPHATASE

Inorganic phosphate apparently inhibits the DTT-dependent activation of sedoheptulose-bisphosphatase (Woodrow et al., 1983). DTT-thioredoxindependent activation of maize sedoheptulose-bisphosphataseis enhanced in the presence of sedoheptulose biphosphate, but fructose bisphosphate has no effect (Nishizawa and Buchanan, 1981). Ca2+enhances the DTTdependent activation of maize sedoheptulose-bisphosphatase and inhibits the activity of the enzyme (Wolosiuk et al., 1982). Ethylene glycol bis(paminoethy1 ether)N,N ,N’ ,N’-tetraacetate (EGTA), which chelates Ca2 , completely inhibits activation. Mg2+ and sedoheptulose bisphosphate (presumably both are required) enhance the DTT-dependent activation of wheat leaf sedoheptulose-bisphosphatase (Woodrow and Walker, 1982; Woodrow et al., 1983). Clearly the availability of metal cations, as well as substrate, could modulate light activation of both phosphatases in uiuo. Light activation of sedoheptulose-bisphosphatase in wheat chloroplasts is stimulated by the transportable metabolite dihydroxyacetone-P(Woodrow and Walker, 1980). Presumably the dihydroxyacetone-P is con+

LIGHT/DARK MODULATION

27

verted to sedoheptulose bisphosphate in the chloroplast which enhances activation of the enzyme. D . RIBULOSE-5-PHOSPHATE KINASE

Ashton (1983b) has examined the effects of ATP on DTT-dependent activation of maize ribulose-5-P kinase . Since ATP inhibits DTT-dependent activation and also inactivation by GSSG or alkylating reagents, the regulatory disulfide is probably located near the ATP binding site. Activation of this enzyme in broken pea leaf chloroplast preparations is inhibited by AMP, NADPH, NADH, and 6-P-gluconateand stimulated by NADP (Anderson et al., 1981). E. GLUCOSE-6-PHOSPHATE DEHYDROGENASE

The DTT-dependent inactivation of this enzyme in crude extracts of Anabaena uariabifis is reversed by the substrate, glucose-6-P (Cossar et al., 1984). Glucose-6-P, sedoheptulose bisphosphate, fructose bisphosphate, Pi, NAD, NADH, ADP, and NH4Cl inhibit light inactivation of this enzyme in broken pea leaf chloroplast preparations (Anderson et d . , 1981). In intact spinach chloroplasts light inactivation of glucose-6-Pdehydrogenase is inhibited by Pi, which causes transport of metabolites out of the chloroplast (Huber, 1979). Inhibition is reversed by P-glycerate. F. GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

Activation of the NADP- and NAD-linked enzyme activities in broken chloroplast preparations is inhibited by ATP, ADP, NADPH, NADH, NAD, 6-P-gluconate, 3-P-glycerate, sedoheptulose bisphosphate, and fructose bisphosphate (Anderson et al., 1981). Activation of the NADPlinked activity (but not of the NAD-linked activity) is inhibited by AMP, NADP, fructose bisphosphate, and NH4Cl. Glucose-6-P inhibits activation of the NAD-linked activity, but not activation of the NADP-linked activity. The pH dependency and Mg2+requirements for activation and for inactivation are different for each light modulated enzyme (Anderson et al., 1981). It seems likely that these differences reflect direct influence on, or interaction with, the target enzyme rather than the enzymes of the modulating system. Stepwise and/or differential activation by a single modifying system may be made possible by the sensitivity of the target enzymes to effectors and to pH.

28

LOUISE E. ANDERSON

IV. DARK MODULATION Aerobic conditions appear to be required for dark modulation in uiuo (Nakamoto and Edwards, 1983~).Under anaerobic conditions reversal of dark modulation (mimicking light modulation) has been observed both in intact leaves (Nakamoto and Edwards; 1983c) and in chloroplasts (Leegood and Walker, 1981a). A . THIOREDOXIN IN DARK REVERSAL OF LIGHT MODULATION

Oxidized thioredoxin is required for dark modulation of NADP-linked malic dehydrogenase and glucose-6-P dehydrogenase in the reconstituted pea leaf chloroplast modulation system (Scheibe and Anderson, 1981). Fructose-bisphosphatase is rapidly inactivated in the dark following activation with highly purified components of the ferredoxin-thioredoxin system and illuminated thylakoid membranes (Schurmann, 1983). Oxidized thioredoxin f also has been shown to inactivate DTT-activated fructosebisphosphatase (Soulib et al., 1981) and sedoheptulose-bisphosphatase (Meunier et al., 1983; Gontero et al., 1984a). Antibody to E. coli thioredoxin, which inhibits the activity of purified pea leaf thioredoxin, inhibits dark modulation in broken chloroplast preparations (Scheibe and Anderson, 1981). Clearly thioredoxin is necessary for the reversal of light modulation. Kagawa and Hatch (1977) earlier showed that a small heat stable regulatory protein catalyzed the inactivation of NADP-linked malic dehydrogenase from maize. This factor was probably thioredoxin. The specificity of thioredoxins as dark mediators has not been determined. Scheibe (1981) has shown that levels of oxidized thioredoxin are high in the dark and decrease markedly in the light (Fig. Il), but it is not clear how reduced thioredoxin is oxidized in the chloroplast. The rate of inactivation of fructose-bisphosphatase is decreased if an 02 trap is included in the system. Oxidized ferredoxin or oxidized thioredoxin, added when the light is turned off, increases the rate of inactivation (Schiirmann, 1983; Schurmann and Kobayashi, 1984). Although oxidation of reduced thioredoxin is catalyzed by ferredoxin-NADP oxidoreductase, which transfers electrons from thioredoxin to NADP (SouliC et al., 1981), this reaction seems too slow to account for the rapid oxidation of reduced thioredoxin in intact chloroplasts. A cysteine oxidase which might function in dark modulation has been identified in a variety of photosynthetic organisms ranging from angiosperms to purple nonsulfur photosynthetic bacteria (Schmidt and Kramer, 1983, 1984). According to the scheme postulated by these workers cystine would oxidize either the light form of the target enzyme or thiore-

LIGHT~DARKMODULATION

29

Fig. I I. NADP-linked malic dehydrogenase activity (0)and level of oxidized thioredoxin From Scheibe (1981) with permission.

(m)in chloroplast suspensions upon light/dark/light transitions.

doxin. Since catalase inhibits dark modulation (Brennan and Anderson, 1980) (see also Charles and Halliwell, 1981c), there is also a possibility that peroxides are involved in the oxidation of thioredoxin. Figure 12 depicts a possible scheme for the thioredoxin-dependent dark reversal of light modulation. B. OTHER POSSIBLE MEDIATORS

Various naturally occuring oxidants including dehydroascorbate and oxidized glutathione will reverse reduced thioredoxin-mediatedenzyme activation and it has been suggested that they function as dark modulators

Fig. 12. Scheme for dark modulation. When the light goes out the target enzyme (TE) is in the reduced, light form and thioredoxin (Td) becomes oxidized (frame I). The enzyme passes its electrons to thioredoxin and changes conformation as it changes to the dark form (frame 2). Thioredoxin in turn finds an electron acceptor and returns to its active form as dark modulator (frame 3).

30

LOUISE E. ANDERSON

(Nishizawa and Buchanan, 1981; Schurmann and Wolosiuk, 1978, Wolosiuk and Buchanan, 1979). Since in all of these experiments oxidized thioredoxin would have been generated, it seems likely that the effect of these compounds was mediated by thioredoxin. Udvardy et al. (1984) have shown that various nonprotein Fe3+chelates mimic the effect of dark modulation with partially purified chloroplast and cyanobacterial enzymes. They note that this property is consistent with a role in dark modulation in uiuo. C. REGULATION OF DARK MODULATION

Dark modulation is subject to pH and metabolite control. Fructose bisphosphate, added to the ferredoxin-thioredoxin reductase activation system, accelerates light activation of fructose-bisphosphatase, and slows down or, at high concentrations, totally blocks dark inactivation at a pH approximating to that of the stroma in the illuminated chloroplast (pH 7.9) (Schurmann, 1983). At pH 7.2 (dark stromal pH) fructose bisphosphate has no effect on dark inactivation. The effect of a series of metabolites and of pH and Mg2+concentrations on dark modulation in broken chloroplast preparations has been reported (Anderson et al., 1981). As is the case for light modulation, no clear-cut effect of any single metabolite on dark modulation is evident. It therefore seems likely that the effects are mainly on the target enzyme which becomes more or less susceptible to dark modulation in the presence of the effector.

V. CHANGES IN THE TARGET ENZYME Light modulation probably involves a posttranslational covalent modification of proteins already modified by oxidation of cysteine residues. As yet there is no direct evidence for target enzyme reduction by the light modulation system. It is known that the conformation of the enzyme and its kinetic parameters are altered by light modulation and, except in the case of transaldolase (Anderson, 1981), by DTT-dependent reduction. A. CHANGE IN THE TARGET ENZYME AS A RESULT OF LIGHT MODULATION

1. Oxidation State

There are several reasons to suspect that light modulation is reductive and dark modulation is oxidative. (1) The effects of DTT and light modulation

LIGHT/DARK MODULATION

31

(Anderson, 1979) and of dark modulation and the strong oxidant diamide (Ashton and Anderson, 1981) on enzyme activity are similar. (2) Schiirmann (1983) has shown that reduced ferredoxin is oxidized in the presence of fructose-bisphosphatase, ferredoxin-thioredoxin reductase, and thioredoxin. (3) There is a marked DCMU-sensitive increase in total stroma1 thiol groups when spinach chloroplasts are irradiated (Slovacek and Vaughn, 1982; Slovacek and Monahan, 1984). The increase in the activity of fructose-bisphosphatase and NADP-linked malic dehydrogenase is essentially coincident with the increase in stromal thiol groups. (4) There is a significant increase in both soluble and membrane-bound thiols in an illuminated activation system consisting of thylakoids and partially purified ferredoxin-thioredoxin reductase, fedredoxin, thioredoxin, and NADP-linked malic dehydrogenase (Jacquot et a f . , 1983). An earlier suggestion (Anderson et al., 1978) that light modulation might result in intrapeptide thiol-disulfide exchange rather than in net reduction was based on the assumption, later shown to be incorrect, that no soluble intermediate was involved in light modulation. Nevertheless, modulation of the soluble chloroplast enzyme could involve thiol-disulfide exchange or some other alteration in enzyme structure rather than net disulfide reduction. Activation of the chloroplast coupling factor, CFI, by light and DTT involves reduction of a disulfide on the y subunit (Ketcham et al., 1984; Moroney et al., 1984; Nalin and McCarty, 1984). It is thought that in vivo activation is the result of light-dependent energization which makes the redox-active disulfide accessible to light-dependent reduction by the reducing component of the light modulation system. 2. Conformation There is good evidence that light modulation is accompanied by a change in conformation. Although the light and dark forms of pea leaf chloroplastic glyceraldehyde-3-P dehydrogenase are the same size, they differ in electrophoretic mobility (Anderson and Lim, 1972). This indicates that the exposed charged groups on the two forms differ. Light modulation involves a change in the susceptibility of four enzymes (fructose-bisphosphatase, glucose-6-P dehydrogenase, NADP-glyceraldehyde-3-P dehydrogenase, and ribulose-5-P kinase) to proteolytic degradation in crude extracts (Anderson et af., 1979) and, in the case of glucose-6-P dehydrogenase, in a highly purified system (Srivastava and Anderson, 1983). The exposure of protease-susceptible bonds is apparently altered as a result of the change in conformation associated with modulation. Differences in the thermal denaturation patterns of the light and dark forms of glucosedP dehydrogenase also suggest that light/dark modulation involves confor-

32

LOUISE E. ANDERSON

mational changes in the modulated proteins (Srivastava and Anderson, 1983). Glyceraldehyde-3-P dehydrogenase from some species forms aggregates (Wolosiuk and Buchanan, 1976; Cerff, 1978; Pupillo and Faggiani, 1979; Wara-Aswapati et al., 1980). The relationship of the aggregation state of the enzyme to light modulation is unclear, but it seems likely that it reflects the change in conformation which accompanies modulation. B. CHANGE IN THE TARGET ENZYME AS A RESULT OF THIOREDOXIN-DTT-CATALYZED MODULATION

Jacquot et al. (1984) report that two thiols per 38 kDa subunit appear when maize NADP-linked malic dehydrogenase is thioredoxin-DTT activated, but DTT treatment (48 hr) produces only 1.5 thiols per subunit. According to Scheibe (1984), two thiols per subunit appear when pea leaf NADP-liked malic dehydrogenase is thioredoxin-DTT or DTT activated. In contrast there is, according to Buchanan (1981), no change in the thiol content of fructose-bisphosphatase as a result of thioredoxin-DTT activation, but DTT activation results in an increase in 16 thiol groups. It should be noted that this enzyme reportedly has a very high Cys content (Buchanan et d . ,1971) and that it might be difficult to detect a change in one disulfide in such a protein. Pradel et al. (1981) report an increase of 24 sulfhydryl groups following activation of fructose-bisphosphatase by DTT. Wolosiuk and Buchanan (1976, 1979) have isolated DTT-thioredoxinsensitive and -insensitive forms of ribulose-5-P kinase and glyceraldehyde-3-P dehydrogenase. These apparently differ in molecular weight. The basis for the differences in aggregation and thioredoxin-DTT sensitivity has not been investigated. C. CHANGE IN THE TARGET ENZYME AS A RESULT OF DITHIOTHREITOL TREATMENT

There is an increase of two thiol groups per subunit when pea leaf NADPlinked malic dehydrogenase is reduced with DTT (Scheibe, 1984). Activity lags behind the appearance of the thiol groups (Fig. 13). Analyses of the kinetics of activation and enzyme reduction indicate that all four subunits of the tetramer must be reduced before the enzyme is activated. Activation can, however, also be achieved by treating the enzyme with guanidineaHC1, which would seem to indicate that activation results from a conformational change rather than from reduction per se (Scheibe and Fickenscher, 1985).

LIGHT/DARK MODULATION

I

I

I

0

10

2'0

33

30 6 0

Activation time (Mia)

Fig. 13. Appearance of thiol groups and activity during D'IT-dependent activation of NADP-linked malic dehydrogenase. From Scheibe (1984) with permission.

Fertk et al. (1984) find no free thiols on purified spinach NADP-linked malic dehydrogenase, and three per subunit after reduction. They suggest that at least one Cys per subunit is involved in an interprotomeric disulfide bond. Ashton (1983a) has used Procion Rubine MXB as an affinity label for maize NADP-linked malic dehydrogenase. The dye reacted both with a group in or near the active site and with a reactive thiol, presumably reduced when the enzyme is activated with DTT. Potentially the light and dark forms of the enzyme could be separated and the redox-active dithiol involved in light modulation could be identified with this dye. This is an exciting new approach. It is now important to determine whether the dye interacts with the light-activated form of the enzyme. In a similar set of experiments Ashton (1984) used Procion Red MX2B as an affinity label for the regulatory thiol of maize ribulose-5-P kinase. The DTT-activated form of the enzyme is inhibited by the dye and by sulfhydryl reagents while the nonactivated form is not. Since ADP and ATP inhibit the dye inactivation, the reactive thiol is probably positioned near the active site. Experiments with alkylating reagents give similar results (Omnaas et al., 1985). The ATP-protected thiol appears to be extraordinarily reactive. There is an increase of one thiol per subunit (four per mole enzyme) when maize P-enolpyruvate carboxylase is treated with DTT (Iglesias and Andreo , 1984). The kinetics of thermal denaturation of the DTT-inactivated glucose-6P dehydrogenase are the same as those of the dark form of the enzyme and distinct from those of the light form (Srivastava and Anderson, 1983).

34

LOUISE E. ANDERSON

Likewise proteolytic degradation of the DTT-inactivated enzyme resembles that of the dark form. Clearly the light form of this enzyme is not identical with the DTT-inactivated form. The DTT-activated form of fructosebisphosphatase appears to differ conformationally from the DTTthioredoxin-activated form, since the latter is much more sensitive to trypsin (Schurmann and Wolosiuk, 1978). Apparently the posttranslational modification produced by the light modulation system is not necessarily equivalent to the modification produced by DTT treatment alone or by DTT treatment in the presence of thioredoxin. The light modulation system is probably a considerably more specific reductant than is DTT or DTT- thioredoxin. D. CHANGE IN KINETIC PARAMETERS OF THE TARGET ENZYME

Shifts in pH dependency of pea leaf chloroplastic glucose-6-P dehydrogenase (Ben-Bassat and Anderson, 1981) , of spinach chloroplastic fructose-bisphosphatase (Slovacek and Vaughn, 1982), and of P-enolpyruvate carboxylase from Sedum praealtum (Manetas, 1982) and Mesembtyanthemum crystallinurn (Winter, 1980) as a result of light/dark modulation of activity have been observed. The dark forms of P-enolpyruvic carboxylase from Salsola soda (Karabourniotis et al., 1983) and pea leaf chloroplastic fructose-bisphosphatase (Marques and Anderson, 1985a) exhibit biphasic kinetics, while the kinetics of the light form are linear. This behavior suggests that there are two conformers of the enzyme and that the dark conformer is converted to the light conformer, which has a higher apparent affinity for substrate, when the plant is irradiated. This would be consistent with experiments indicating that light modulation effects conformational change (see above). An alternative explanation for biphasic kinetics, i.e., that there are two different carboxylases and two different phosphatases, cannot be ruled out since crude extracts were used in both sets of experiments. P-enolpyruvatecarboxylase in the CAM plant Sedum praealturn is light inactivated (Manetas, 1982; Manetas and Gavalas, 1983). The dark form of this enzyme has apparent Michaelis-Menten kinetics, while the light form shows positive cooperativity. The differences, also, are indicative of two conformers or of two enzymes. Both forms are inhibited by malate. For most of the enzymes which have been studied, maximal velocity rather than Km changes as a result of light modulation (Table IV). This may simply reflect the fact that most surveys for light-modulatable enzymes have been done with conventional assays containing excess substrate, in which case those enzymes responding to light modulation by a change in apparent affinity for substrate will escape detection.

35

LIGHT/DARK MODULATION

TABLE IV w e c t of Light Modulation on Kinetic Parameters of the Target Enzymes Change Enzyme"

Source

V,,

Km

Glyceraldehyde-3-P dehydrogenase

Pea leaves

Increase

None

Ribulose-5-P kinase

Spinach

Increase

None

Fructosebisphosphatase Glucose-6-P dehydrogenase Glucose-6-P dehydrogenase

Pea leaves

Increase

Decrease

Pea chloroplast Pea cytosol

Decrease

None

Decrease

None

Reference Melandri et al. (1970); Anderson and Duggan (1976) Gardemann er al. (1983) Marques and Anderson (1985a) Anderson and Duggan (1976) Anderson and Duggan (1976)

For changes in P-enolpyruvate carboxylase kinetic parameters, see text.

The effects of inhibitors on the activity of the light and dark forms of these enzymes have received almost no attention, possibly because of the complications that might arise in inhibitor experiments with crude extracts. Winter (1980) has shown that the light (less active) form of Penolpyruvate carboxylase from M.crysralliniurn is more sensitive to inh:bition by malate than is the dark form. Interestingly, the maize enzyme is activated to the same extent by either glucose-6-Por by DTT and glucose6-P (Iglesias and Andreo, 1984), which would seem to indicate that reduction and binding of the modifier glucose-6-Paffect the same group(s) in the active site. Ashton and Hatch (1983b) propose that light/dark conformational changes shift the equilibrium of the enzyme-substrate/product complex away from unity. (Efficient catalysis is thought to require that the equilibrium for the substrate/product complex be close to unity.) Such a change must be reflected in a change in substrate binding since the equilibrium constant for the overall reaction cannot be altered. Consistent with this proposal, marked differences in binding of NADP and NADPH to the DTT-active and -inactive forms of NADP-linked malic dehydrogenase are observed. Whether or not light/dark transitions are entirely identical with the DTT-induced changes in conformation, this is a very neat kinetic explanation for the effect of thiol modulation. Differences in the binding of Mg2+and fructose bisphosphate to the inactive and DTT-activated forms of fructose-bisphosphatase have been

36

LOUISE E. ANDERSON

reported by Meunier et al. (1981). Mixed cooperativity is observed with the inactive form. After DTT activation the substrate and the cation are bound with positive cooperativity. VI. THE FUNCTION OF LIGHT MODULATION A. INDUCTION OF PHOTOSYNTHETIC CARBON DIOXIDE FIXATION

There is considerable controversy about the exact function of light modulation in metabolic regulation in the green plant (Anderson et al., 1981, 1982, 1983; Charles and Halliwell, 1981a; Hiber, 1978, 1980; Leegood and Walker, 1981b; Robinson and Walker, 1980; Wirtz et al., 1982). Photosynthetic carbon dioxide fixation in intact chloroplasts is characterized by an initial lag period. This lag is thought to be related to the time required for the build-up of Calvin cycle intermediates when the chloroplast is illuminated. Arguments have been put forth for (Leegood and Walker, 1981b; Leegood et al., 1982; Anderson et al., 1980, 1981, 1982,1983;Heldt et al., 1981; Huber, 1978)and against (Charles and Halliwell, 1981a-c; Robinson and Walker, 1980; Leegood and Walker, 1980c) a causal relationship between light modulation and induction. Since light activation of the two phosphatases never follows induction, and since inhibition of light modulation prolongs induction (Anderson et al., 1983) and preactivation with DTT shortens induction time (Leegood and Walker, 1981b), it seems clear that the two phenomena are related. Leegood et al. (1982) point out that the great variation in induction times in intact chloroplasts is consistent with an interaction between autocatalysis and light activation. The increase in enzyme activity resulting from light activation must accelerate the build-up of reductive pentose phosphate cycle intermediates in the chloroplast. With each turn of the cycle, metabolite levels, and hence enzyme rates, will be increased. Without this acceleration the lag period will be extended to infinity. This is seen experimentally when chloroplasts are poisoned with arsenite (Gibbs and Calo, 1960; Marques and Anderson, 1985b). On the other hand, if carbon intermediates were added when light activation is blocked, carbon dioxide fixation might be expected to occur, but at a reduced rate, since some rate-limiting enzymes would not be fully active. This has been observed in experiments with arsenite-poisoned chloroplasts (Bamberger and Gibbs, 1965). It would seem then that light activation is involved in the autocatalytic build up of carbon intermediates in the chloroplast. It still remains to be shown experimentally that light activation is required for the build-up of photo-

LIGHT/DARK MODULATION

37

synthetic intermediates in the light and that this build-up is related to induction. A preliminary computer simulation of the reductive pentose phosphate cycle is consistent with this hypothesis (J. B. Anderson and L. E. Anderson, unpublished). The availability of ATP and NADPH, and the light-dependent rise in stromal pH and increase in stromal Mg2+ concentration, will also undoubtedly be involved, along with light modulation, in determining the extent of the lag phase. Experiments with intact maize leaves indicate that light activation is not rate limiting during induction (Usuda et al., 1984). Computer simulation probably represents the only means of estimating the relative contribution of each of these factors (metabolite levels, pH, cation concentrations, light modulation) in the regulation of the activity of the reductive pentose phosphate pathway in the intact chloroplast. Woodrow et al. (1984)have analyzed the effects of reductive activation and of pH and Mg2+ concentrations on the activity of sedoheptulosebisphosphatase. They conclude that in the dark less than 1% of the enzyme molecules will be in the active form. Heldt and co-workers (Gardemann et al., 1983) have measured ribulose-5-P kinase activity levels under conditions which simulate a light/ dark transition. In the presence of metabolites the effect of light activation is dramatically increased. Clearly, modulation of the activity of the light and dark forms of the enzyme is important in chloroplast metabolism in the intact leaf. B.

CFI-CFo Mg ATPASE

The thiol-related light activatioddark inactivation of CFI-CFo is thought to allow rapid responses to illumination and darkness. CFI-CFo is also pH activated, but presumably pH changes in the chloroplast would be slow compared to redox thiol changes (Mills and Mitchell, 1982b). C. STOMATAL OPENING

Three epidermal enzymes which are thought to be involved in metabolism related to stomatal movement, P-enolpyruvate carboxylase, NADPlinked malic dehydrogenase, and NADP-linked isocitric dehydrogenase, are light activated, and one, aspartate amino transferase, is light inactivated. Arsenite and sulfite inhibit light-dependent stomatal opening and light activation of the epidermal enzymes (Rao and Anderson, 1983b). There is an increase in the measureable free thiols when epidermal strips are irradiated which appears to parallel stomatal opening (Rao and Anderson, 1983a). The thiol content of the epidermis is decreased when intact

38

LOUISE E. ANDERSON

plants are exposed to SO2in the light (Rao et al., 1983). Apparently light modulation of the activity of these epidermal enzymes is involved in stomatal opening. VII. EFFECT OF SULFUR DIOXIDE ON LIGHT MODULATION The extreme sensitivity of the LEM to sulfite (Anderson and Avron, 1976) and the fact that plants, and not animals, are SO2 sensitive suggests that light modulation might be the primary process affected by SO2 pollution. In support of this hypothesis it was found that treatment of intact seedlings with SO2 results in inhibition of the system for light inactivation of glucose-6-P dehydrogenase (Anderson and Duggan, 1977). Apparently, inactivation of the light modulation system can and does occur in uiuo when the plant is exposed to SO*. Prolonged exposure to low levels of SO2 almost certainly will disrupt the regulatory system for control of carbon metabolism by light, leading to metabolic chaos and eventually to death or extreme damage to the plant. Recently Alscher (1982a, 1983) has found that light activation of fructose-bisphosphatase in an SO2-sensitivesoybean cultivar is sulfite sensitive, while light activation in an SO2-insensitivecultivar is not affected by sulfite. Muschinek et al. (1985) report that the thylakoids in a more SOZsensitive variety of Pisum is more sensitive to S032-(and to AsOz-) than in a less SO2-sensitivevariety. These experiments suggest that SO2 susceptibility may be related to light modulation. These experiments were done with crude broken chloroplast preparations and with plants fumigated in growth chambers and in the field. They indicate that SO2 susceptibility is related to light modulation. What is not yet known is how the light activation system differs in the two varieties. VIII. OSMOTIC STRESS AND LIGHT MODULATION Even though light modulation was first observed in dehydrated specimens of a resurrection plant (Ziegler and Ziegler, 1965), light activation of fructose-bisphosphatase and sedoheptulose-bisphosphataseis severely inhibited when isolated spinach chloroplasts are subjected to osmotic stress (Boag and Portis, 1984). Metabolite levels indicate that the light-activated enzyme fructose-bisphosphatase does not function normally in stressed chloroplasts (Berkowitz and Gibbs, 1982). The effects of osmotic stress and desiccation on light modulation in resurrection plants have not been studied.

LIGHT/DARK MODULATION

39

IX. CONCLUSIONS Lighddark modulation is an important regulatory process in the green plant. Work in progress in a number of laboratories should soon lead to a resolution of the differences in the proposed mechanisms for modulation, to positive proof that the result of modulation is posttranslational modification and that the modification catalyzed by the LEM system or the ferredoxin-thioredoxin system involves reduction of a disulfide bond, and to a better understanding of the function of modulation in photosynthetic carbon metabolism. There will probably be more surprises for those who, like Irmgard and Hubert Ziegler, have the good luck to do the right experiment and the wit to interpret it correctly.

ACKNOWLEDGMENTS Work in the author’s laboratory was supported by grants from the United States National Science Foundation, Department of Agriculture, and Department of Energy. Myhanh Hoang and Loretta Moore helped with the processing of the manuscript. I thank Drs. Peter Jablonski, Ivan0 Marques, Gyorgi Muschinek, and Duane Ford for critical reading of the manuscript.

REFERENCES Alscher, R. (1983). I n “Light-Dark Modulation of Plant Enzyme Activity” (R. Scheibe, ed.), pp. 154-163. University of Bayreuth, Bayreuth. Alscher-Herman, R. (1982a). Enuiron. Pollut. 27, 83-96. Alscher-Herman, R. (1982b). Plant Physiol. 70, 728-734. Anderson, L. E. (1974). Biochem. Biophys. Res. Commun. 59,907-913. Anderson, L. E. (1979). I n “Encyclopedia of Plant Physiology, New Series” (M. Gibbs and E. Latzko, Eds.), Vol. 6, pp. 271-281. Springer-Verlag, Berlin. Anderson, L. E. (1981). Biochem. Biophys. Res. Commun. 99, 1199-1202. Anderson, L. E. (1983a). I n “Molecular Models of Photoresponsiveness” (G. Montagnoli and B. F. Erlanger, eds.), pp. 153-173. Plenum, New York. Anderson, L. E. (1983b). I n “Light-Dark Modulation of Plant Enzyme Activity” (R. Scheibe, ed.), pp. 47-52. University of Bayreuth, Bayreuth. Anderson, L. E., and Avron, M. (1976). Plant Physiol. 57, 209-213. Anderson, L. E., and Ben-Bassat, D. (1981). In “Photosynthesis IV. Regulation of Carbon Metabolism” (G. Akoyunoglou, ed.), pp. 373-378. Balaban International Science Services, Philadelphia. Anderson, L. E., and Duggan, J. X . (1976). Plant Physiol. 58, 135-139. Anderson, L. E., and Duggan, J. X . (1977). Oecologia 28, 147-151. Anderson, L. E.,and Lim, T.-C. (1972). FEES Lett. 27, 189-191. Anderson, L. E., and Nehrlich, S. C. (1977). FEBS Lett. 76,64-66.

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Wan-Aswapati, O., Kemble, R. J., and Bradbeer, J. W. (1980). Plant Physiol. 66, 34-39. Whittaker, M. M., and Gleason, F. K. (1984). J . Biol. Chem. 259, 14088-14093. Wildner, G. F. (1975). Z . Naturforsch. 3Oc, 756-760. Winter, K. (1980). Plant Physiol. 65, 792-796. Wirtz, W., Stitt, M., and Heldt, H. W. (1982). FEBS Lett. 142, 223-226. Wolosiuk, R., and Buchanan, B. B. (1976). J . Biol. Chem. 251, 6456-6461. Wolosiuk, R. A., and Buchanan, B. B. (1979). Arch. Biochem. Biophys. 189, 97-101. Wolosiuk, R. A., Buchanan, B. B., and Crawford, N. A. (1977). FEBS Lett. 81, 253-258. Wolosiuk, R. A., Crawford, N. A., Yee, B. C., and Buchanan, B. B. (1979). J. Biol. Chem. 254, 1627-1632. Wolosiuk, R. A., Perelmuter, M. E., and Chehebar, C. (1980). FEBS Lett. 109, 289-293. Wolosiuk, R. A., Hertig, C. M., Nishizawa, A. N., and Buchanan, B. B. (1982). FEBS Lett. 140931-35. Woodrow, I. E., and Walker, D. A. (1980). Biochem. J. 191,845-849. Woodrow, I. E., and Walker, D. A. (1982). Arch. Biochem. Biophys. 216,416-422. Woodrow, I. E., Murphy, D. J., and Walker, D. A. (1983). Eur. J . Biochem. 132, 121-123. Woodrow, I. E., Murphy, D. J., and Latzko, E. (1984). J. Biol. Chem. 259, 3791-3795. Yee, B. C., de la Torre, A., Crawford, N. A., Lara, C., Carlson, D. E., and Buchanan, B. B. (1981). Arch. Microbiol. WO, 14-18. Ziegler, H., and Ziegler, I. (1965). Planta 65, 369-380. Ziegler, H., and Ziegler, I. (1967). Planta 72, 162-169. Ziegler, H., Ziegler, I., and Schmidt-Clausen, H.4. (1968). Planta 81, 169-180. Ziegler, I. (1972). Proc. Int. Congr. Photosyn. Res., 2nd pp. 1847-1853.

Algal Toxins

WAYNE W. CARMICHAEL Department of Biological Sciences Wright State University Dayton, Ohio, U.S.A.

I. Introduction.............................................................

. .. . .. .... .. ... . . .... .. .. . . In. Occurrence, Growth, and Toxicity.. . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . A. Chrysophyta ... ...................................................... B. Pyrrophyta. .. . . . . ... .. . .. . . .. . .. . . . . . .. .. .. .. ... . . .. .. .... . .. .. .... . . C. Cyanophyta .......................................................... IV. Isolation, Characterization, and Toxinology . . . . . . . . . . . .. .. . . . .. . . . . . . .. .. . . . A. Prymnesium Toxins.. . .. .. . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . ... .. . . . B. Freshwater Cyanophyte Toxins . . . . . , . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . C. Marine Cyanophyte Toxins ............................................ D. Dinoflagellate Paralytic Shellfish Poisons (PSP) . . . . . . . . . .. . . . . . . . .. . . . , . . E. Dinoflagellate Ciguatera Toxins . . . . . .. . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . F. Other Dinoflagellate Toxins . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . . . V. Environmental Role of Algal Toxins . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . References . . . . . . . . . .. , . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 11. Naming Algal Toxins. ... .. .. ... .. ... .. .. . .. .. .

47 52 52 52 59 64 67 67 68 78 82 86 89 91 93

I. INTRODUCTION Chemicals of secondary biosynthesis are important to the growth and development of all organisms, plant, animal, and microbial. The term “secondary” derives from the observation that these chemicals are not needed for primary metabolism, i.e., respiration or photosynthesis. The term also serves to suit the fact that for many of the chemicals no obvious functional role exists, hence the term secondary would seem to catalog them well. Secondary chemicals were first suggested by Stahl (1888) as ADVANCES IN BOTANICAL RESEARCH, VOL. 12

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Copyright 8 1986 by Academic Press Inc. (London) Ltd. All rights of reproduction in any form reserved.

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perhaps having evolved for protection against attack by herbivorous animals. Numerous other functions have been suggested for these chemicals, including the following: Regulators of plant growth or biosynthetic activity Storage forms of plant growth regulators Energy reserves Transport facilitators Waste products 6. Detoxification products of environmental poisons (Chew and Rodman, 1979) 1. 2. 3. 4. 5.

While some debate still exists concerning the adaptive significance of the secondary chemicals, evidence continues to accumulate to support an ecological role for these metabolites both in interactions among plants and their associated biota and as protective agents against physical environmental stresses (Rhoades, 1979). Secondary chemicals having a marked biological effect, usually negative, are often called biotoxins. While the role of biotoxins for animals is often clear, i.e., prey gathering or defense, the role of microbial biotoxins is not. Although they may act in some capacity as defense chemicals against herbivores or to improve an organism’s competitive advantage for a food source, it is also possible to find nontoxic forms of a potentially toxic species. Algae responsible for producing toxins are found in three of the eight divisions which make up the algae (Scagel et al., 1982). These are the Chrysophyta (class Prymnesiophyceae), Cyanophyta (cyanobacteria or blue-green algae), and the Pyrrhophyta (class Dinophyceae, also known as the dinoflagellates). In the Chrysophyta the toxic species is the flagellated unicellular Prymnesium paruum. It is a euryhaline chrysophyte widespread in brackish and marine habitats, producing toxins best known for causing mass mortalities of fish (Shilo, 1971, 1981). In the Cyanophyta both marine and freshwater toxic species are found. Marine toxic forms are in the filamentous genera Lyngbya, Schizothrix, and Oscillatoria. Toxins produced are of the type responsible for the contact dermatitis called “swimmers itch” (Moore, 1981b, 1984). Toxic freshwater cyanobacteria include the unicellular colonial Microcystis and the filamentous Anabaena, Aphanizomenon, (Figs. 1-3) Nodularia, and Oscillatoria. These freshwater algae can form thick surface accumulations of cells as the water bloom develops within the water body. They can be found in many eutrophic lakes and ponds at temperate latitudes and are responsible for sporadic but widespread outbreaks of wild and domestic animal illness or death (Gentile, 1971; Carmichael, 1981; Carmichael and Mahmood, 1984; Carmichael and Schwartz, 1984). They are also implicated in

Fig. 1. Scanning electron micrograph of Microcystis aeruginosa. Gelatinous matrix, which holds the cells in a colony form, is embedded with numerous rod-shaped bacteria. Micrograph by M. M. Ecker.

Fig. 2. Scanning electron micrograph of Anabaena circinafis.Numerous rod-shaped bacteria are embedded in the matrix surrounding the individual cells. Anabaena circinafiswas recently identified as the probable cause of animal deaths from a toxic water bloom in Illinois (Beasley et al., 1983). Micrograph by M. M. Ecker.

Fig. 3. Scanning electron micrograph of Aphanizornenon flos-aquae from New Hampshire. Cell in center of filament not covered by the sheath is a heterocyst-primary site of nitrogen fixation. Micrograph taken by M. M. Ecker.

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WAYNE W . CARMICHAEL

human poisonings in certain municipal and recreational water supplies (Carmichael et al., 1985). The Pyrrhophyta contain most of the algae documented as causing outbreaks of algal disease. They are almost exclusively from the marine environment. Two major disease types are recognized. The first, paralytic shellfish poisoning (PSP), refers mainly to toxic species of Protogonyaulax (formerly Gonyaulax) (Figs. 4 and 5), which are concentrated by filter-feeding molluscs. Recently the tropical dinoflagellate species Gymnodinium catenatum (Morey-Gaines, 1982) (Fig. 6) and Pyrodinium bahamense var. compressa, and the tropical red alga Jania sp. have also been found to produce PSPs (Oshima et al., 1984). The molluscs are in turn eaten by humans, resulting in yearly episodes of illness and death. The second disease type is called ciguatera seafood poisoning. It refers to the concentration by many reef-feeding fish of toxic dinoflagellates recently identified as belonging to the genera Gambierdiscus (Fig. 7), Prorocentrum, Gymnodinium, and Gonyaulax. The fish are in turn eaten by humans (Ragelis, 1984).

11. NAMING ALGAL TOXINS As with most natural toxins, algal toxins have been named from the organisms that produce them or the vectors by which the toxins move through food chains. Only occasionally is a toxin name based on its chemical structure. This seems logical to the investigator doing the research and to his colleagues to whom he must communicate information about the toxin and the organism producing it. Coincidentally, it also reinforces the interest, respect, and image of mystique that the general public accords natural toxins. As more chemical information is known about toxins and when more than one toxic compound is isolated from a given toxic species, the use of this type of nomenclature system has become confusing. However, since most toxins are investigated and the findings published before their structure is entirely understood, the naming of toxins based on the organisms involved will continue. Table I lists the algal toxins and, where known, the origins of their names. 111. OCCURRENCE, GROWTH, AND TOXICITY A . CHRYSOPHYTA

The chrysophyte Prymnesium parvum was first identified as the causative agent of fish deaths by Carter (1938). Most problems have been reported from brackish water environments in England, Holland, Denmark, Bul-

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53

Fig. 4. Scanning electron micrograph of Protogonyaulax (Gonyaulax) tarnarensis. This species is the primary offender in outbreaks of PSP along the eastern coast of the U.S.A., Western Europe, and the western Pacific. Micrograph by L. Firtz.

Fig. 5. Scanning electron micrograph of Protogonyaulax (Gonyaulax) catenella, a PSP-producing dinoflagellatefrom the west coast of the U.S.A. Strands covering the cells are trichocysts extruded by the organism. Micrograph by G . Gaines.

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Fig. 6. Scanning electron micrograph of Gymnodinium catenatum, a paralytic shellfish poison-producing dinoflagellate from Mazatlan, Mexico (Morey-Gaines, 1982). Micrograph by G . Gaines.

TABLE I Algal Toxins-Terms Used and Main Species Responsible Organism A. Chrysophyta Prymnesium parvum

2

B. Cyanophyta 1. Marine Lyngbya majuscula

Schizorhrix calcicola Oscillatoria nigrouiridis 2. Fresh water Microcystis aeruginosa

Anabaena jlos-aquae (different strains of cyanobacteria)

Toxin

Chemical group

Origin of toxin name

Reference

Ichthyotoxin Cytotoxin Hemolytictoxin

Unknown

Fish toxin Cell toxin Red blood cell toxin

Shilo (1981)

Lyngbyatoxin A Debromoaplysiatoxin Aplysiatoxin Debromoaplysiatoxin Oscillatoxin A

Indole alkaloid Phenolic bislactone Phenolic bislactone Phenolic bislactone Phenolic bislactone

Genus Lyngbya

Moore (1984)

Microcystin Cyanoginosin

Peptide Peptide

Genus Microcystis

Anatoxin-a Anatoxin-b Anatoxin-c Anatoxin-d Anatoxin-b(s) Anatoxin-a(s)

Alkaloid Unknown Peptide Unknown Unknown Unknown

Genus Anabaena

Genus Oscillatoria Carmichael and Mahmood (1984); Botes et al. (1984) Carmichael and Gorham (1978)

Aphanizomenon Jos-aquae

Oscillatoria agardhii rubescens C. Pyrrophyta Gonyaulax (Protogonvaulax) catenella

u -4 l

tamarensis acatenella phoneus Pyrodinium bahamense var. compressa Ptychodiscus brevis (Gymnodinium breve) Dinophysis fortii acuminata Gambierdiscus toxicus

Aphantoxins

Alkaloid

Genus Aphanizomenon

Oscillatoria toxin

Peptide

Genus Oscillatoria

Paralytic shellfish poison (PSP) Saxitoxin

Alkaloid

Sasner et a/.( 1984); Carmichael and Mahmood (1984) Skulberg e f 01. (1984)

Hall and Reichardt (1984) Genus Saxidomas (Alaskan butter clam)

Neosaxitoxin Gonyautoxin 1-4 Cryptic (B1-2.C14)

Genus Gonyaulax

Brevetoxins

Pol ylactone

Genus P . brevis

Baden et al. (1984)

Dinophysistoxin Pectenotoxin

Okadaic acid-like Polyether lactone

Genus Dinophysis

Yasumoto el a/. (1984)

Ciguatoxin

Alkaloid

Cigua-from Cuban name for the marine snail Turbo Livona pica

Ragelis (1984)

Maitotoxin Scaritoxin

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WAYNE W. CARMICHAEL

Fig. 7. Scanningelectron micrograph of Gumbierdiscus toxicus, collected from the Virgin Islands. Gumbierdiscus toxicus is the primary organism currently identified as being responsible for ciguatera seafood poisoning. Micrograph by G . Gaines.

garia, and especially Israel. The primary concern in Israel comes from the great number of commercial, brackish water fish ponds in which P. paruum was found to grow (Shilo, 1971, 1981). Prymnesium toxin consists of three toxins, hemoly sin, ichthyotoxin, and cytotoxin. Laboratory cultures on modified S50 medium (Shilo, 1967) produced the greatest amount of toxins during the late stages of logarithmic growth and continued at a high level into stationary phase. In contrast to other algal toxins the toxins of P. paruum are secreted into the environment, especially in the late

ALGAL TOXINS

59

stages of growth. Culture conditions can have a significant effect on toxin production. In the absence of light, a marked reduction of toxin production occurs when a carbon-enriched (glycerol) medium is us_ed.Cell multiplication continues during this time (Shilo, 1967). In contrast a 10- to 20fold increase in all the toxins is found in phosphate-starved cells with even more toxin than normal being found in the culture medium. Although the greatest production of the toxins occurs under stress conditions which would imply some protective (ecological)role for these secondary chemicals, Shilo (1981) feels that their formation has no direct role or importance in the life of P . paruum. B. PYRROPHYTA

Most reported PSP outbreaks have occurred in northern latitudes. Protogonyaulax (Gonyaulax) catenella and P . polyedra (Taylor, 1984) are implicated on the west coast of North America while Protogonyaulax tamarensis var. excauata (Schmidt and Loeblich, 1979) is involved in Northern Europe, Great Britain, Japan, and Western Pacific. These marine dinoflagellates exist in three cell types: 1. A resting zygote (cyst, 25-30 X 45-50 pm)(Dale et al., 1978; Turpin et al., 1978; Anderson, 1984) 2. A baagellate motile cell (35-50 pm in diameter) which is the bloomforming stage 3. An asexual temporary cell (cyst, 30-40 p m in diameter) called a pellicle or ecdysal cyst (Anderson and Wall, 1978; Turpin et al., 1978).

Blooms of these dinoflagellates are generally defined as occurring when cell populations are between 104-106 dm-3. Lower cell concentrations of lo3dm-3 can occur at any time during the April-October growing season. When cell concentrations reach about lo6 dm-3 the water can be discolored and referred to as a “red tide.” Red tides are not all toxic, however, since (1) red tides can form from the presence of other, nontoxic dinoflagellates (Yentsch et al., 1978); (2) shellfish can accumulate toxins at levels below those necessary to discolor the water (Hurst and Yentsch, 1981); and (3) not all Protogonyaulax species are toxic when isolated, cultured, and tested (C. M. Yentsch, personal communication). Dense concentrations of toxic marine dinoflagellates can result from several factors (Yentsch and Incze, 1980) in addition to the direct rapid growth of the algal population. These physical factors apparently have a great deal to do with frontal circulation patterns (Seliger et al., 1979). Active phytoplankton growth is situated along or close to these frontal systems that form between well-mixed and season-

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WAYNE W . CARMICHAEL

ally stratified water masses. Holligan (1979) defines three stages of bloom formation in these frontal areas, as measured by chlorophyll a. The first stage, which depends on upward mixing of nutrient-rich waters, involves growth of phytoplankton at the base of the thermocline. The second stage occurs in the upper part of the thermocline at a depth of about 10 m. The third stage, which often finds the blooms patchy in appearance, may be initiated by upward mixing of the subsurface bloom. While Holligan (1979) does not find evidence to suggest that vertical migration of dinoflagellates occurs, Dugdale (1979) and others find evidence to indicate that migration occurs in response to nutrient gradients, especially nitrates. These studies indicate that dinoflagellates swim downward in the afternoon and evening to take advantage of higher nutrient levels than those near the surface where higher photosynthetic rates have been taking place during the day. There is also some evidence that toxic dinoflagellates exhibit an annual rhythm. Yentsch and Mague (1980) report that Protogonyaulax excuuatu, cultured under constant conditions over a 5-year period, exhibited a circannual rhythm. Growth rates varied from a maximum of two doublings per day for the months of July and August to 0.10 doubling per day in the month of January. Hydrographical mechanisms are also important in the formation of toxic blooms. These mechanisms require no rapid reproduction and provide a means for delivery of an existing population to an area where biological behavior such as phototaxis can result in dense concentrations. These mechanisms can be triggered by meteorological events such as rainfall and wind or the formation of discontinuities between water masses, called frontal zones. These fronts which result from tide or wind-generated convergences and/or density discontinuities are some of the locations most likely to generate red tides (Yentsch and Incze, 1980). Blooms and cultures of toxic dinoflagellatescan vary in toxicity. When cultured in the laboratory, variations are seen in quantity and type of toxin produced. Points to remember when studying dinoflagellate toxicity include the following: 1. The mouse bioassay used to detect toxicity has an inherent variability of about k 20%. 2. Toxicity is a measure of total toxins produced by the population in culture and does not distinguish between the different toxin types.

Growth would seem to be the most obvious regulator of toxicity with optimal growth conditions reponsible for optimum toxin production. However, with some dinoflagellates this may not be so. Hall (1982) studied the effects of nutrient deprivation using modified seawater with Guillard’s “F/2” enrichment (Guillard and Ryther, 1962), on cell growth and

ALGAL TOXINS

61

toxicity of selected Alaskan coast Protogonyaulax clones. The omission of nitrate and phosphate from the culture medium was tested for its effect on growth and toxicity. Despite growth reductions to a level of 3200 and 3000 cells ~ m for - ~minus nitrate and minus phosphate, respectively, compared to 16,300 (over 27 days) for controls, the toxicity per cell was slightly better then the control for minus nitrate and over 5 times as much for the minus phosphate cultures. Growth of the clone over a series of temperatures ranging from 6 to 12°C showed a reduction in growth rate at lower temperatures accompanied by an increase in toxin content per cell. This growth attenuation accompanied by increased toxicity is supported by DuPuy (1968) and Proctor et af. (1975), who concluded that toxin content is inversely related to growth rate. Perhaps this increase in a secondary metabolite under stress conditions can be likened to the production of secondary compounds by higher plants when they are grown under nutrient stress conditions (Janzen, 1974). Hall (1982) also rightfully points out that the conditions that induced higher toxin content, viz., low phosphate and low temperature, are closer to the range of these parameters found under natural conditions. Hall (1982) also investigated toxin composition under the attenuated growth conditions which increased overall toxin composition. The results of his limited experiments in this area showed that toxin composition among strains isolated from different regions of the Alaskan coast had different toxin compositions while those from close neighboring areas were uniform through successive culture runs. He found that the sulfamate toxins (Fig. 11) were dominant across the entire geographical range sampled. It should also be said that toxicity of dinoflagellates has been found to decrease as the cultures approach the end of log growth phase (White and Maranda, 1978). The economic impact of toxic dinoflagellates is felt mainly when the toxins are concentrated by shellfish. Although many different shellfish can accumulate dinoflagellate toxins (Carmichael et a f . , 1985) the complex pattern of toxification and detoxification has been best studied using the blue mussel Mytilus edulis. This is based on the organism’s widespread occurrence, ease of collection, and rapid rate of intoxification and detoxification. In the laboratory M. edulis will toxify in proportion to the filtration rate and the number of cells in the water. Along the coast of Maine (U.S.A.) during the summer when optimal cell densities of about lo6 ~ m are - ~found, M. edulis can toxify at the rate of 10 pg toxin g-I tissue per day. These are the times when Protogonyaulax is in the motile stage (Gilfillan and Hansen, 1975). Under these conditions the lethal levels for humans of 10 mg of toxin are often reached. In a 4-year study (1976-1979) involving shellfish intoxication patterns along the coast of Maine, Hurst and Yentsch (1981) found that the peak toxicity occurred in

62

W A Y N E W. CARMICHAEL

1978. Toxin maxima were found in May and ranged from 25 to 114 pg g-' tissue. These high levels of toxin accumulation are possible only because shellfish are not harmed by the toxins they accumulate (Twarog and Yamaguchi, 1975). Detoxification of the mussel tissue depends on the amount of toxin present and the amount of melanin pigment in the tissue (Price and Lee, 1971). Decay constants can in some cases be determined and used to help determine when to reopen contaminated shellfish beds (Hurst and Gilfillan, 1977). Hurst and Yentsch (1981) concluded from their survey that 1. Considerable variability exists for toxicity between stations a few kilometers apart. However, appearance of toxicity is simultaneous over wide areas. 2. Major occurrences of toxicity are during spring or late summer when water temperatures are changing. During this time toxic cysts and motile cells in the water provide the inoculum for a new bloom. 3. Considerable variation between years is found at all stations. Most stations are characterized by one major outbreak per year. 4. Rates of intoxification and detoxification are rapid, being several hundred micrograms of shellfish toxin per 100 g shellfish tissue per day.

Another important factor influencing the apparent toxicity of PSP-producing dinoflagellates is biotransformation by the shellfish (Shimizu and Yoshioka, 1981; Shimizu et al., 1984). By using homogenates of the east coast scallop Placopecten magellanicus, it was shown that gonyautoxins and neosaxitoxin decreased while saxitoxin content increased. Bioconversion of neosaxitoxin to saxitoxin in Mya arenaria and Mercenaria mercenaria was also demonstrated. Dinoflagellates causing ciguatera disease are found to be most prevalent in the tropics between 35"N and 34"S, especially in the South Pacific islands and the Caribbean. In these areas the disease affects more than 10,000 individuals per year and appears to be increasing in frequency (Halstead, 1967; Ragelis, 1984). Ciguatera has been caused by over 400 species of fish, but especially the herbivorous surgeon fish and parrot fish, and the larger carnivorous reef sharks and red snappers (Banner, 1976). Randall (1958) concluded that all such fish were tied to the coral reef through the food chain. The possibility that ciguatera originates as a result of toxic algae was first mentioned by a British physician, Colin Chisholm, in 1808 (Halstead, 1967). Evidence gathered during the 1960s and 1970s pointed to certain dinoflagellates that grow in close association with macroalgae and/or other bottom structures as progenitors of ciguatera toxins. It was not until a publication by Bagnis et al. (1977) that a causative organism was isolated from the biodetritus layer of waters surrounding

ALGAL TOXINS

63

the Gambier Islands in the South Pacific. It was initially identified as a Diplopsalis sp. by Yasumoto et al. (1977) but later it was decided that this benthic dinoflagellate belonged to a new genus and was named Gambierdiscus toxicus (Adachi and Eukuyo, 1979). Other species of dinoflagellates inhabiting the same microhabitats in the Pacific have been found to produce toxins which may also contribute to the ciguatera syndrome. These include Amphidinium carteri, Amphidinium klebsii, Ostreopsis ovata, Ostreopsis siamensis, Prorocentrum concavum (Nakajima, 198 l), and Prorocentrum lima (Yasumoto et al., 1980; Nakajima et al., 1981; Murakami et al., 1982). Current studies on ciguatera seem to center around those of Tindall and co-workers working in the Caribbean, Yasumoto, Bagnis, and others who are working in the Pacific island area, and Kimura, Hokama, and coworkers in Hawaii. Tindall et al. (1984) surveyed over 70 sites in the Virgin Islands. From these collections 65 strains representing 18 species of dinoflagellates are being cultured at the University of Southern Illinois. These species have all been proved to culture best using the ES medium of Provasoli (1968) to which was added 1-5% soil extract. Initial screening for toxicity showed that all possessed some toxic component and studies to date have concluded that Prorocentrum concavum and Gambierdiscus toxicus are the major contributors of ciguatera toxins to these Caribbean reef and inshore ecosystems. These studies also concluded that no one dinoflagellate or toxin is the major cause of ciguatera. It was also found that the major ciguatera toxin found in the contaminated fish, ciguatoxin, was not the major toxin produced by cultured toxic strains. This implies that ciguatoxin may be formed by biotransformation processes in the fish, that laboratory cultures of toxic dinoflagellatesproduce different toxins in the laboratory than in nature, or that ciguatoxin-producing dinoflagellates have not been successfully isolated and cultured. A recent study by Carlson et al. (1984) examined the effect of extracts of macroalgae on the growth of G . toxicus, P . concauum, and Prorocentrum mexicanum. The macroalgae chosen were those commonly found in association with these toxic dinoflagellates in the Caribbean, viz., Chaetomorpha linum (Chlorophyta), Dictyota dichotoma (Phaeophyta), and Turbinaria turbinata (Phaeophyta). Water-soluble extracts from field collections of these macroalgae were aseptically added to axenic cultures of the toxic dinoflagellates. It was found that soil and macroalgal extracts enhanced the growth of bacterized cultures of G . toxicus and P . concauum but inhibited that of bacterized P . mexicanum. Generation times of axenic cultures of G. toxicus and P. concavum were lengthened. Evidence was found to indicate that bacteria could contribute to variation in toxin production. Variation of toxicity independent of growth due to bac-

64

WAYNE W. CARMICHAEL

teria has also been demonstrated in freshwater toxic cyanobacteria (Carmichael and Gorham, 1977). The dinoflagellate study concluded that enhanced algal growth in the presence of bacteria was not due to increased C02production. It was also speculated that hormones such as cytokinins may play a role in the stimulation of growth by the macroalgal extracts. C. CYANOPHYTA

Cyanobacteria producing toxic substances have been reported since the late 1800s (Carmichael, 1981).The first laboratory cultures of toxic strains were obtained from the colonial species Microcystis aeruginosa by Hughes et al. (1958) using a modified Fitzgerald medium (Fitzgerald et al., 1952). Later, cultures of Microcystis utilized the medium ASM (Artificial Seawater McLachlan), which is a modified ASP (Artificial Seawater Provasoli) (McLachlan and Gorham, 1961). Growth experiments on several single-cell isolates (clones) of Microcystis revealed that toxicity varied between clones and that there were toxic and nontoxic cells within a given colony. A natural consequence of this varying toxicity was that only the most toxic clones were retained in culture for future work (Gorham, 1964). The optimal temperature for growth of the toxic clone M . aeruginosa NRC-1 was not the same as that for optimal toxin production. The same toxic clone was grown in 9 dm3 batch cultures for isolation and characterization of the toxic principle (Bishop et al., 1959). Since the late 1960s, however, NRC-1 has not been grown for toxicity studies and when tested in 1971by this author the toxic characters differed from those when the strain was originally isolated. Recent tests for toxicity in 1980 indicated that overall toxicity was also declining. This has resulted in the use of another toxic strain, M . aeruginosa 7820 (Codd and Carmichael, 1982). It should be noted that over the period of time NRC-1 was cultured, the medium in which it was grown was changed from modified Fitzgerald to ASM to ASM-1 (Gorham et al., 1964), with the current medium being BG11 (Stanier et al., 1971). These media have progressively higher levels of nitrate as NaN03. BG-11 has 18 times the amount of nitrate as ASM. Phosphate concentration as orthophosphate is equal in all three media. These culture media were chosen for their growth-promoting potential and only secondary consideration was given to whether toxin production was affected with extended subculturing. In retrospect it is important to ask whether the decline in toxicity is due to the fact that the cells are now in a nutrient-rich medium and no longer “stressed,” resulting in lowered toxin production. This same pattern is emerging with other genera of toxin-producing cyanobacteria similar to those of Microcystis, including Anabaena (Carmichael, unpublished data) and Oscillatoria (0. M.

ALGAL TOXINS

65

Skulberg, personal communication). Sirenko (personal communication) also finds that culture toxicity declines when Microcystis isolated from

waters in the U.S.S.R. is maintained in the laboratory over a number of months to years. In the early 1960s toxic strains of Anabaena flus-aquae were isolated for laboratory studies by Gorham and co-workers (Gorham et al., 1964). The basic ASM medium was modified by doubling the nitrogen, phosphorus, calcium, and iron contents while increasing by four times the boron, zinc, cobalt, and copper contents to produce a medium termed ASM-1, Most studies on the chemistry and toxicology of Anabaena toxins have been done using the clonal isolate NRC-44-1 (Carmichael, 1981). Toxicity has remained quite stable in this strain over the period of 1961 to 1980. At this time, for reasons of convenience, NRC-44-1 was transferred to BG-11 for maintenance in this author’s culture collection. Growth continued to be good in this strain but a routine check of toxicity in 1982 revealed that the strain had become nontoxic. A check of 25 subcultures of NRC-44-1 maintained as backup cultures in the laboratory revealed that only 1 had a level of toxicity detectable by mouse bioassay. Transfer of this toxic subisolate back to ASM-1 medium has restored its normal level of toxicity, i.e., 40-60 mg kg-1 (intraperitoneal injection in mouse). Subcultures maintained by P. R. Gorham (University of Alberta) on ASM-1 medium and 0. M. Skulberg (Norwegian Institute for Water Research) on Z-8 medium (Skulberg, 1983), have retained their toxicity throughout this time period. Both ASM-1 and Z-8 media contain the same level of nitrate. Light levels, temperature, and pH were maintained the same throughout this time period. At present, investigations are underway to see if nontoxic NRC-44-1 returned to ASM-1 medium will regain its former toxicity levels. Another instance of apparent change in toxicity has been observed in Anabaena flos-aquae S-23-g, a strain isolated by Carmichael from Saskatchewan, Canada in 1975 (Carmichael and Gorham, 1978). This strain, at the time of initial isolation, produced a toxin, anatoxin-d, with neurotoxic properties. Repeated subculturing of this strain on ASM-1 and then changing to BG-11 medium has resulted in the loss of neurotoxin production and expression of a hepatotoxin like that of Microcystis (Jones, 1984). It will be important to examine whether cultures of toxic cyanobacteria now being cultured from Norway (Skulberg, 1983; Skulberg et al., 1984) and South Africa (Eloff el al., 1982; Botes et al., 1982a; Siegelman et al., 1984) will also undergo a decrease in toxin production with time when grown on the standard nutrient-rich culture media. Toxicity of the filamentous c yanobacterium Aphanizomenonflos-aquae

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WAYNE W. CARMICHAEL

has proved to be the most difficult to study. This is largely due to the difficulty in culturing compared to other toxic cyanobacteria. Phinney and Peek (1961) were among the first to report that blooms of Aph.Jos-aquae could be toxic. Toxicity was not verified, however, by laboratory cultures from the bloom. Gorham (1964) reported some success in culturing Aphanizomenon from sources in Ontario and Saskatchewan but toxicity in the mouse bioassay was negative in all cases. Gentile (1971) reported some success in culturing toxic Aph. Jos-aquae from blooms collected at Klamath Lake, Oregon in 1968. However, no work was done on toxicity or toxicology of the isolates to verify toxicity beyond the standard mouse assay. Toxic isolates were cultured and examined from blooms collected at Kezar Lake, New Hampshire (Gentile and Maloney, 1969). In growth studies toxin production was not increased independently of growth when light or temperature was changed. Toxicity did decrease independently of growth, however, when Aph. flos-aquae was cultured at stress temperatures of 30”C, but not at the more normal environmental temperatures of 15, 20, or 26°C. No experiments were conducted to investigate possible toxin increases by stressing the cells through lowering nitrate or phosphate concentrations. These toxic isolates were subsequently discarded and no toxic Aphanizomenon existed in culture until new isolates from New Hampshire were made by Carmichael in 1980 (Ikawa et al., 1982; Carmichael and Mahmood, 1984). These isolates produce toxins similar to the earlier New Hampshire isolates but as yet no growth studies have been done to investigate possible regulation of toxin production. Toxic marine cyanobacteria have not been studied in laboratory cultures. Instead, all chemical and toxicological studies have used field collections. Toxic marine cyanobacteria are all found in the Oscillatoriaceae family, occupying benthic habitats in warm water areas such as Hawaii and Okinawa (Moore, 1981a,b). The secondary chemicals produced cause a severe “swimmer’s itch” or contact dermatitis among swimmers who come in contact with the filaments. Most reports of these contact irritations come during the summer months and it is not clear whether toxins are produced at other times of the year, although it would seem that they would be. While direct comparisons between marine and freshwater cyanobacteria toxins are difficult to make, at present there is some indication that freshwater cyanobacteria produce contact irritants. This comes from field observations on certain species of Anabaena, Aphanizomenon, Oscillatoria, and Gloeotrichia in the U S A . and Europe over the past 4 years (Skulberg et al., 1984; Carmichael et al., 1985; Codd and Carmichael, unpublished data). When these toxins are characterized and studied it should become clear that the freshwater cyanobacteria are capable of producing secondary chemicals with neurotoxic, hepatotoxic, and dermatotoxic properties.

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67

Overall there has been little work on the effects of growth on toxin production for any of the toxic algae. Most investigators have been kept busy just maintaining a few strains in culture in order to isolate, purify, and characterize the toxins present. It is possible to outline, however, several studies which could yield information on qualitative and quantitative aspects of toxin production and the role of these secondary chemicals in the ecology of the organism. These include 1. The role of nutrient depletion on toxin production, especially major nutrients such as nitrogen and phosphorus. At least some evidence points to a possible stimulation of toxin production as growth decreases due to a shortage of nitrogen and phosphorus. This should be analyzed over several generations of subculturing. 2. Although the few studies that have been done do not indicate that temperature or light affect toxin production independently of growth, longer term studies could prove useful in examining this possibility. 3. Possible role of zooplankton herbivores through selective feeding of nontoxic over toxic cells. Zooplankton presence could also stimulate production of toxins under herbivore stress, as is now being revealed for higher plants. This possibility does not seem as likely as nutrient stress, however, since it would imply that some chemical communication exists between cells of a bloom. 4. Possible role of extrachromosomal DNA (plasmids) in regulation of toxin production (Hauman, 1981). Expression of the plasmid regulating toxin production (if present) could be part of nutrient or predator stress.

IV. ISOLATION, CHARACTERIZATION, AND TOXINOLOGY A. PRYMNESIUM TOXINS

The toxic principles of P.paruum have been purified and characterized by Ulitzur (1969) and Ulitzur and Shilo (1970). The toxin has a broad spectrum of different biological activities in uiuo and in uitro (Shilo, 1971, 1981). It seems that there is a family of compounds having similar composition but different toxic effects, rather than a single compound with different toxic effects. These effects are generally classed as being cytotoxic, hemolytic, and ichthyotoxic. Since the toxic effects are primarily to fish the main component is often referred to as an ichthyotoxin. Intoxication of fish consists of two stages. Initially there is reversible damage to the gill tissues, resulting in the loss of their selective permeability. The second stage, which leads to death, is the response of the sensitized fish to any of a number of toxicants in the environment including the Prymne-

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WAYNE W . CARMICHAEL

sium toxin (Shilo, 1981). The gill epithelium of the fish is the primary site of action for the toxin. Extraction of the toxic compounds indicates that they are acidic, polar lipids. Analysis of the toxin revealed 15 amino acids, a number of fatty acids, 0.47% phosphate, and 10 to 12% hexose sugars (Ulitzur and Shilo, 1970). In its composition of fatty acids together with protein and phosphate, the purified toxin resembles proteolipids (Shilo, 1981). More recently isolation and purification of Prymnesium hemolytic toxin and ichthyotoxin have been done by Kim and Padilla (1977) and Kozakai et al. (1982).

B. FRESHWATER CYANOPHYTE TOXINS

1 . Neurotoxins Known chemical groups of toxins from freshwater cyanobacteria include alkaloids and peptides. The alkaloid toxins are the most rapidly acting. They function as neurotoxins paralyzing peripheral skeletal muscles, then respiratory muscles, with death due to respiratory arrest occurring between a few minutes to a few hours. These toxins are produced by various strains and species of Anabaena and are generally referred to as anatoxins (Table I). Anatoxin-a (antx-a) is the only alkaloid toxin from this group which has been chemically characterized (Fig. 8). It is a potent depolarizing neuromuscular blocking agent (Carmichael et al., 1975, 1979; Spivak et al., 1980). Chemically the compound is the secondary amine, 2-acetyl9-azabicyclo[4.2.l]non-2-ene,and is isolated from the filamentous strain Anabaena flos-aquae NRC-44-1 (Huber, 1972; Devlin et al., 1977). Synthesis of antx-a has been done through a ring expansion of cocaine (Campbell et al., 1977) from 1,5-cyclooctadiene (Campbell et al., 1979), and by intramolecular cyclization between an iminium salt and a nucleophilic carbon atom (Bates and Rapoport, 1979).

4

Fig. 8. Structure of anatoxin-a hydrochloride, a depolarizing neuromuscular blocking agent produced by the filamentous cyanobacteriumAnabaena flos-aquae clone NRC-44-1.

ALGAL TOXINS

69

Poisoning of domestic and wild animals by anatoxins comes from ingestion of the toxic cells and extracellular toxin from a water bloom. Given the LD5o for intraperitoneal bioassay in mouse for purified anatoxin-a as 200 pg kg-I body weight and the approximate oral lethal dose for certain animals (Carmichael et af., 1977; Carmichael, 1981), it is estimated that a lethal bolus of water bloom ranges from a few cubic centimeters to a few cubic decameters, depending upon animal species, toxicity of the bloom, and amount of food material in the animal’s gut. The presence of more than one toxin in the bloom can also result in different signs of poisoning and survival times. Known toxins of Aphanizomenon Jos-aquae are also neurotoxic alkaloids, generally referred to as aphantoxins. Sasner and his colleagues presented evidence that aphantoxin from water blooms of Aph. Josaquae was similar to the paralytic shellfish poisons saxitoxin and neosaxitoxin (Sawyer et al., 1968; Jackim and Gentile, 1968; Alam et a f . , 1973, 1978; Alam and Euler, 1981; Sasner et af., 1981; Adelman et al., 1982). Current research with aphantoxin is concerned with a toxic strain isolated from a small pond near Durham, New Hampshire in 1980 by Carmichael, referred to as NH-1 and NH-5 (Carmichael, 1982; Ikawa et a f . ,1982). The toxins of NH-5 are primarily neosaxitoxin (80%) and saxitoxin (15%). Depending on extraction procedure it appears that aphantoxin also contains some precursors to neosaxitoxin and saxitoxin (Carmichael and Mahmood, 1984; Sasner et al., 1984). At present the structures of the aphantoxins have not been determined but assuming that they are the same as PSPs, and all evidence to date supports this, they will have structures like those in Fig. 11. Detection methods for these toxins in water supplies have not been adequately developed but high-performance liquid chromatography (HPLC) is being used in some cases. Both Astrachan and Archer (1981) and Wong and Hindin (1982) have used HPLC for the detection of anatoxin-a. In our laboratory we routinely use HPLC to purify other neurotoxins of Anabaena and Aphanizornenon (Carmichael and Mahmood, 1984). These methods could be modified to detect low levels of the toxins in water reservoirs or recreational waters. 2 . Hepatotoxins The peptide toxins of the freshwater cyanobacteria are primarily found in various strains of Microcystis aeruginosa. These toxins are responsible for most poisonings by cyanobacteria since M. aeruginosa is more common in its worldwide distribution than the other toxigenic cyanobacteria. Although other cyanobacteria, including Anabaena, Aphanizomenon, and Oscilfatoria (Carmichael, 1981, 1982; Skulberg et af., 1984) are

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thought to produce peptide toxins, Microcystis peptides have been studied most. Toxic blooms have been found in ponds and lakes thoughout the world including the U.S.A., Canada, the U.S.S.R., Europe, South Africa, India, Japan, the Middle East, and Australia. In temperate zones most toxic blooms occur in mid to late summer (Carmichael et al., 1985). Rats and mice injected either intravenously or intraperitoneally with acutely toxic doses of the cells or toxin extract (LDso 25-100 pg/kg, intraperitoneally in mouse) die within 1 to 3 hr. Death is preceded by pallor and prostration, with episodes of unprovoked leaping and twitching, Upon necropsy, the animals show grossly enlarged livers engorged with blood, with the remainder of the carcass being exsanguinated. Liver weight is increased, and at death composes about 8 to 10% of body weight of mice as compared to about 5% in controls (Slatkin et al., 1983; Falconer et al., 1981; Theiss, 1984). The blood content of livers of mice poisoned by Microcystis increases from 66 mm3g-' liver in controls to 534 mm3g-l for mice killed 45 min after toxin injection (Falconer et al., 1981). Histological examination of the liver reveals extensive centrilobular hemorrhagic necrosis with loss of characteristic architecture of the hepatic cords. Transmission electron microscopic examination indicates that both hepatocytes and hepatic endothelial cells are destroyed. The only alterations noted prior to cell rupture are slight mitochondria1 and cell swelling. Damaged cells have extensive fragmentation and vesiculation of the membrane (Runnegar et al., 1981). Gross and histological examination of intestine, heart, spleen, kidneys, and stomach show no consistent abnormalities; lungs are mildly congested with occasional patches of debris. Thrombi thought to contain platelets are found in the lungs of affected animals (Slatkin et al., 1983; Falconer et al., 1981). It is suggested that these thrombi may be a direct effect of the toxin and may secondarily cause the liver effects by creating sufficient pulmonary congestion to cause right heart failure which, in turn, could cause blood pooling and congestion in the liver (Slatkin et al., 1983). However, in time course studies, Falconer et al. (1981) reported that the pulmonary thrombi did not appear in histological preparations taken at 15 and 30 min after toxin injection and were present only in later preparations (while liver damage was noted as early as 15 min). This evidence, along with other evidence of effects on isolated hepatocytes and the rapid onset of the liver effects in uiuo, have led other researchers to believe that the liver damage is a direct effect of the toxin on the hepatocyte membrane and that the immediate cause of death in acutely dosed animals is hemorrhagic shock (Falconer et al., 1981; Runnegar and Falconer, 1981; Theiss, 1984). Occasionally, hemorrhages are noted in organs other than the liver (Ostensvik et al., 1981). This could possibly be due to coagulation problems associated with the liver damage.

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71

The action of Microcystis toxin on isolated rat hepatocytes was investigated by Runnegar et al. (1981), Runnegar and Falconer (1981), and Foxall and Sasner (1981). In cell suspension, normal hepatocytes viewed under the scanning electron microscope show a rounded appearance with a surface covered with microvilli. Within 5 min of incubation with the toxin, obvious lumps can be seen on the cell surface, which increase with time to present a distorted lumpy cell without microvilli. These changes occur only in live cells and are found to be dose dependent, with the minimum concentration of toxin causing deformation being directly related to the LDso for the toxin. There is no difference in trypan blue exclusion by hepatocytes incubated with or without the toxin, the toxin causes no cell lysis, and there is no release of aspartate amino transferase (ASAT) into the medium. The toxin appears to be transported into the cell via the bile acid transporters in the cell membrane. Deformation of the cells by Microcystis toxin is blocked by addition of sodium deoxycholate to the medium. The blocking effect also shows a dose response, with increasing concentration of toxin requiring a higher bile acid concentration. In another study by Grabow et al. (1982) the effect of Microcystis toxin was tested on isolated liver, lung, cervix, ovary, and kidney cell cultures. Cells of all cultures were damaged or disintegrated after overnight incubation in the presence of Microcystis toxin. While no mechanism of action was proposed it was suggested that the toxin may act on cell membranes. The effect on isolated hepatocytes of the presence and absence of calcium in the medium was also investigated (Runnegar and Falconer, 1982). It has been suggested by Farber and his co-workers that calcium entry into the cell is the final common step in the death of cells injured by membrane toxins and that cells can be protected by excluding calcium from the medium (Farber, 1981). Conversely, it has been reported by other researchers that toxic injury to hepatocytes is not dependent on calcium and in some cases calcium in the medium actually protects cells from injury by various toxins (Smith et al., 1981). Tests with Microcystis toxin and isolated hepatocytes revealed that the presence or absence of calcium in the medium makes no difference to the toxic effect (Runnegar and Falconer, 1982). It was reported that toxic extracts from algal blooms would agglutinate red blood cells (Carmichael and Bent, 1981). However, Runnegar and Falconer reported no red blood cell agglutination with extracts isolated in their laboratory (Runnegar and Falconer, 1982). The effects of the toxin on mouse liver slices, isolated mitochondria, and microsomes were investigated and no specific effects were noted (Runnegar and Falconer, 1981). The toxin did not significantly affect the incorporation of labeled leucine, uridine, or methylthymidine into trichlo-

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WAYNE W. CARMICHAEL

roacetic acid-insoluble precipitates from incubated mouse liver slices, indicating that it exerts no major effect on protein, RNA, or DNA synthesis. The toxin did not affect oxygen consumption of liver slices and only slightly increased that of isolated mitochondria. Measurements of glycogen degradation in liver slices incubated with toxin showed a variable increase in glycogen loss and glucose appearance in the incubation medium (Runnegar and Falconer, 1982). Runnegar and Falconer (1981) noted similarities in both structure and gross pathological effects of Microcystis toxin and phalloidin, a bicyclic peptide from the poisonous mushroom Amanita phalloides. Phalloidin produces a hepatic hemorrhagic necrosis similar to that produced by Microcystis, but these similarities do not extend to the cellular level. Phalloidin produces its toxic effects by inhibiting the depolymerization of F actin to actin; Microcystis does not. The molecular mechanism of action of Microcystis toxin is, at this time, unknown. Time course studies of dosed rats and mice have been done by several researchers (Slatkin et al., 1983; Ostensvik et al., 1981; Runnegar and Falconer, 1981). Liver damage can be noted in histological sections as early as 15 min after dosing, with the centrilobular hemorrhagic necrosis steadily progressing outward with time. Platelet counts taken during the course of the toxic reaction show a steady decrease in the circulating platelet count that is inversely proportional to the increase in liver weight (Slatkin et al., 1983; Jones, 1984). Runnegar and Falconer (1981) noted changes in aspartate amino transferase (ASAT) and lactic dehydrogenase at 15 min, and by 30 min had increased to 50 times control levels. Ostensvik et al. (1981) found changes in ASAT commencing at 30 min, but no changes in alanine amino transferase (ALAT) nor bilirubin during the course of toxicity. Ostensvik et al. (1981) monitored the effect of an extract of a predominantly Microcystis water bloom on blood pressure of rats. The blood pressure decreased markedly immediately after intravenous administration, but began increasing at 1.5 min and was back to normal levels within 10 min of injection. During the next 40 min, the blood pressure decreased slowly to very low levels consistent with hemorrhagic shock and remained constant at these low levels until the rats died at about 90 min. Theiss (1984), using purified toxic peptide of M. aeruginosa strain 7820, also found that blood pressure responses, both arterial and venous, were indicative of a direct hepatotoxin which caused death by hemorrhagic shock. Jackson et al. (1984) inoculated sheep intraruminally with M. aeruginosa water bloom suspension. The majority of the lethally poisoned sheep died within 18 to 23 hr. The carcasses of the sheep showed that the

ALGAL TOXINS

73

primary site of toxicity was the liver, which had centrilobular to near massive hepatocyte necrosis. Small hemorrhages were also noted in many other areas of the body. Lungs were mildly edematous, but other organs were normal except for the hemorrhages. It was noted that neutrophils were attracted into the lumen of bile ductules without a cholangitis. This has been noted in other animals poisoned with Microcystis and may have diagnostic significance in differentiating algal poisoning from other plant hepatotoxicities. These sheep experiments revealed a sharp dose-response curve in that up to 90% of the lethal dose of bloom could be ingested in a single administration without measurable effect. From the animal response it was estimated that the 27-kg sheep used would have to ingest about 650 cm3of the thick algal bloom as it occurred on the pond to provide the approximately 30 g of dried cells necessary to cause acute lethal toxicity. Foxall and Sasner (1981) reported that cladocerans, amphibians, crustacea, and teleosts were not affected by the toxin, but all mammals and birds tested to date are sensitive to it. Some effects have been reported on cardiac muscle and blood hemostasis (Kirpenko and Kirpenko, 1980). No effect has been reported on skeletal muscle or isolated nerve preparations. Foxall and Sasner (1981) reported that young hepatocytes in uitro are not affected and neither are young mice nor rats. These mice were not affected by lethal doses of the toxin until they had reached the age of approximately 20 days. This would suggest a possible activation of the toxin by the liver enzyme systems, but to date no one has investigated this possibility. Female mice were slightly more sensitive to the toxin than male mice. Foxall and Sasner (1981) reported no antibiotic activity against green algae, yeast, or bacteria and no toxicity to certain zooplankton, crayfish, amphibians, and teleosts. However, Grigor’yeva et al. (1977) reported a wide spectrum of antimicrobial activity against Escherichia coli, Shigella jlexneri, Salmonella typhimurium, Staphylococcus aureus, Enterococcus, and Candida. The mechanism involved was a decreased thiamine content and inhibition of dehydrogenase activity. Kirpenko et al. (1982) found that Microcystis toxin extracts inhibited the development of saprophytic microflora in artificial ponds. Kirpenko et al. (1981) found that toxic Microcystis caused embryolethal, teratogenic, and gonadotoxic effects in the rat. Mutagenesis involving anomalies of chromosome and chromatid apparatus was also reported. However, Runnegar and Falconer (1982) reported no mutagenicity when a purified extract was tested by the Ames Salmonella assay.

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Reports on Microcystis poisoning are often conflicting. The only consistent pathological findings in Microcystis toxicity are the swollen, blood-engorged liver with hemorrhagic necrosis and the mildly edematous lungs. These conflicting reports may be attributed to possible differences in the effects of different strains of toxic Microcystis, the use of extracts from mixed blooms that contain other algal or bacterial species, or differences in research technique. Microcystis has been linked to an outbreak in 1979 of hepatoenteritis among an aboriginal population on Palm Island (Queensland, Australia) (Bourke et al., 1983). An epidemiological investigation revealed that 139 children and 10 adults were affected shortly after a copper sulfate treatment of a dense algal bloom in Solomon Dam, the source of the island’s reticulated water supply. All of the affected individuals were using water from Solomon Dam and no incidence of disease was reported among persons not receiving water from this supply. The disease had three welldefined phases which presented in the following order: hepatitis phase-2 days; lethargic phase with severe electrolyte derangement-1 to 2 days; diarrheal phase-5 days. Repeated sanitary assessments of the environment of Palm Island failed to uncover any other possible cause for the outbreak. Falconer et al. (1983a) examined the results of routine assays for hepatic enzymes in plasma of persons who obtained drinking water from a reservoir (Malpas Dam, Armidale, New England, Australia) containing a heavy bloom of toxic M . aeruginosa during periods before, during, and after the algal bloom. These results were compared with corresponding assays from an adjacent population which did not use water from this source. The residents supplied with water from the bloom-infested Malpas Dam reservoir showed a significant rise in y-glutamyltransferase (GGT) during the bloom period, while no such increase occurred in residents not receiving their water from the Malpas Dam. ALAT also showed an increase during this period, but it was not statistically significant (p I 0.10). ASAT and alkaline phosphatase showed no significant increases. GGT is characteristically released after alcohol or toxin damage to liver cell membranes and is a more sensitive indicator of liver damage than alkaline phosphatase or ASAT. The toxin of M . aeruginosa is normally contained within the algal cells and is released only when the cell is damaged, either by poisoning the algae with copper sulfate, by mechanical rupture of the cell, by breakdown in the stomach, or age-related death of the cell. Damage could occur to the cell when algae in a reservoir are transported through a municipal water system; therefore presence of the blooms in a water supply reser-

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voir might be expected to result in the presence of the toxin in drinking water. Some toxins and odor and flavor organics associated with algal blooms can be removed by filtration of reservoir water through sand topped by a layer of granular activated carbon (Falconer et al., 1983b). The exact composition and structure of the Microcystis toxin have remained elusive, despite the efforts of many scientists since the attempts of Louw (1950) to identify the toxic principle from a Microcystis bloom in South Africa. Considerable variation exists in the toxins from different strains of the algae. The toxin also appears to have an unusual structure which does not lend itself to classical techniques. Working with different strains and utilizing different procedures for purification, most workers now agree that the toxin is a pentapeptide, with some common and some variable amino acids. On hydrolysis the toxin is found to contain five amino acids and methylamine in approximately equimolar amounts. Amino acids isolated from all various toxin preparations invariably include aspartic acid (or P-methylaspartic acid), glutamic acid, and alanine. Glutamic acid and alanine, the invariant amino acids of Runnegar and Falconer (1981) and Elleman et al. (1978), and methionine, have no free carboxyl groups while the a-carboxyls of glutamic and P-methylaspartic acid were shown to be free. The toxin does not react with ninhydrin, nor were any amino groups dansylated in the intact toxin, indicating the absence of any free amino groups (Bishop et al., 1959; Murthy and Capindale, 1970; Rabin and Darbre, 1975; Toerien et al., 1976; Elleman et al., 1978; Botes et al., 1982a; Eloff et al., 1982; Santikarn et al., 1983). From amino acid analysis, a minimum molecular weight of 654 was derived by Runnegar and Falconer (1981). Other proposed molecular weights, derived by different researchers from various strains and utilizing different techniques, have also been reported (Runnegar and Falconer, 1981). The existence of the toxin as dimers, trimers, or even larger groups of identical or similar subunits could explain the wide variability of proposed molecular weights of the toxin (Eloff et al., 1982). The lack of free amino groups has led to the speculation that the toxin is cyclic (Bishop et al., 1959; Santikarn et al., 1983; Williams, 1983; Botes et al., 1984) and/or has a blocking group on the terminal amide group (Botes et al., 1982a,b). The extreme hydrophobicity of Microcystis toxins, as exemplified by their chromatographic behavior on paper, cannot be accounted for in terms of their peptide composition and could logically reside in the properties of such a blocking group. The UV spectrum of the toxin shows an absorption maximum at 240 nm; again, this cannot be accounted for by the peptide portion of the molecule, since Botes et al. (1982a) found no

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WAYNE W. CARMICHAEL

aromatic amino acid present. They do suggest that this absorbance peak could be due to the presence of a conjugated diene chromophore in the blocking group. Eloff et al. (1982) found that the toxins present in different strains were very similar, but variations in toxin composition were found. In some organisms, as many as six different toxins were obtained, but generally one or two toxins accounted for >90% of the toxin in a single isolate. In all cases the toxin contained P-methylaspartic acid, glutamic acid, alanine, methylamine, and two other amino acids in equimolar ratios. The additional amino acids present in major toxins were as follows:-leucine and arginine, leucine and alanine, tyrosine and arginine, methionine and arginine, leucine and tyrosine, alanine and tyrosine, or arginine and arginine. The peptide containing leucine and arginine was present in 9 of the 13 toxic isolates. Botes et al. (1982a) isolated four toxin variants (BE-2 to BE-5) from a laboratory strain of Microcystis aeruginosa (WR-70). The strain was cultured in a modified Volk and Phinney medium (1968) with the trace element mix of Stanier et al. (1971). All variants were composed of five amino acid residues with one residue each of P-methylaspartic acid, glutamic acid, and alanine common to all. For the remaining two residues combinations of leucine and arginine, tyrosine and arginine, leucine and alanine, and tyrosine and alanine were found. Methylamine was detected in acid hydrolysates in all cases. Configuration assignments (Botes et al., 1982b) of the a-carbon atom of the amino acid residues have been made by stereospecific enzymatic transformations, showing that the constant residues are in the D-form, whereas the L-configuration could be assigned to all the variant residues. The relative configuration of the @carbon atom of P-methylaspartic acid could be made by comparison of the electrophoretic mobility of the toxin-derived residue with literature values for the authentic compound. The presence of N-methyldehydroalanine, which gives rise to methylamine upon acid hydrolysis, has been confirmed by identifying N-methylalanine in the acid hydrolysates of the toxin variants, after reduction of the toxin with sodium borohydride. The use of 400 MHz proton NMR spectroscopy showed the toxins to be more complex than suggested by amino acid analysis alone. Apart from the unambiguous assignment of the amino acid residues, an apolar side chain of 20 carbon atoms was demonstrated. The toxin is thought to exist in dimers, trimers, and other larger aggregates with each subunit consisting of a pentapeptide plus an apolar side chain. In this study the molecular weight of each subunit was estimated at 909. Based on mass spectrometry, using the BE-4 toxin, Santikarn et al. (1983) and Williams (1983) proposed that the blocking group is a highly

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unsaturated hydrocarbon with a molecular weight of approximately 3 13. This would be in addition to the 909-Da subunit of BE-4 toxin. The structure of this side group was reported to be a novel p-amino acid. There is some new evidence that the peptide may be cyclic. This would explain the resistance of the intact toxin to Edman degradation and degradation by proteolytic enzymes. Neither fast atom bombardment mass spectrometry nor the electron impact mass spectrum of methylated Microcystis toxin BE-4 shows evidence of the sequence of ions normally observed in the spectra of linear peptides (Santikarn et al., 1983; Williams, 1983). Botes et al. (1984) showed that the p-amino acid residue was a part of the linear amino acid sequence. The molecular weight of the entire toxin was concluded to be 909. They also proposed the term cyanoginosin for the monocyclic heptapeptides. To designate the variable Lamino acids they propose a two-letter suffix after cyanoginosin. Thus the BE-4 toxin becomes cyanoginosin-LA (Fig. 9). A low-toxicity strain of M. aeruginosa collected by Watanabe and Oishi (1980, 1982) and Watanabe et al. (1981) from Lake Suwa in Japan, in contrast to reports of toxins obtained from other world-wide sources, was ninhydrin positive, inactivated by proteases, and not toxic when administered orally to mice. The main amino acids composing this toxin were glutamic acid, aspartic acid, alanine, glycine, arginine, and leucine. The molecular weight was reported to be approximately 2950, with a minimum of 770, as determined by HPLC. There have been no reported livestock deaths from Microcystis in Japan. Siegelman et al. (1984) presented a method for microdetection of Microcystis toxins. This involves extraction of 20 mg of lyophilized cells with 1 cm3of 38% ethanol, 5% n-butanol, 50 mM ammonium acetate for 1 hr, followed by centrifugation for 5 min at 12,000 g . One cm3 of n-butanol followed by 1 cm3 of water are added, with vortexing, to the supernatant. The sample is then centrifuged (10 min at 500 g), and the upper phase of nbutanol is collected and dried by evaporation. The residue is extracted with I cm3 of 26% acetonitrile, 500 mM ammonium acetate, stored at -10°C for 16 hr, and then centrifuged for 10 min at 12,000 g . These partially purified extracts are stable for several months when stored at - 10°C.

r

o-Ala-m-Masp-B-Adda

- o-Glu-

Mdha

1

Fig. 9. Proposed general structure for cyclic heptapeptide toxins of Microcystis aeru1984). X and Y, variable L-amino acids which can differ between strains. Masp, P-methylaspartic acid; Adda, p-amino acid residue of 3-amino-9-methoxy2,6,8-trirnethyl-10-phenyldeca-4,6-dienoic acid; Mdha, methyldehydroalanine. ginosa (Botes er a/.,

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W A Y N E W . CARMICHAEL

Aliquots of 5 to 25 mm3 of these extracts are examined by HPLC as follows : Precolumn-pellicular octadecyl (C-18) beads (SynChropak, RSC) (0.4 x 5.0 cm) Column-3-pm octadecyl (C-18) silica (Hypersil, Shandon) (0.46 x 5.0 cm) Solvent-26% acetonitrile, 500 mM ammonium acetate, pH 6.0 Flow- 1.O cm3 min-I Detection-238 nm at 0.04 AUFS (absorbance units full scale)

In our laboratory we are using a modification of this procedure to purify and compare toxic peptides of Microcystis, Anabaena, and Oscillatoria. These modifications employ C- 18 semipreparative columns, Sephadex G-25 gel filtration columns, and Bond Elut C-18 preparative cartridges (Theiss, 1984; Carmichael, unpublished data). Kirpenko et al. (1976) have reported on the use of an enzyme photometric assay to detect Microcystis toxins. The method uses cholinesterase and acetylcholine in the presence of the toxin to yield a product which can be measured with the color indicator bromothymol blue.

C. MARINE CYANOPHYTE TOXINS

Marine cyanobacteria have not presented as many problems as the freshwater cyanobacteria, but several species of the family Oscillatoriaceae (unbranched filamentous, lacking heterocysts) produce toxic compounds that cause dermatitis in man and animals and promote tumors in laboratory animals. These toxins may also have toxic affects on some forms of marine life (Moore, 1981b). The most common and best documented toxic reaction to marine cyanobacteria is a severe contact dermatitis known as “swimmer’s itch.” This is a cutaneous inflammation characterized by erythema, followed by blisters and deep desquamation within 12 hr of exposure to the algae. The dermatitis is induced by exposure to two compounds, debromoaplysiatoxin and lyngbyatoxin A (Fig. lo), which have been isolated from Lyngbya majuscula (Moikehai and Chu, 1971; Moikehai et al., 1971; Kato and Scheuer, 1975; Hashimoto et al., 1976; Mynderse et al., 1977; Cardelh a et al., 1979). Debromoaplysiatoxin has been isolated from deep water strains of L . majuscula; while lyngbyatoxin A is found in a Hawaiian shallow water variety of L . majuscula. The dermatitis produced by lyngbyatoxin A is the same as that from debromoaplysiatoxin. However, the shallow water variety of Lyngbya majuscula has never been impli-

ALGAL TOXINS

79

cated in an outbreak of swimmer’s itch, mainly because it grows on the leeward side of Oahu and swimmers rarely come into contact with the broken filaments of the alga in the water, since the normal tradewinds blow them out to sea rather than toward the shore. Debromoaplysiatoxin, along with several closely related compounds, has also been isolated from other Oscillatoriaceae (Mynderse and Moore, 1978; Moore, 1981b). Debromoaplysiatoxin and oscillatoxin A, for example, are major toxic constituents of a mixture of cyanobacteria from Enewetak identified as Schizothrix calcicola and Oscillatoria nigroviridis. Recent work (Moore et al., 1984) shows that oscillatoxin A has a structure similar to the aplysiatoxins. Solomon and Stroughton (1978) reported reactions to debromoaplysiatoxin occurring in humans at concentrations of 5.6 mg ~ m and - ~in making it one of the most potent dermatotoxins animals at 0.5 pg known. Since swimmers who come in direct contact with toxic strains of the algae develop erythema, followed by blisters and deep desquamation, within 12 hr of exposure, the active agent is classed as a primary irritant rather than an allergen. During animal testing, a second application of the debromoaplysiatoxin produced a similar reaction to the first, which is not suggestive of sensitization. Debromoaplysiatoxin, a phenolic substance, has been isolated from the midgut gland of the sea hare (Stylocheilus Eongicauda) along with a bromine-containing analog bromoaplysiatoxin (Kato and Scheuer, 1976). Lyngbya majuscula is a favorite food of the sea hare, and the digestive tract of this animal appears to be unaffected by the toxin. The sea hare, in fact, utilizes an unknown metabolite in the algae for metamorphosis (Switzer-Dunlap and Hadfield, 1977). Hashimoto et al. (1976) observed rabbitfish (Siganus fuscescens) feeding on sea grass entangled with L . majuscula, but observed no harm to the digestive tract of the fish. However, he suggested that ingestion of L . majuscula might be related to an epidemic of rabbitfish poisoning among the natives in the Ryukyus Islands in 1969, but no further study was made of this. Other marine life infesting toxic Lyngbya may not be so fortunate as the sea hare and the rabbitfish. Lightner (1978) suggested that Lyngbya spp. might be responsible for mass mortalities of raceway-reared blue shrimp (Penaeus stylirostris) in the large shrimp culture facility in Penasco, Mexico. The affected shrimp had severe necrosis in the epithelia lining the midgut, dorsal cecum and hindgut gland, along with subsequent hemocytic enteritis. However, Moore (1981a) could detect no mouse toxicity or activity against P-388 lymphocytic mouse leukemia in lipophilic extracts of a sample of the toxic algae obtained from Lightner’s laboratory in 1979. The appearance of the necrosis in the shrimp was slow and Moore sug-

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WAYNE W . CARMICHAEL

gests that perhaps a very small contaminant in the algae that normally grow in the rearing tanks may have been responsible. Debromoaplysiatoxin and lyngbyatoxin A, isolated from the lipophilic extract of Lyngbya majuscula, show activity against P-388 lymphocytic mouse leukemia at sublethal doses (Mynderse et al., 1977; Cardellina et al., 1979), as do the aqueous extracts of some marine Oscillatoriaceae (Kashiwagi et al., 1980). Water-soluble toxins are also present in several other cyanobacteria belonging to the Oscillatoriaceae, but none of these has been isolated and characterized (Moore, 1981b). Cooper (1964) reported that the natives in Marakei atoll associate toxic fish with an alga they called “tan-tan.” This cyanobacteria was identified as Schizothrix calcicola, which contains both lipid- and water-soluble toxins which had a small amount of anticholinesterase activity (Banner, 1967). Lyngbya majuscula also possess water-soluble toxins (Kashiwagi et al., 1980)but little is known of these toxins at the present time. The gross structure of lyngbyatoxin A, as determined by chemical and spectral data, is the same as that of teleocidin A (Fig. lo), a poisonous substance associated with several strains of Streptomyces (Cardellina et al., 1979; Sakabe et al., 1966). Teleocidin was first isolated from the mycelia of Streptomyces mediocidicus (Takashima and Sakai, 1960). It is now known that teleocidin is composed of two compounds, teleocidin A ( M , = 437) and teleocidin B ( M , = 451) (Fujiki et al., 1982a, 1984). Teleocidin A produces dermatotoxic effects that are the same as those of lyngbyatoxin A and both are highly toxic to certain types of fish. Lyngbyatoxin A, bromoaplysiatoxin, debromoaplysiatoxin, and teleocidin are also reported to be tumor promoters in mice (Fujiki et al., 1981, 1983, 1984). The in uitro effects of these substances are identical to tetradecanoylphorbal 13-acetate (TPA), the tumor-promoting constituent of croton oil. To screen for the tumor-promoting activity of these compounds a three-test procedure was used. The first test is on irritation of mouse ear, the second on induction of ornithine decarboxylase (ODC) in the skin of the back of mice, and the third on adhesion of human promyelocytic leukemia cells (HL-60) (Fujiki et al., 1984). Using these tests it was shown that lyngbyatoxin A, bromoaplysiatoxin, and teleocidin were all potent promoters, but debromoaplysiatoxin was much weaker. In addition to the above, lyngbyatoxin A and teleocidin show a number of effects on mammalian cells in culture that are similar to those of TPA. These include induction of differentiation of human promyelocytic leukemiacells (HL-60) (Nakayasu et al., 1981; Fujiki et al., 1982b), aggregation of human lymphoblastoid cells (Hoshino et al., 1980), and stimulation of prostaglandin production and choline turnover in HeLa cells. All of these effects are similar to those produced by the structurally dissimilar phorbol ester tumor promoter TPA (Sakamoto et al., 1981).

81

ALGAL TOXINS

OH

bh

B

Fig. 10. (A) Debromoaplysiatoxin and bromoaplysiatoxin (R = Br), a dermatotoxic phenolic bislactone produced by several genera of marine cyanobacteria. (B) Lyngbyatoxin A, a dermatotoxic indole alkaloid, is produced by Lyngbya majuscula. This toxin has the same structure as teleocidin A , which is produced by strains of the filamentous bacterium Streprornyces.

The discover of these tumor-promoting properties suggests that, in addition to the problem posed by the dermatotoxicity of L y n g b y a majuscula, the toxins produced by these cyanobacteria could conceivably be involved in the development of human cancer, even though there is no direct evidence that L y n g b y a majuscula has caused human cancer. Freshwater cyanobacteria have not been investigated for the production of these dermatitis toxins with tumor-promoting activity. Some evidence exists that they are being produced however. This includes reports of contact irritation from freshwater recreational waters (Billings, 1981; Carmichael et al., 1985; Carmichael and Codd, unpublished data). It appears that freshwater cyanobacteria are capable of producing three

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WAYNE W . CARMICHAEL

groups of toxins: neurotoxic alkaloids, hepatotoxic peptides, and the as yet unidentified dermatitis toxins which may have some similarity to marine cyanobacteria toxins. These three groups are in addition to the presence of lipopolysaccharide endotoxins, which are a part of the cyanobacterial cell wall and do not apparently contribute to their environmental toxicity (Raziuddin et al., 1983). D . DINOFLAGELLATE PARALYTIC SHELLFISH POISONS

(PSP)

PSP has been known as mussel or clam poison depending on the source of extracts. Occasionally it has been called plankton poison and mytilotoxin. The poison was later named saxitoxin (STX), a name derived from Saxidomus gigenteus (Alaskan butter clam), which was then thought to be the only toxic material responsible for all PSP along the Pacific coasts (Schuett and Rapoport, 1962). From the early work of Meyer et al. (1928) and Muller (1939), it was observed that the poison was water soluble, acid stable, and alkaline labile. Using these observed properties, Schantz et al. (1957) was able to purify saxitoxin using carboxylate cation exchange resins. Further analysis of the purified poison as the dihydrochloride.salt (white hygroscopic powder) indicated a molecular weight of 300 to 400 (diffusion coefficient of 4.8 X cm2 sec-I; pK, of 8.2 and 11.6; specific optical rotation of 130 2"). Saxitoxin was also found to be insoluble in lipid solvents, could be detoxified by hydrogen reduction, and showed no absorption above 220 nm (Schantz et al., 1957, 1966; Schantz, 1960). The correct structure of saxitoxin (Fig. 11) was shown to be an alkaloid plus a purine (Schantz et al., 1975). Schantz (1960) and hvans (1970) found out that a large component of toxins isolated from North Atlantic mussels was not being retained by the carboxylate cation exchange resins. With modifications in the purification method involving gel filtration on polyacrylamide gel (Shimizu et al., 1975) and TLC (Buckley et al., 1976), they were able to identify the unretained toxins as derivatives of saxitoxin. It was obvious then that saxitoxin was not the sole cause of toxicity. Ghazarossian et al. (1974) suggested that the saxitoxin derivatives were n-oxides. Subsequently Shimizu et al. (1978) and Boyer et al. (1978) demonstrated structurally that the derivatives are n-1-hydroxysaxitoxin and 1 1-hydroxysaxitoxin sulfates, which are commonly known as neosaxitoxin and gonyautoxins. Hall et al. (1980) and Koehn et al. (1982) isolated and identified other toxins including saxitoxin from Pacific isolates of cultured Protogonyaulax spp. This array of 12 toxins can be divided into 2 groups (Fig. 11):

*

ALGAL TOXINS Carbamate

- R4

83

(H)

&l& H H H

OH OH OH

H H OS03H H OSO,

-

H OSO, H H OSO, H

N Sulfocarbamoyl

STX GTXz GTX 3 neoSTX GTXl GTX4

H

- Rg (SO3-)

R1R2b H

H H OH OH OH

H H OS03H

H OSO,

H OSO, H H OSO, H

B 1(GTX5) C1 C2(GTXe) BP(GTX6) C3 c4

Fig. 1 1 . Structure of paralytic shellfish poisons produced by several genera of marine dinoflagellates. At least some of these toxins have been found in species of Protogonyuulux, Pyrodinium, the red alga Juniu, and the freshwater cyanobacterium Aphanizomenon (Carmichael er al., 1985).

1 . The carbamate toxins-H is the substituted group at &. This group includes saxitoxin, neosaxitoxin, gonyautoxins 1 , 2, 3, and 4. 2. The N-sulfocarbamoyl toxins-sulfite (SO3-) is the substituted group at R4 and includes B1 (gonyautoxin 5 ) , B2 (gonyautoxin 6), C1, C2 (gonyautoxin 8), C3, and C4.

Another naturally occurring toxin that had been reported by Hsu et a f . (1979) and Wichmann et al. (1981) (as an unknown) is gonyautoxin 7. A similar array of toxins has been reported from sources found in subtropical and tropical waters (Yasumoto et a f . , 1983). Oshima et a f . (1984) demonstrated that the tropical marine dinoflagellate Pyrodinium bahamense var. compressa can produce STX, neoSTX, GTXS, GTX6, and decarbamoylsaxitoxin (dcSTX). In addition they showed that the red algal genus Jania produced gonyautoxins which were ingested by coral reef crabs and gastropods. In preliminary tests they found that these gonyautoxins could be bioconverted in the digestive organs of these crabs to STX. In addition, Harada et al. (1982) reported the presence of unidentified new toxins from turban shells (actually top shells) which were designated as TST (turban shell toxin). The relative potencies of these toxins were found to be different. Sax-

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WAYNE W . CARMICHAEL

itoxin at an LDsoof 10 pg kg-' or 5000 MU mg-' (1 MU is defined as the amount of toxin needed to kill a 20 g mouse in 15 min) is the most potent (Schantz et al., 1966). Hall (1982) and Proctor et al. (1975) showed that the potencies of some of these toxins change upon hydrolysis (0.2 M HCl, 100°C for 5 min) indicating transformations. Toxins B1, C1, C2, B2, C3, and C4 after hydrolysis were found to convert to STX, GTX2, GTX3, neoSTX, GTXI, and GTX4, respectively, indicating hydrolysis of the R4 group (SO3-) (Hall et al., 1980). Neosaxitoxin is as potent as saxitoxin (Shimizu et al., 1978; Hsu et al., 1979; Hall, 1982) even though Genenah and Shimizu (1981) reported a much lower value (2363 ? 101 MU mg-I). Potencies, according to Hall (1982), are STX and neoSTX > GTX3 > C2 > B2 > B1 > C1 (no data given for GTX,, C3, GTX4, and C4; while according to Genenah GTX3 was > STX > GTXl > neoSTX > GTX2 > GTX4 > B1 (GTX5). As mentioned earlier, characterization studies of STX indicated two dissociable groups with pK, values of 8.3 and 11.6 -+ 0.1. Rogers and Rapoport (1980) reported values of 8.22 and 11.28. A similar titration of neoSTX by Shimizu et al. (1978) revealed a third dissociable group with a pK, of 6.75. These pK, values correspond to the dissociation of N-lhydroxyl (6.75), C-8 guanidium (8.5), and C-2 guanidium (1 1.6). The observed differences in the binding to carboxylate cation exchange resins are due to the presence of charges under the acidic condition. STX and neoSTX have a net charge of + 2 whereas GTXs (1 through 4) have a net charge of + 1 (Boyer et al., 1978). Hence it was once postulated that the net charge was the sole determinant of potency. However, structural variation studies on potency of synthetically derived material indicated otherwise. Manipulations of C-I2 OH groups (Fig. 11) made the molecule completely devoid of activity (Kao et al., 1981; Kao and Walker, 1982). Removal of the carbamoyl group (-CONHz) reduces potency to less than 70% of that of STX (Kao and Walker, 1982). Also effective in reducing potency (in uiuo by 15 times that of STX) is the substitution of the carbamate group with a sulfo group (SO3-) (Hall, 1982). Symptoms of paralytic shellfish poisoning are primarily neurologic and appear rather quickly, often within 30 min after ingestion of contaminated shellfish. Most deaths occur from respiratory paralysis within 12 hr of illness. The prognosis is considered good if one survives after 24 hr (Schantz, 1971). The amounts of toxins ingested to cause illness vary with the individual but ranges from 3000 to 20,000 MU. The illness is characterized by paresthesia of lips, mouth, face, and extremities, often followed by nausea, vomiting, weakness, and incoherence. As the illness progresses, respiratory distress and muscular paraly-

ALGAL TOXINS

85

sis become more severe and death results from respiratory paralysis. In a large outbreak of PSP in England in 1968, the common symptoms reported were paresthesia, weakness of extremities, lightheadedness, and floating sensation (McCollum et al., 1968). Since the toxins leave the system quickly, resuscitation is recommended for treatment. But in severe cases, treatment should consist of a cathartic or enema to remove the unabsorbed toxins from the intestinal tract. Gastric lavage should be considered if vomiting has not occurred (Hughes and Merson, 1976). Artificial respiration should be supplied if respiratory difficulties appear (Schantz, 1971). Evans (1965) and Kao (1975) reported some hypotensive effects of the toxins with high doses. Potency varies widely depending on the recipient and routes of administration. From experiments with mice, rats, and rabbits, intraperitoneal (ip) or intravenous (iv) administration of STX is far more potent than oral administration, ranging from 10 to 50 times in potency (Evans, 1972). Thus substances that increase intestinal absorption or peripheral circulation will definitely intensify the symptoms of illness. The mechanism of action for saxitoxin is interference of the transmission of electrical impulses along the nerve and muscle membranes. The toxin causes a reversible blockage of the permeability to sodium ions without affecting permeability to potassium ions. Thus the propagation of nerve-muscle action potentials is blocked (Evans, 1964, 1970; Kao and Nishiyama, 1965). Kao and Walker (1982), using analogs of STX in their studies of Na+ channel blocking, showed that active groups of STX were the C-12 OH groups, the 7,8,9 guanidinium and carbamyl group, but not the N-1 of 1,2,3 guanidinium. They proposed that STX binds the receptor located in the outside of the membrane very close to the opening of the Na+ channels. The guanidinium group is then thought to be electrostatically attracted by the fixed anionic charges around the opening of the Na+ channel and the bulk of the molecule (STX) covers up the channel. The idea of having a toxin-binding site distinct from the Na+ channel was recognized by Twarog and Yamaguchi (1975). Later Spaulding (1980) showed that frog muscle fibers subjected to trimethyloxonium ion (which derivates carboxylate groups) became less susceptible to STX blockade. Cohen and Barchi (1981) noted the binding sites to have glycoprotein characteristics. Detection of paralytic shellfish poisons has been a concern for public health and fisheries people for many years. The officially employed method of detection, worldwide, is the mouse bioassay (Horwitz, 1980; Houser, 1965). Because of the variable nature of this assay method and the inability to distinguish types of toxins present other methods are being developed. Carlson et al. (1984) have developed a radioimmunoassay for

86

WAYNE W . CARMICHAEL

saxitoxin but it does not bind to neosaxitoxin. Other immunoassay methods are also being developed including a rabbit antiserum which has successfully been used as a therapeutic agent in mice to prevent death from saxitoxin (Davio and Pickering, 1983). HPLC is also proving a powerful tool in the assay of PSPs. This method uses the fluorescence property of the PSP derivatives when oxidized under alkaline conditions. Initially problems existed in the detection of neosaxitoxin and gonyautoxin 1 and 4 due to its poor fluorescence (Sullivan and Iwaoka, 1983; Sullivan et al., 1983). These problems have been corrected, making it possible to detect by fluorescence all of the known carbamate and N-sulfocarbamoyl toxins of PSP (Sullivan and Wekell, 1984). E. DINOFLAGELLATE CIGUATERA TOXINS

Ciguatoxin (CTX) is the name given to the first active component isolated which was felt to be responsible for ciguatera poisoning. It is a lipidsoluble neurotoxin. It imparts no different taste or color to the fish and is not affected by cooking or freezing. It was isolated from contaminated red snapper (Lutjanus),shark (Carcharhinus), and the moray eel (Gymnothorux) by a series of organic solvents, liquid-liquid partition, and chromatography. The isolated toxin is a transparent, yellow oil. This final product had an MLD (minimum lethal dosage) of 0.5 mg kg-' and was identified as having the molecular formula CuH65NOs,with the nitrogen being quaternary (Scheuer et al., 1967). Since then it has been further purified and found to have a polyether structure similar to okadaic acid or brevetoxin C, with a molecular weight of about 1111 (Nukina et al., 1983), a crude extract LD5o of 28.1 mg kg-', and a purified toxin LDw of 0.25 pg kg-' intraperitoneally in mouse (Hokama et al., 1984). Research on ciguatera toxin at the University of Southern Illinois found that there is more than one ciguatoxin-like compound. From cultures of Gambierdiscus toxicus grown in the laboratory, three distinct lipid toxins, GT-1, GT-2, and GT-3, were isolated, which all act as competitive antagonists against acetylcholine and histamine sites on guinea pig ileal smooth muscle, and whose effects are not offset by calcium (Miller et al., 1984). It may be that these toxins are individual cyclic polyether residues similar to okadaic acid isolated by Tachibana et al. (1981). Another toxin thought to contribute to ciguatera poisoning is the watersoluble substance known as maitotoxin (MTX), which is found in the liver and gut of affected fish (Yasumoto et al., 1971) and from laboratory cultures of G . toxicus (Dickey et al., 1984). It has been suggested that MTX is a precursor of CTX (E. P. Ragelis, personal communication). On the isolated guinea pig atrium, MTX shows positive inotropic effects similar

ALGAL TOXINS

87

to CTX, but only at low concentrations; and a decrease in contactibility leading to complete suppression with higher concentrations (Miyahara et al., 1979). In anesthetized cats, MTX induced hyperventilation, then respiratory depression, slight bradycardia, with hypertension at low doses and cardiac arrest prior to respiratory arrest. MTX seemed to be more potent in these experiments than CTX (Legrand et al., 1982). MTX has been reported as having a lethal dose ranging from an MLD of 0.2 pg kg-l (Takahashi, 1982) to 1.1 mg kg-' (Dickey et al., 1984). It has also been demonstrated to cause an increase in calcium ion influx into cells, a release of norepinephrine (Takahashi et al., 1982), and to inhibit Na+- and K+-ATPase from microsomes of cat and human kidneys (Bergmann and Nechay, 1982). MTX has been isolated from cultures of G. toxicus and following a latent period it produces irreversible contraction of the guinea pig ileum (Dickey et al., 1984). A third, and less well-studied, toxin thought to contribute to ciguatera poisoning was isolated from the parrotfish (Chungue ef al., 1977) and termed scaritoxin (Bagnis et al., 1974). Individuals having eaten this toxic fish showed initial symptoms similar to ciguatera poisoning but then after 5 to 10 days progressed into a second phase which persisted for more than a month, consisting of equilibrium disorders and kinetic tremors. Like CTX, it is lipid soluble and distinguished from it by its chromatographic properties. Likewise, many of its physiological properties are similar and it has been suggested that it might be a metabolite of CTX. Several attempts have been made to find more reliable methods of detecting the presence of ciguatoxin than the sometimes used cat, mongoose, or mouse bioassays (Halstead, 1967). A radioimmunoassay (RIA) using CTX-human serum albumin to produce anti-CTX in sheep was developed by Hokama et al. (1977). Testing has shown that this method is relatively accurate but economically feasible only in fish weighing greater than 9 kg (Kimura et al., 1982a). This RIA method shows close correlation with mouse bioassay and the in uitro guinea pig atrium assay (Miyahara et al., 1979), and cross-reacts with okadaic acid, a toxic component of sponge (Kimura et al., 1982b). The mouse bioassay involves injecting a fish extract intraperitoneally into mice and watching symptoms for a 48-hr period. Symptoms have been shown to be dose dependent and include lowered rectal temperature, reduced pain reflexes, reduced grip and tightrope responses, reduced locomotor activity, and death (Hoffman et al., 1983). An enzyme immunoassay has been developed and is being used in parts of Hawaii to survey fish (Hokama et al., 1983). This enzyme immunoassay is reported to be as sensitive as the radioimmunoassay but is easier to run, economically feasible for screening all sizes and varieties of fish, and could be used for testing liver in addition to flesh samples. The

88

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enzyme assay uses the sheep anti-ciguatoxin used for the radioimmunoassay but instead of iodine employs horseradish peroxidase to couple with the antibody (Hokama et al., 1984). The symptoms of ciguatera disease begin within minutes of ingestion to 30 hr afterward. As there are no definitive tests used to diagnose ciguatera poisoning, it is made solely from symptomatology and history. It is often differentiated from other types of poisoning by the presence of paresthesia of the extremities. The first symptoms present are usually gastrointestinal, lasting a few hours; followed by neurological disturbances which last up to several weeks or even months (Halstead, 1967). Additional symptoms can be dermatological and neuromuscular (Morris et al., 1982; Lawrence el al., 1980). Ten to 12% of the intoxications lead to death by respiratory paralysis (Rayner, 1972). With severe poisoning, bradycardia was observed and an electrocardiogram showed inverted T waves in some patients, indicating myocardial ischemia, which was reversible upon recovery (Hanno, 1981). Symptoms have been known to vary in different locations and with ingestion of different fish (Chretien et al., 1981). An unexplained sensitization has often been observed after ciguatera poisoning and patients often experience the same symptoms of poisoning after the ingestion of nontoxic fish or after alcohol ingestion (Barr, 1982). Banner (1967), however, has suggested that this may be psychosomatic. Treatment mostly involves supportive care, but may include gastric lavage, cathartics, atropine, and protopam chloride (Dembert and Pearn, 1982; Bagnis, 1967). Alkaloids from the plant Duboisia myoporoides have been used by natives of New Caledonia as an antidote, and are thought to contain atropine, nicotine, hyoscyamine, and scopolamine (Dufva et al., 1976). Ciguatoxin has been shown to have anticholinesterase activity in uitro (Li, 1965) but not in uiuo (Rayner, 1968). Kosaki and Anderson (1968) observed depressed respiration, a fall in arterial pressure, bradycardia, arrhythmias, and neuromuscular responses in rats after administration of lethal doses of ciguatoxin extracted from various fish, and suggested that the toxicity may be due to demyelination of peripheral and central nervous tissue. In anesthetized cats, ciguatoxin caused respiratory depression, leading to respiratory arrest with bradycardia. An ECG indicated ventricular extrasystole and idioventricular rhythm (Legrand et al., 1982). Setliff et al. (1971) demonstrated that ciguatoxin increases the passive permeability of the cell membrane of frog skin, thus increasing the influx of sodium ions by competitively inhibiting calcium binding at receptor sites which regulate steady state sodium ion permeability. This suggested that possibly ciguatoxin inhibited Na+,K+-ATPase activity. However, when highly purified extrakts of ciguatoxin were used, no inhibition of

ALGAL TOXINS

89

Na+,K+-ATPase was seen, indicating that impurities may have had an effect on some previous results (Rayner and Szekerczes, 1973). A biphasic response is observed on isolated atria from rats and rabbits, with an initial inhibition followed by stimulation of atrial contraction, indicating both cholinergic and adrenergic action and possibly the release of catecholamines (Ohshika, 1971). CTX was later shown to exhibit both positive inotropic and chronotropic effects on the isolated atria of the guinea pig (Miyahara et al., 1979). CTX has also been shown to induce contraction of the guinea pig vas deferens, by releasing stores of norepinephrin, which is probably due to the increased permeability to sodium ions (Shizumi et al., 1981). Possible effects of the ciguatera disease on an unborn infant were observed by Pearn et al. (1982). A cesarean section was performed 2 days after a full-term gravid woman became poisoned with ciguateric fish. The woman experienced fetal movements which began simultaneously with her own symptoms. The infant was born with left-sided facial palsy, but with no heart abnormalities. This suggest that the toxin can cross the placenta, which would not be surprising due to its small molecular size. F . OTHER DINOFLAGELLATE TOXINS

While several other dinoflagellate genera have some species producing various toxic factors, the main genera other than Protogonyaulax (Gonyaulax) involved in poisonings are Ptychodiscus (formerly in the genus Gymnodinium) and Dinophysis (Taylor, 1984). The best known toxin producer in Gymnodinium is the fish killer G . breve. Recently it was transferred to the genus Ptychodiscus by Steidinger (1979). Baden et al. (1984) was able to isolate and purify two toxins from laboratory cultures of P . breuis termed T34 and T17. Both polyether toxins are neurotoxic and ichthyotoxic. One of the toxins also caused bronchioconstriction in anesthetized guinea pigs, which suggests it is responsible for the airborne respiratory irritant in humans associated with these toxic blooms along the Florida coast. The structure of these two “brevetoxins” is given in Fig. 12. The Dinophysis toxins are collectively referred to as diarrhetic shellfish poisoning (DSP). While the difficulty in culturing these dinoflagellates has slowed definitive studies it is thought that D . fortii and D . acuminata are the main toxic species (Taylor, 1984; Yasumoto et al., 1984). DSP produces gastrointestinal disturbances but not fatalities in humans ingesting various shellfish, i.e., Mytilus, which have concentrated the dinoflagellate. DSP toxins isolated from shellfish in Japan are all polyether-based compounds referred to as okadaic acid, dinophysistoxin, and pecteno-

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a

b

Key: a=T34

b=T17

Fig. 12. Structure of brevetoxins, T34 and T17, produced by the Florida red tide dinoflagellate Ptychodiscus brevis (Baden et al., 1984).

okadaic acid dinophysistoxin- 1 dinophysistoxin-3

: :

:

R 1 = H , R,=H R, = H, R, = CH, R, = acyl, R, = CH,

47

R 40

pectenotoxin-1 pectenotoxin-2

: :

R =OH R=H

Fig. 13. Structure of polyether lactone toxins involved in cases of diarrhetic shellfish poisoning (DSP). The toxins are produced by the dinoflagellate Dinophysis acuminata found in the shellfish areas of northern Japan (Yasumoto et a / . , 1984).

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toxin (Yasumoto er al., 1984). Figure 13 illustrates the structure of these DSP toxins. V. ENVIRONMENTAL ROLE OF ALGAL TOXINS Since so little is known about the ecological or metabolic roles of algal toxins it is important to consider the possible roles in the light of what is known about plant toxins. Since the 1960s evidence has been accumulating to show that vascular plant secondary chemicals have distinct ecological roles (Fraenkel, 1959, 1969). These plant secondary chemicals provide predation protection throughout the growing and dormant seasons. Evidence is now accumulating which shows that chemical protection can be regulated and their production quite rapid. For example, leaves of red oak trees defoliated by gypsy moth larvae show higher secondary chemical production in subsequent years (Schultz and Baldwin, 1982). Responses have also been observed in which the length of time for chemical response has been days to months. Time response can be even more rapid, however, as in the studies of Carroll and Hoffman (1980). They found that damaged leaves of Cucurbita moschata mobilize substances to the damaged region within 40 min. There is even some evidence that damaged plants can chemically communicate the need for increased chemical defense to undamaged plants within the nearby area (Baldwin and Schultz, 1983). This indicates the presence of two broad classes for chemical defenses of plants: those present before herbivore attack and those that change in response to herbivore attack (Levin, 1976). In these cases of secondary chemical production it has been in response to environmental stress. This stress can come from predation pressures as just presented, or from growth pressures such as would be provided by secondary chemical production in response to low soil nutrients (Janzen, 1974; McKey, 1979; Chew and Rodman, 1979). Central to the concept of an ecological role for secondary chemicals is the concept of cost and benefit for the defense of the organisms. If these secondary chemicals are serving an ecological rather than a metabolic (i.e., nutrient storage) role, then the energy cost involved in producing them should be less than the protective value obtained from their presence. While hypotheses abound supporting this idea within secondary plant chemicals, there are no careful quantitative models of costs and benefits (McKey, 1979). With all of these concepts in mind, some points worth considering if algal toxins are to be considered as having an ecological role include the following:

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1, Evidence is accumulating which shows that zooplankton herbivores of algae do respond to the toxins (Snell, 1980; Lambert, 1981; Ransom, 1978; Porter and Orcutt, 1980). What is not clear is just how much, if any, avoidance of toxic cells or filaments occurs relative to nontoxic cells or filaments. If there is a selective advantage to being toxic then it should be possible to show this in laboratory experiments. An important need in the testing of this idea will be the development and use of sensitive assays for toxicity of cells. This would also make it possible to check for increases or changes in toxin production due to predation pressures. Since these algae exist largely as single cells, or in some cases as filaments, there is a need to show chemical communication between the cells if production of toxins as opposed to simple selection of toxic cells is to be demonstrated. For the present it is perhaps too much to envisage that toxin production could be influenced by such mechanisms. At present it is hard to see why many water blooms of cyanobacteria and even dinoflagellates are apparently nontoxic if predation pressures are to have a significant influence on toxin production. 2. What is more tempting to envisage is the possible role of growth conditions on toxin production. This includes the possibility that nutrient composition, pH, temperature, or light may independently or in combination influence toxin production. The algae may in turn produce higher levels or different kinds of toxins in order to minimize predation pressure or to store the toxins for some metabolic need. This seems to be supported by the preliminary work of Hall (1982), who demonstrated higher specific toxin levels per cell in dinoflagellates grown on decreased nitrogen or phosphorus. It also could explain decreases observed in toxin producticin in our laboratory for almost all toxic cyanobacteria when they are grown in nutrient-rich media. This reduction in toxin production is most common among the peptide toxin-producing strains and almost never observed among the alkaloid toxin-producing strains. The decrease in toxin production is also slow, requiring months or more of repeated subculturing before significant reductions are observed. This implies that if toxin production is to be influenced by growth conditions it will take more than a few subcultures. Just as important, however, could be the qualitative changes in toxin production that growth changes could influence. 3. Finally, it has been pointed out that the costs of storing some kinds of secondary compounds are higher than those of others (McKey, 1979). Substances such as tannins and saponins exert their toxic effect by reducing digestion of proteins in the gut of the herbivore. Other toxins such as alkaloids are absorbed from the gut and are active against specific metabolic processes. Rhoades and Cates (1976) argue that the digestion inhibi-

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tors impose higher costs on the plants since they can influence plant processes and thus must be segregated from other cellular components. The more specific metabolic toxins such as alkaloids are effective against herbivores in lower concentrations and are less likely to influence plant metabolism. This perhaps is part of the reason why algae produce the more toxic, more metabolically specific alkaloids, peptides, and related toxins. Whatever the role for algal toxins, it should be evident that (1) they are diverse groups of physiologically potent secondary chemicals that are more chemically related to higher plant toxins than they are to bacterial or animal toxins; (2) their production should not be considered fortuitous or accidental but rather part of evolutionary processes by which the organisms have adapted to particular needs and requirements of their environment; and (3) studies on production of the toxins including possible environmental and genetic influences will serve to aid an overall understanding of secondary chemical production.

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Plant Transposable Elements

PATRICIA NEVERS, NANCY S. SHEPHERD* AND HEINZ SAEDLER Mux-Plrnc.k-In.stitiltjlir Ziic,htiln~.sforsc.hunC:Egelspfud Kiiln. Federril RC.pilhlic of Gcrtnany

I. The Phenomenon of Variegation.. ......................................... A. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Examples of Variegation Excluded from Detailed Consideration . . 11. Recognizing Mutations due to Transposable Elements by ... Classical Genetic Means.. .... A. Reports of Mutable Alleles ... B. Segregation Ratios and Wh ................ C. Reversion Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Reverse Variegation . . . . . . . . . . . . . . ............. E. Variegation Associated with Chromosomal Anomalies .................... F. Stable and Unstable Allelic Variants of Mutable Alleles G. Transposition: The Most Characteristic Pr 111. Transposable Elements at a Molecular Level. A. Cloning Strategy. ...................... B. Structural Features of Plant Transposable Elements ...................... C. Multiple Copies of Plant Transposable Elem D. Detection of Transposition at the Molecular E. How a Transposable Element Influences Ge F. Proteins Encoded by Transposable Elements . . . . . . G . Reversion Events at the Molecular Level.. . H. The Molecular Basis for Allelic Derivatives IV. A Model of the Mechanism of Transposition.. . A. Observations on Which the Model is Based. B. Excision of a Transposable Element in Plants C. Integration of a Plant Transposable Element. . . . . . . D. Predictions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Present address: E.I. Dupont de Nemours & Company, Inc., Central Research & Development Department, Wilmington, Delaware 19898, U.S. A. 103 ADVANCES IN BOTANICAL RESEARCH, VOL. 12

Copyright 0 1986 by Academic Press Inc. (London) Ltd. All rights of reproduction in any form reserved.

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E. Large-Scale Rearrangements Induced by Transposable Elements in Plants F. Specificity of Integration .............................................. V. Interaction between Transposable Elements A. General Remarks ..................... B. Dosage Effects ..... C. Interaction with Alte D. Other Modifiers .... ................................ E. Possible Interaction between Two Transposable Elements of Antirrhinurn

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majus ............................................................... 185 VI. Induction of Genomic Instabilities .................. . . . . . . . . . . . . . 188 V11. Addendum ..................................... ........ 193 References .............................................................. 194

I. THE PHENOMENON OF VARIEGATION A. INTRODUCTION

Where uniformity is the rule, irregularities like a flaw in a woven pattern or a spot on a plain-colored surface immediately strike the eye. Thus among living organisms the phenomenon of variegation, the unusually spotted, striped, or otherwise mosaic appearance of some individuals as opposed to their normally uniform counterparts, has long been a source of fascination. In plants variegation is most easily recognized as irregularities in pigment patterns on leaves, flowers, and seeds, although other characteristics such as leaf form, flower form, or starch content (in maize kernels) may also be subject to variegation. As early as 1588 Jacob Theodor von Bergzabern described with great enthusiasm the multicolored and variegated kernels of maize from the New World. And variegation in tulips, illustrated in the still-life paintings of many Dutch masters, became almost a craze in the seventeenth century in the Netherlands. Indeed, variegated tulips were so coveted that a single bulb was worth as much as several pigs, sheep, tons of grain, or even 1000 lb of cheese (Dubos, 1958, cited in Pollard, 1959). Although present scientific interest in variegation is certainly more rational, it is no less animated. Researchers hope that variegation may contribute to our understanding of an organism’s capacity for change and provide a starting point for unraveling the complex mechanisms underlying its development. As discussed below, variegation can be caused by several different factors. The focal point of this article will be variegation in plants due to genetic alterations implemented by transposable elements. The distinguishing feature of this kind of variegation, as opposed to other examples

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discussed below, is that the two or more different kinds of tissue in the variegated plant differ genotypically, that is, with respect to the DNA of their nuclear genomes. In the simplest case one kind of tissue contains a transposable element at a given locus and the other does not. The terminology used in connection with transposable elements reflects historical developments in this field of research. Prior to the discovery of transposable elements as discrete physical entities, their existence was predicted on the basis of classical genetic evidence. In this period a premolecular terminology arose, some terms of which are still relevant today while others may require redefinition. The term “mutable allele” was used to refer to a special mutational condition of a locus characterized by frequent reversion to the wild type or to a new, intermediate condition in somatic and/or germinal cells. Likewise the term “mutability” most commonly was used to designate the capacity of an allele to revert to the wildtype condition. Since the phenotypes of plants with mutable alleles are usually variegated, these alleles were given such names as variegata, mutabilis, marmorata, maculata, variabilis, and recurrens. McClintock (1950a) proposed that discrete, transposable genetic elements are responsible for some cases of variegation in maize. She called them “controlling elements” (McClintock 1956a,b) since they seemed capable of influencing the expression of a given locus when integrated in or next to it. Later on, when elements of the same nature were discovered in bacteria, they were classified in two categories according to their size and complexity. The term “insertion sequence” (insertion element, IS element) was assigned to short elements up to about 2000 base pairs (bp) in length while larger, more complex elements containing additional genes unrelated to insertion function were termed “transposons” (Campbell et al., 1977). We have chosen to use the term “transposable element” rather than “controlling element” to avoid confusion with the terminology of other fields of genetic research. Briefly we shall define a transposable element as a discrete segment of DNA that is capable of changing its position in the genome. However, it must be pointed out that the criteria for deciding whether a particular segment of DNA is transposable vary depending upon the experimental methods used. As discussed in Section 111, data from molecular research have provided new criteria beyond those obtained by classical genetic methods. Thus DNA sequence analyses have shown that transposable elements may share certain structural features that might be useful for classification. Furthermore, transposition can now be demonstrated by DNA hybridization techniques that were unknown 20 years ago (see Section 111,D). In general, a transposable element is characterized by its ability to

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integrate into the DNA at some position in the genome and thereby alter the expression of the gene(s) in and around itself. In some instances integration of the element is manifested phenotypically as a mutation. If, for instance, a gene involved in anthocyanin synthesis is affected, integration of an element may result in flowers that lack red/purple anthocyanin pigmentation and are basically yellow or colorless. However, mutations induced by transposable elements are often unstable. In some cells of the mutant plant, for example, the element may be excised from its former position at a given locus. In these cells expression of the locus is then restored. Thus in the case of the anthocyanin gene mentioned above, groups of cells with normal pigmentation appear against a colorless background, resulting in a typical variegated pattern. But although frequent excision events may be the basis for the “mutability” of some alleles, transposable elements can generate genetic instability in other ways. Rearrangements of DNA sequences including deletions, duplications, inversions, and translocations may occur within the element itself or in its vicinity and thus elicit alterations in gene expression and phenotype (McClintock, 1951a, 1956b, 1965a). In some cases chromosome breaks or alterations in heterochromatin structure may also occur adjacent to transposable elements and lead to phenotypic changes (McClintock, 1946, 1947). Therefore we shall extend the term “mutability” to include all genetic alterations induced by transposable elements. Before delving into the intricacies of variegation and transposable elements it is worthwhile briefly considering why this subject has gained such attention in recent years. First of all there is a growing feeling that transposable elements may be important in evolution. The innumerable different levels of expression and control that a transposable element can evoke at a locus reflect an enormous potential of these elements for inducing variability. The rearrangements that occur in connection with transposable elements may be instrumental in restructuring the genome in the course of evolution. Besides evolutionary significance, a possible role in development has also been postulated for transposable elements. Here it is the sensitivity of some elements to changes in their physical and physiological environment, manifested as altered variegation patterns (McClintock, 1965a, 1971; Peterson, 1965b, 1966; Fowler and Peterson, 1978), that suggest that these elements may normally be involved in developmental control. Finally some investigators foresee eventually being able to exploit transposable elements for genetic engineering purposes, particularly in monocotyledons. Two very thorough reviews of maize transposable elements have already been published (Fincham and Sastry, 1974; Fedoroff, 1983). In this

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article we shall present a more basic approach to plant transposable elements and include information on mutable alleles of other plants. In the sections that follow we shall outline the classical and molecular genetic evidence available on transposable elements in plants and try to relate the two. We shall also advance speculations concerning mechanisms underlying the activities of transposable elements in hopes of stimulating a general discussion.

B . EXAMPLES OF VARIEGATION EXCLUDED FROM DETAILED CONSIDERATION

Some of the most common cases of variegation involve no heritable changes in the different tissues of the organism but are due to the direct effects of an external factor operating at random on the organism. The most common example of this kind is variegation caused by virus infection, which often leads to yellow or white blotches on leaves infected by the virus. In these cases the external agent directly destroys tissue in certain parts of the plant and the variegation pattern persists only as long as the virus is active in the organism. They must be clearly distinguished from cases in which viral infection induces mutations secondarily, sometimes even unstable mutations, that persist after the viral agent has disappeared, as discussed in Section VI. Another example of nonhereditary variegation is that caused by mineral deficiencies. Normal pigmentation can be restored when the deficiency is corrected. More interesting than mosaic patterns due to random effects of an environmental factor are variegation patterns that are heritable. Since plants contain three different genomes, in the chloroplast, mitochondrion, and in the nucleus, mutations can in principle occur in any one and lead to a variegated phenotype. Since we are limiting our article to mutations of the nuclear genome, let us first consider how chloroplast or mitochondria1 mutations can be distinguished from nuclear ones. Kirk and Tilney-Bassett (1978) have compiled and classified many examples of greedwhite variegation patterns that all arise from chloroplast defects in some plant cells. Some of these are the consequence of nuclear mutations while others are due to mutations of the chloroplast genome itself. Plastid mutations can be identified on the basis of the following criteria (Kirk and Tilney-Basset, 1978): (1) Segregation of the trait in crosses is non-Mendelian. (2) Inheritance is maternal, as demonstrated by nonreciprocity in crosses. (3) Cells containing both defective and nonde-

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fective chloroplasts (mixed cells) can be observed. (4) The distribution of colorless cells corresponds to what is expected by random sorting out of chloroplasts during cell division. Essentially the same criteria apply to mitochondrial mutations, although mixed cells and sorting out cannot be observed directly as with chloroplasts. Our decision to exclude cases of variegation due to chloroplast and mitochondrial mutations is a purely economical one, and we are aware that mutable alleles due to transposable elements very likely also occur in the genomes of these organelles. Indeed, some cases have been reported that resemble nuclear activation of an unstable chloroplast allele (Potrykus, 1970; Sears, 1984; other cases are summarized in Kirk and TilneyBassett, 1978). Plasmid-like DNAs with structures resembling transposable elements (see Section 111) have been found in mitochondria of S cytoplasmic male-sterile maize (Pring et al., 1977; Levings and Pring, 1980; Kim et al., 1982; Weissinger et al., 1982; Timothy et al., 1983). Reversion of male sterility in maize has been attributed to instability of mitochondrial DNA, possibly associated with transposition of such plasmids (Levings et al., 1980; Kemble et al., 1983). The most obvious indication that a mutation of the nuclear genome is involved in inducing a variegated phenotype is Mendelian segregation of this trait. However, not all mutations induced by transposable elements exhibit strict Mendelian segregation ratios (see Section II,C), and not every mutation that causes a variegated phenotype and segregates in Mendelian fashion is due to a transposable element. As indicated by Kirk and Tilney-Bassett (1978), there are many cases of greedwhite variegated mutants that exhibit Mendelian segregation ratios but cannot be unequivocally attributed to the presence of a transposable element. In these cases the mutated gene is apparently one required for normal chloroplast development, but its protein product may be labile and become inactivated in response to some physiological or environmental factor. Alternatively the mutation may be a non-transposon-induced one that causes expression of the gene to be inhibited in certain parts of the plant. Two aspects distinguish these cases from most mutants caused by transposable elements. First, the phenotypic switch observed here is from the wild-type state (green) to the mutant condition (white). Second, these mutants differ in breeding behavior compared to those harboring transposable elements (see Section 11,C-F); they breed true and rarely or never throw off genetically stable white or green progeny or other unusual offspring. This suggests that the tissue of the variegated plants is uniform in genotype, as has been verified in a number of cases summarized in Kirk and Tilney-Bassett ( 1978).

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109

11. RECOGNIZING MUTATIONS DUE TO TRANSPOSABLE ELEMENTS BY CLASSICAL GENETIC MEANS A . REPORTS OF MUTABLE ALLELES

Mutations attributable to transposable elements were analyzed as early as the middle of the nineteenth century (Darwin, 1868). In Tables I, 11, and I11 we have compiled detailed lists of such mutations. Many examples may have been overlooked, especially those cited in languages other than French, German, or English or published in journals with limited circulation. We have included only examples of mutable alleles in higher plants, omitting a few cases known in algae, ferns, and fungi (Anderson, 1923; Andersson-Kotto, 1930; Bgrresen and Fjeld, 1977; Clutterbuck, 1970). As mentioned earlier, only mutable alleles of the nucleus are considered. We have included only examples for which some genetic data are available, although there are certainly many more candidates including several under investigation at the John Innes Institute in Norwich (B. J. Harrison and R. Carpenter, personal communication). Since it is tempting to anticipate a transposable element behind many different kinds of genetic anomaly, we have tried to establish a set of criteria for identifying transposable element-induced mutations in the nucleus. These will be elucidated in Section I1,B. B. SEGREGATION RATIOS A N D WHAT CAN BE DEDUCED FROM THEM

In order to demonstrate that a variegated phenotype is due to a mutation of the nuclear genome, segregation of the variegated trait must be analyzed in crosses. In the most common case, integration of a transposable element at a locus causes a mutation that is recessive to wild type and exhibits monoallelic segregation, as described below. A hybrid carrying a dominant wild-type gene involved in anthocyanin synthesis in one homolog and a transposable element inserted at the same locus in the chromosome homolog will have fully colored flowers. The progeny of this hybrid will include wild-type and variegated plants in a ratio of 3 : 1 . When the same mutable allele is combined with a stable recessive allele in a heterozygote, the variegated phenotype of the mutable allele will prevail since it is formally dominant to the stable recessive one. In the following generation, after selfing, the hybrids segregate three variegated plants to each stable recessive one. In backcrosses of the hybrid with a homozygous stable recessive tester strain, 50% of the progeny will be variegated and 50% stable recessive. An example of F2 progeny showing monoallelic segregation of a mutable allele is outlined in Fig. 1 .

TABLE I Examples of Mutable Alleles in Plants“

Plant Aquilegia vulgaris L. (common columbine) Avena sativa L. (common oat) Bullota nigru L. (black horehound) Brassica campestris L. (field cabbage)

Z

Mutable alleleb variegata yellow-striped variegata purple variegated War)

Callistephus chinensis (China aster, Queen Marguerite)

Capsicum annum (red pepper)

variegata

Celosia cristata L. (cock’s comb, woolflower)

yellow mutable (a) apm

Chrysanthemum sinense (chrysanthemum)

mutable magenta

Phenotype Yellow-green leaves with a few large green spots Leaves have yellow and green stripes Yellow-white leaves with many green areas of variable size Foliage, stem, and seed pods contain patches of purple or pinkish-white which may bleach to white White petals among yellow ones; pink petals among salmon-colored ones ; bright blue petals among light blue ones; some flowers also spotted in same colors Green leaves with whitish or white patches of variable size; green “ticks” on white patches also observed Flowers have yellow background with red sectors Flowers have a light pink-dark pink variegation Flowers variegated with white and intense magenta

Germinal reversions

+ + + + +

New alleles (S,I,V)c

Modifier‘

References‘ Baur (1911, 1912) Christie (1922)

(S)V

S

SJ,V

S

+

Kirk and Tilney-Bassett (1978) Orakwue and Crowder (1983)

Hondelmann (1959)

Imai (1936, 1938)

Terasawa (1922)*; Kihara (1979) Kihara (1979)

S

Miyake and Imai (1934-1935)

Dahlia variabilis Desf. (dahlia) Delphinium ajacis L. (rocket larkspur)

rose-alpha lavender-alpha uariegated pink (P*)

mottling (m)

--

Elymus junceus (Russian wild ryegrass)

U

Epilobium paruijorum

marmorata

Fagopyrum sagittatum (buckwheat) Glycine max (soybean)

C"

Hosta minor

dotted (dr)

Impatiens balsamina L (impatiens, garden balsam)

pm

Y ,grn

Stems and ray florets have a mosaic distribution of anthocyanin pigmentation Flower has dark purple spots on a rose background Flower has small purple spots on a lavender background Flowers have blue spots, streaks, and sectors on a pink background Flowers have pink sepals with rose markings which may be distinct streaks and sectors White leaves have green stripes when the independently segregating factor, G , is present White leaves with many green areas of variable size

+

Yellow sectors of variable size on a green background Yellow sectors of variable size on a green background (pale green sectors also observed)

+

Leaves have green spots on a white background Flowers are stable white; if the independent controlling element, M, is present, pale and dark sectors of anthocyanin color appear

+

+

Lawrence (1931); R. Carpenter (personal communication) Demerec (1931)

+

Demerec (1931)

+

Dawson (1955)

-

Dawson (1955)

+

+

Michaelis (1957)*; Kirk and Tilney-Bassett ( 1978) Ah-Khan (1971)

s.v

(+)

+

Lawrence (1974)

s,v

+

Peterson and Weber (1969); Sheridan and Palmer (1977); Kirk and Tilney-Bassett (1978) Vaughn and Wilson (1980) Sastry et a / . (1980)

(continued)

TABLE I (Continued)

Plant Lathyrus odoratus L. (sweetpea) Malva parviJora

Mirabilis jalapa L. (four-o'clock flower, marvel of Peru)

Mutable alleleb

Ci laciniata

chlorina (chf)

L

t4

P

RU

Nigella damascena L. (love in a mist, devil in a bush)

striata L

Phenotype White flower with purple flakes of variable size Leaf form changes from laciniata (narrow leaf) to ''hypernormat' ' Homogeneous yellowish-green leaves unless the unlinked V, regulatory element is present. If V is present, green flecks and sectors of variable size occur. Homogeneous white flower becomes variegated with yellow sectors, stripes, and spots in presence of the unlinked regulator, V (also assuming rr) Homogeneous yellow flowers (if dominant Y allele is present) become variegated with red sectors, stripes, and spots when the unlinked regulator V is present Striped flowers Dark blue flowers with large, light blue sectors; flowers with varying shades of blue from pale to dark blue on one plant

Germinal reversions

New alleles (S,I,V)c

Modifier'

References'

+

Punnett (1922, 1932, 1935) Lilienfeld (1929)*; Kihara (1979)

+

Correns (1909, 1910); Delool and Tilney-Bassett (1984)

+

+

Engels et al. (1975); Spitters et al. (1975)

+

+

Engels et al. (1975); Spitters et al. (1975)

(+I

+

+ +

V

I

Correns (1908, 1909) ToxopCus (1928)

Nicotiana tabacum L. (tobacco)

v(s1)

v(Ds1)

Oryza sativa L. (rice)

white striped

Pelargonium zonale (horse-shoe geranium)

variegata

Petunia hybrida (garden petunia)

anlsii

anl+‘*

“Speckled” flower [v(sl) + homozygous]: scattered, small clusters of anthocyanin pigmentation on a uniformly colorless background “Sectorial” flower [ v ( S l ) / v ( s l ) ] : frequent, clearly defined sectors of speckled tissue on a solidly pigmented background “DsLsS” flower [v(Dsl)lv(sl)]: similar to “sectorial” except that frequent sectors of light speckling are on a dark speckled background White plant with large green areas when the modifier is present

+

V

+

Smith and Sand (1957); Sand (1969, 1976)

+

V

+

Smith and Sand (1957); Sand (1969, 1976)

-

V

+

Smith and Sand (1957); Sand (1969, 1976)

+

S

+

Mitra (1932)*;Mitra and Ganguli (1934)*; Kirk and Tilney-Bassett (1978) Imai (1936)

+

SJ,V

+

White-dark red spots on pale colored flowers

+

S,LV

Red and pink spots on white flowers

t

S,I

Bianchi et a!. (1978); Mulder et al. (1981); Gerats et al. (1982, 1983) Gerats et al. (1984a); A. G . M. Gerats (personal communication) Bianchi et al. (1978); Doodeman et al. (1984a)

Green leaves with white patches; green “ticks” on white patches also observed White flowers with red spots

(+)

+

(continued)

TABLE I (Continued)

Plant Petunia hybrida (cont.)

Mutable alleleb anll"+

White flowers with red spots

+

ad-1

White, intermediate, and red spots on pale-colored flowers Red spots on white flowers, some white sectors Red spots on white flowers, some white sectors White, intermediate, and red spots on white background Red spots on pale background, some white sectors Yellow-green leaves with green spots Branches with normal leaves on mutant plants Pale flowers with red-gray spots

+ + + + + +

an2-14 an2-16 rt-3 rt-38 Yg3' alp ad"

dw5 p1-m

Pharbitis nil (Japanese morning glory)

Phenotype

Germinal reversions

willow leaf ( m ' )

Dwarf plants White flowers with purple stripes, leaves striped and slightly corrugated Two kinds of variegation depending on modifier: green leaves with white edges or white leaves with green spots Willow leaf is a slender, three-lobed leaf that rarely but consistently gives rise to a maple leaf bud sport

New alleles (S,I,V)'

Referencesc Doodeman et al. (1984b) Cornu (1977); Farcy and Cornu (1979) Cornu (1977)

SJ

Cornu (1977)

SJ

Cornu (1977)

s.1

Cornu (1977)

+

V

+

SJ s.1

+

Modifier'

Doodeman et al. (1984a) Doodeman et al. (1984b) A. G . M. Gerats (personal communication) Bianchi ef al. (1974)* Maizonnier and Cornu (1971)

+

Potrykus (1970)

Imai (1926)

Yellow-green leaves with a few large green areas

+

yellow-inconstant (Y i )

Yellow leaves with a few green areas of variable size

+

xanthic (x)

Yellow leaves with a few green areas

Pecked (p)

White flower with anthocyanin flecks of variable size Flowers have normal colored flecks or parts on a "duskish" or dilute colored background Flowers have anthocyanin spots on a yellowish or white background Yellow leaves with green areas of variable size

duskish (dk)

speckled (sp) Phaseolus vulgaris L. (French bean, green bean) Pisum sativum L. (garden pea) Portulaca grundiJlora (rose-moss, sun plant)

yellow mutable (us)

(S)V

+

S

+

+

A2

White flowers with purple spots

+

inconstant creumish

Creamish colored flower with yellow stripes; may contain orange stripes superimposed on the yellow

+

s,v

Imai (1934)*; Miyake and Imai (1934)*; Kirk and Tilney-Bassett (1978) Imai (1926,* 1927, 1930, 1934)*; Miyazawa (1929*, 1932)*; Miyake and Imai (1934)*; Kirk and Tilney-Bassett (1978) Miyake and Imai (1934)*; Kirk and Tilney-Bassett (1978) Imai (1931)

+

Imai (1931, 1934*, 1934- 1935)

+

Imai (1921, 1931)

+

Coyne (1966, 1967, 1969); Kirk and They-Bassett (1978) De Haan (1930) Ikeno (1929)*; Imai and Kanna (1935)

(continued)

TABLE I (Continued)

Plant

Mutable alleleb

Phenotype

recessive j a k e (e)

Magenta-colored spots and streaks of various sizes on the petals, stems, and leaves Dwarf plants with some normal branches White flower with magenta flakes

Portulaca grandifora (cont.)

Primula sinensis

--

Sorghum bicolor (chocolate corn) Verbena hybrida (common garden verbena)

Variegated seed coat of fine red stripes on a white background White flower with purple spots

Germinal reversions

New alleles (S,I,Vp

Modifield

References‘

+

Ikeno (1929)*; Fabergt and Beale (1942)

+ +

Blakeslee (1920) De Winton and Haldane (1935); Harrison and Fincham (1964) McWhirter (1973)

+ -

I,V

Eyster (1928)

a We have assembled a list of plants where unstable nuclear genes are thought to be involved in producing a variegated phenotype. Mutable alleles of Antirrhinum majus and Zea mays are not included, but are given in Tables I1 and 111, respectively. Many examples listed are discussed in Kirk and They-Bassett (1978). Other examples of plant variegation are mentioned in the literature, but there are insufficient published data to determine the nature of the instability (see for instance the 1930 review by De Haan). These are not included in the table. Imai (1936) lists many plants having a chlorophyll variegation pattern-some of which may be due to unstable nuclear mutations. However, due to the difficulty of analyzing greedwhite variegation patterns (see text and Kirk and Tilney-Bassett, 1978) only a few of these plants are listed in this table. Most forms of variegation listed involve a recessive to dominant phenotypic change. Only four cases of green + white (yellow) variegation are listed. These cases of “reverse variegation” are given as examples and are more dimcult to explain in terms of transposable elements (see Section 11,D). Note that examples of “rogues” and “eversporting” lines have not been included in thk table. Cases where variegation seems to be associated with chromosomal anomalies (see text) have also been excluded. Although only flower color variegation may be mentioned, other plant parts may also show a variegated pigmentation. Derivatives of the original unstable mutable allele are denoted by S (stable recessive allele), I (stable allele showing an intermediate phenotype), and V (new variegated forms). The presence of a modifier is indicated by +. The modifier may be necessary for variegation or may only alter the pattern of variegation (see text). References with a * indicate that the reference was not available to us.

TABLE I1 Mutable Alleles of Anthirrhinum majusa Reversionb Mutable allele albostriata (stri)

bicolor (bic)

--

Phenotype

S

G

Allelic derivatives

Green spots on white leaves; some normal green leaves Large green sectors on yellow-white leaves

+

+

Forms with different degrees of spotting

+

+

Interaction with other mutable alleles and/or modifiersc. Codominance of alleles observed in homozygotes

Yellow-green leaves

-

+

cupuliformis (cup)

Plants with cup-shaped leaves and some shoots with normal leaves Symmetrical, radial

+

+

-

+

Forms with flowers with varying degrees of radial symmetry

-

+

Forms with varying degrees of altered petals and anthers

4

dejiciens-globifera (def-gli) delilah-recurrens (del-rec)

flowers Flower petals reduced to sepals; male tissue missing Tube of corolla basically colorless with purple-red streaks

+

Baur (1910, 1924); Stubbe (1933, 1966) Kuckuck and Schick (1930); Schick (1933); Stubbe (1933, 1966) Schick and Stubbe (1932); Schick (1933, 1935); Stubbe (1933; 1966); Baur (1932) Stubbe (1933; 1966)

chamaeleon (cham)

cycloidea-radialis recurrens (cyc-rad-rec)

References

No interaction with niu-rec and pal-rec

Isolated by Baur and Stubbe, cited in Kuckuck (1936); Stubbe (1966) Baur (1918, 1924); Hertwig (1926); Stubbe (1966) Harrison (1965, 1971)

(continued)

TABLE I1 (Continued) Reversionb Mutable allele depauperatamutabilis (dep-mut) discolor (dis)

-m

eluta-recurrens (el-rec)

flavostriata Mast)

galbana (ga) gilvostriata (gi)

graminifoliamutabilis (gram-mut)

Phenotype

S

G

Small, pointed undulated leaves; fewer smaller flowers Leaves with green spots on white background; blisters on some leaves due to unequal growth; plants with some normal leaves el'lel+ flowers redd; el-lel- flowers ivory with pale red flush; el-rec flowers red with ivory spots Leaves with yellow background and green spots

-

+

+

+

+

AUelic derivatives

+

Yellow-green leaves with green sectors Cream-colored leaves with light green spots

+

+

+

+

Very narrow leaves, sepals, and petals

-

+

References Stubbe (1974)

Schick and Stubbe (1932); Kutscher (1935); Schick (1935); Stubbe (1936, 1966) Derivatives with different numbers of spots

+

Interaction with other mutable alleles a n d o r modifiers'

No dosage effects; effects of different el-rec alleles additive

Doring (1941); Stubbe (1966)

Kuckuck and Schick (1930); Stubbe (1933, 1966); Bergfeld (1958) Stubbe (1974) Derivatives with varying degrees of variegation Derivatives that exhibit different degrees of germinal reversion

Scherz (1927); Stubbe (1933, 1966) Mechelke and Stubbe (1954); Stubbe (1966)

illustris (ill)

incolorata 2recurrens (inc2)

-

incolorata recurrens (inc)

\o

lucida (luc)

microfolia (mic)

mutabilisrecurrens (mut-rec)

Young plants with narrow, upright leaves; less branching, yellow spot on lower lip enlarged and surrounded by red Mottled leaves, red seedlings

Flowers with red spots and streaks on ivory background Young plants with lighter colored leaves; smaller flowers; less intensive pigmentation on lower lip Undulated cotyledons; smaller leaves; fewer flowers per inflorescence; growth of main stem inhibited White flowers with red stripes and streaks

Stubbe (1941, 1966)

+

t

+

+

-

+

-

t

+

+

N o dosage effect of allele in homozygous state; codominance observed

Linnert (1972, 1978)

Isolated by Stubbe, cited in Kuckuck (1936); Stubbe (1966) Schick and Stubbe (1932); Stubbe (1966)

Stubbe (1941, 1966)

Derivatives with different frequencies of germinal reversion

Stubbe (1933, 1966); Kuckuck (1936)

(continued )

TABLE I1 (Continued) Reversionb Mutable allele nitens-mutabilis (nit-mut) nivea-recurrens (niv-rec)

Phenotype

S

G

Allelic derivatives

Longer and lighter colored hypocotyl; lighter colored leaves Flowers with red spots and stripes on white background

-

+

+

+

Derivatives with different hypocotyl lengths Forms with stable white and stable intermediate flowers; others with altered patterns of variegation Forms with stable white and stable intermediate flowers; others with altered variegation patterns

pallida-recurrens (pal-rec)

Flowers with magenta flakes and stripes on ivory background

+

+

perlutea-recurrens (per1-rec)

Pale yellow flowers with red spots

+

+

Interaction with other mutable alleles andor modifiersc

References Stubbe (1966)

“Stabilizer” of mutability

“Stabilizer” J, K eosinea

Allele shows no dosage effects in homozygous state

Isolated by Stubbe, cited in Kuckuck (1936); Hamson and Carpenter (1973); Harrison (unpublished results) Baur (191 1); Mather (1947); Harrison and Fincham (1964); Fincham and Hamson (1967); Harrison and Fincham (1968); Harrison and Carpenter (1973); Jeffries and Sastry (1981, 1982); Sastry et al. (1980) Stubbe (1940, 1966); Doring (1941)

perlutea-mutabilis (perl-mut) perluteavariabilis (perl-uar)

punctata (punc)

-!2

retusa (rea)

spectabilis (spec)

+ Yellow flowers with red spots; flowers with narrower lips; leaves rolled inward; reduced growth and viability Some leaves needle-like, others normal Pale-colored flowers with pale red spots on the lower lip Leaves lighter colored and wavy; upper lip extended with pink flush; major yellow and red spots on lower lip with reduced pigmentation Young plants with lighter colored leaves; width and length of main stem reduced; length of branches reduced

+ (+Y

+

Forms with flowers with varying degrees of spotting and deformation and with leaves with different degrees of rolling

Stabilizer that prevents germinal reversion; factor that enhances germinal reversion

Stubbe (1966); Doring (1941) Stubbe et a / . (1981); Meyer ef al. (1981, 1982)

Baur (1926)

+

+

Stubbe (1941, 1%)

-

+

Stubbe (1%6)

-

+

Stubbe (1966)

(continued)

TABLE I1 (Continued) Reversionb Mutable allele striata (str)

tonsa (ton)

-

uaria (va)

N

variicolor (vac) victrir (vic)

Phenotype

S

Smaller plants; smaller flowers with colorless sectors on lower lip Smaller plants; leaves dented small flowers with extremely short lips Light yellow to yellow-green leaves with pale spots Pink flowers with colorless sectors Upper and lower lips of flowers red

+

Stubbe (1966)

-

Stubbe (1933, 1966)

+

Stubbe (1941, 1966)

+

Stubbe (1966)

-

G

Interaction with other mutable alleles and/or modifiers‘

ALlelic derivatives

References

Stubbe (1933, 1941, 1966); Doring (1937)

a Information on many of the mutable alleles described here is derived from Stubbe (1966). and more complete descriptions of the phenotypes can be found in this source. The capital letter “S” stands for somatic reversion and “G” for germinal reversion. Where “codominance” or “no dosage effects” are indicated, the effect of the mutable allele has been shown to be additive when two copies of it are present in homozygotes. Note that in a number of cases no somatic variegation but frequent reversion events in germinal tissue have been observed. There is some discrepancy concerning the designation of mutable and wild-type alleles of this locus. The terminology used here is according to Stubbe (1966). Parentheses indicate that germinal reversion occurs only in the presence of an appropriate modifier.

TABLE 111 Mutable Alleles in Maize“ Chromosome 1 P-RR

Pericarp and cob color are red, brown, or orange Somatic instability is usually expressed as colored pencarp sectors on a colorless background Mutable alleleb p-VV = p-rr-Mp (“variegated’ ’)

-

N

Elementc

Emerson (1914, 1917, 1922, 1929); Eyster (1924, 1925, 1926); Anderson (1924); Brink and Nilan (1952); Barclay and Brink (1954); Brink (1954, 1958); Brink and Barclay (1954); Fradkin and Brink (1956); McClintock (1956a); Wood and Brink (1956); Kedhamath and Brink (1958); Van Schaik and Brink (1959); Greenblatt and Brink (1962, 1963); Orton and Brink (1966); Greenblatt (1%6, 1968, 1974, 1984) Hayes (1917); Anderson (1924); Barclay and Brink (1954) Barclay and Brink (1954)

MP

p-MM (“mosaic”) Q36 (“striped”)

bz2

W

Bronze-2: Bronze aleurone and plant Somatic instability expressed as bronze aleurone or plant exhibiting purple or red sectors

Mutable allele

Chromosome 3 a

Referencesd

Element

bd-m

Ds

bd-S

Ds

References McClintock (1956a); Osterman and Schwarz (1981); Rhoades and Dempsey (1982, 1983) Bianchi et a / . (1969)

Anthocyaninless: absence of anthocyanin pigments in the plant and aleurone, and brown pericarp with P-rr Somatic instability is usually expressed as colored sectors on a pale or colorless background

Mutable allele

Element

a-ml

SPm

a-m2

SPm

References McClintock (1951a,b, 1952, 1953b, 1954, 1955, 1956a-c, 1957, 1958, 1967a, 1968); Peterson (1965, 1966, 1968); Neuffer (1966) McClintock (1951b, 1952, 1953b, 1954, 1956a, 1961b, 1963, 1964, 1965b, 1967a, 1968); Gorman and Peterson (1978) (continued)

TABLE I11 (Continued) Element‘

Mutable alleleb a-m3 a-m4 a-m5 a-standard = a-dt

Ds Ds SPm rDt

a-m = am-1:Cache a-m(dense) = a l - m

rDt En

a-rug a-var a-mrh ap-m a-px Two unstable a alleles

rug En. rMrh (Nonautonomous) (Autonomous)

References” McClintock (1951b, 1952, 1956b, 1964, 1965b, 1978a); Neuffer (1%5) McClintock (1952, 1953a); Neuffer (1965) McClintock (1961b, 1962, 1964) Emerson (1932); Rhoades (1936, 1938, 1941, 1945); McClintock (1951a, 1956b); Peterson (1965, 1966); Doerschug (1976) Nuffer (1955, 1961, 1965, 1966); McClintock (1951a); Doerschug (1976) Peterson (1957, 1961, 1965, 1966, 1970b, 1978b, 1980); Reddy and Peterson (1977); Nowick and Peterson (1981) Friedemam and Peterson (1980, 1982); Peterson and Friedemam (1983) Peterson (1980) Rhoades and Dempsey (1982, 1983) Nuffer (1956, 1962) Nuffer (1962) Langhnan and Rhoades, cited in McClintock (1951a)

Shrunken-2: Kernels are large, sweet, and watery at milk stage and collapse to a shrunken form upon drying. Shrunken-2 may be the structural gene for ADP-glucose pyrophosphorylase (Tuschall and Hannah, 1982) Somatic instability is expressed as nonshrunken patches on an otherwise shmnken kernel Mutable allele sh.2-ml Chromosome 4 Tu c2

Element

References Hannah and Nelson (1976); Hannah et a / . (1980); Tuschall and Hannah (1982)

Ds

Tunicate ear: Long glumes on ear and tassel Instability at the Tu locus was observed by Mangelsdorf in 1948 (cited in McClintock, 1951a) Colorless aleurone: Little or no anthocyanin color in aleurone, reduced plant color Somatic instability is usually expressed as colored sectors on a pale or colorless background Mutable allele c2-ml c2-m2

Element SPm rSpm

c2-m3

c-2

MP

References McClintock (1964, 1967b) McClintock (1964, 1967b, 1978a) B. McClintock (personal communication); Wienand et al. (1982) I. M. Greenblatt (personal communication)

CL-ldf

c2-m4490 c2-rn826019 c2-m82602I c2-m826040 c2-rn826134 c2-m826204 Chromosome 5 a2

Anthocyaninless: Absence of anthocyanin pigments in the plant and aleurone, and red pericarp with P-rr Somatic instability is usually expressed as colored sectors on a pale or colorless background

Mutable allele'

bt

Element

a2-mI

rSpm

a2-rn2 a2-m3 a2-m4 a2-ms a2-mI I511

(Not Ac-Ds) (Not Ac-Ds) Ds rSpm En

a2-m4 1596 a2-rn4 1629 a2-m6 8140 a2-m6 8144 a2-rn7 8018

En En En En En

References McClintock (1948, 1951a, 1952, 1957, 1958, 1971); Reddy and Peterson (1977, 1978) McClintock (1952, 1953a) McClintock (1952, 1953a) McClintock (1952, 1953a) McClintock (1964) Peterson (1963, 1968, 1974, 1978a); Fowler and Peterson (1974, 1978); Reddy and Peterson (1976, 1977, 1978) Peterson (1978a) Peterson (1974, 1976a,b, 1978a) Reddy and Peterson (1976); Peterson (1978a) Reddy and Peterson (1976); Peterson (1978a) Peterson (1976a, 1978a);Reddy and Peterson (1976)

Brittle endosperm: The mature kernel is collapsed and often translucent and brittle Instability is expressed as kernels mosaic for bt and Bt (blistered appearance)

Mutable allele bt-m Unstable bt Pr

Greenblatt (1965, 1975) J. L. Kermicle (personal communication) Peterson (1983) Peterson (1983) Peterson (1983) Peterson (1983) Peterson (1983)

Idj Ds

Element rSpm-P

References R. L. Phillips (personal communication) Rhoades, cited in McClintock (1951a)

Red aleurone: The mutant allele (pr) changes purple aleurone color to red and may also change purple anther color to red Instability is expressed as various grades of anthocyanin intensity varying from red to purple (continued)

TABLE I11 (Continued) Mutable allele* pr-ml pr-m2 pr-m3

EIementc rSpm rSpm

Referencesd McClintock (1951b) McClintock (1965a) McClintock (1965a)

Chromosome 6

Y

Yellow endosperm: Carotinoid pigments are present in the endosperm Instability is expressed as white endosperm with yellow sectors

Mutable allele

Element

Robertson, cited in Neuffer er a / . (1968) McClintock (195la,b)

white mutable (w-m) y mutable

5 m

Chromosome 7 02

References

Opaque-2: In 02 kernels the endosperm is soft, floury, and dull in reflected light and opaque to transmitted light Instability is expressed as sectors of flint tissue on an opaque background in seed endosperm

Mutable allele 02-m(r) 02-h 02-col (=02-COL UMBIAN) 02-AGROCERES 02-ch (= 02-CHARENTES) 02-261 02-mh

Element

References

rBg rB2h rSd

Salamini (1980, 1981); Salamini et a / . (1982); Montanelli et a / . (1984) Salamini (1981) Salamini (1981); Salamini et a / . (1982); Montanelli et al. (1984)

rSd rBg

Salamini (1981); Montanelli er a / . (1984) Salamini et a / . (1982) Montanelli et a/. (1984) Montanelli et a/. (1984)

Glossy: The wild-type allele ( C / )sustains the synthesis of epicuticular waxes in the maize seedling (Bianchi et a/., 1977). The g / seedling is bright green with water spray adhering in fine drops Instability of 12 g / alleles was noted due to a high reversion frequency (0.17 x to GI (Salamini and Borghi, 1%). The reason for this instability is unknown

Chromosome 9 c

Aleurone color: The wild-type allele ( C ) produces anthocyanin color in the aleurone while c is colorless. C' is a dominant colorless allele Instability is expressed as colored aleurone sectors on a pale or colorless background Mutable allele<

c-ml c-m2 C-ml

c-m4 c-m5

C'-mutable c-standard c-ml I702 c-m4 1963 c-m5 5292 ~-m5 5301 c-m5 5320 C-m.5 5351 c-m5 5453 c-m6 8613 c-m6 8655 sh

Element

Ds Ds (Not Ac-Ds) Ds SPm En En

En En En

En En

En En

References McClintock (1948, 1949, 1950a. 1951a.b, 1952, 1953a) McClintock (1948, 1950a, 1951a, 1952, 1953a) McClintock (1952, 1953a) McClintock (1952, 1953a, 1964) McClintock (1963, 1978a) McClintock (1951a.b) McClintock (1951a) Peterson (1963, 1978a); Gorman and Peterson (1978) Peterson (1976, 1978a) Peterson (1976, 1978a); Reddy and Peterson (1983) Peterson (1978a); Reddy and Peterson (1983) Peterson (1976, 1978a) Peterson (1978a); Reddy and Peterson (1983) Peterson (1978a); Reddy and Peterson (1983) Peterson (1978a); Reddy and Peterson (1983) Peterson (1978a); Reddy and Peterson (1983)

Shrunken endosperm: Structural gene for the enzyme sucrose synthase. In s h kernel the endosperm collapses to form an indented crown Instability is expressed as germinal reversions to sh' Mutable allelef

sh-m(5933)

Element

Ds

References McClintock (1952, 1953b); Burr and Burr (1980, 1981a,b, 1982, 1983); Doring et al. (1981, 1983, 1984); Geiser et al. (1982); Fedoroff et a / . (1983a,b); Courage-Tebbe et a/. (1983)

(continued)

TABLE I11 (Continued) Mutable alleleb

sh-m(6233)

Ds

sh-m(6258)

Ds

sh-m(6795)

Ds

sh-5582 sh-5584 sh-5586 sh-5596 m

bz

Elementc

Referencesd McClintock (1952, 1953b); Burr and Burr (1980, 1981a,b, 1983); Doring et a / . (1981, 1984); Geiser et al. (1982); Fedoroff et a / . (1983~);Weck e f al. (1984) McClintock (1952, 19853b); Burr and Burr (1980, 1981a, 1982, 1983); Fedoroff et a / . (1983b) McClintock (1952, 1953); Burr and Burr (1981a,b, 1982); Geiser et a / . (1982); Fedoroff et a / . (1983a) Mottinger et a / . (1984b) Mottinger et al. (1984b) Mottinger et al. (l984b); S. Dellaporta (personal communication) Mottinger et al. (1984b)

Tz86

Bronze: A bz mutation changes a purple aleurone to brownish pale, the plant becomes reddish brown, and the tassel anther will fluoresce under UV light Somatic instability is recognized by a phenotypic bz + Bz change in aleurone and plant tissue Mutable allele

Element

bz-ml bz-m2 (=Acbz-mZ)

Ds Ac

bz-mil bz-m4

(Not Ac-Ds) Ds

bz-mut (=bz-m-rh) bz-ml3 bz-m805137 bz-m826301 bz-m826302

rMut rSpm = Rs

References McClintock (1951a,b, 1952); Dooner and Nelson (1977) McClintock (1951a,b, 1952, 1955, 1956a-c, 1962); Dooner and Nelson (1977); Fedoroff et a / . (1984) McClintock (1952, 1953a) McClintock (1953a, 1956~); Dooner and Nelson (1977); Dooner (1980); Gerats et a / . (1983) Rhoades and Dempsey (1982, 1983) Klein and Nelson (1983) Peterson (1983) Peterson (1983) Peterson (1983)

wx

Waxy endosperm: The wild-type allele wx+ determines the amylase content of endosperm tissue and pollen (Sprague et al., ( 1943) Somatic instability is observed by 12-KI treatment of endosperm tissue

Mutable allele wx-ml

Ds

wx-m2 wx-m3

wx-m5 wx-m6

(Not Ac-Ds) (Autonomous) (Not Ac-Ds) (Autonomous) (Not Ac-Ds) Ds Ds

wx-m7

AC

wx-m8

rSpm = Spm-I8

Acwx-m9

AC

WX-BJ WX-B~

AC Ds

wx-m

(Autonomous)

wx-m4 d

W N

Element

References McClintock, 1948; 1950a, 1951a,b, 1952, 1953a); Nelson (1968); Echt and Schwartz (1981); Rhoades and Dempsey (1983) McClintock (1948, 1951a, 1952, 1953a) McClintock (1951a,b, 1952, 1953) McCIintock (1951a. 1952, 1953) McClintock (1952, 1953a, 1964, 1965b) McClintock (1952, 1953a, 1964); Nelson (1968); Echt and Schwartz (1981); Fedoroff et al. (1983b); Shure et al. (1983) McClintock (1953a, 1964, 1965b, 1978a); Peterson (1974); Schwartz-Sommer e f a / . (1984); Behrens et al. (1984) McClintock (1961b, 1978a); Nelson (1968); Echt and Schwartz (1981); Shure et al. (1983); Schwartz-Sommer et al. (1984) McClintock (1963, 1964); Fedoroff et al. (1983b); Shure et al. (1983); Pohlman et al. (1984) Nelson (1968); Echt and Schwartz (1981) Nelson (1976) cited in Echt and Schwartz (1981); Echt and Schwartz (1981) Sager (1951)

TABLE I11 (Continued) Chromosome 10 R’

Aleurone andplant color: Controls the expression of anthocyanin pigment in aleurone and certain plant parts Somatic instability is usually expressed as sectors of anthocyanin pigment on a colorless background

Mutable allele8

Element

R-st (“stippled”)

I-R

mR”J(“R-Navajo”) R-mb (“marbled”)

MP

R-r#2

rFcu and rSpf

r#IO

rFcu and rSpf rFcu

r-cu

R% r-ml r-m3 r-m9

Ds DS

Ds

References Fogel (1950)*;Ashman (1960, 1965); Kermicle (1970, 1973, 1984); Dooner and Kermicle (1971); Williams e r a / . (1984) Williams and Brink (1972); Brink and Williams (1973) Brink (1956); Brink and Mikula (1957); Brink and Weyers (1957); Weyers (1961) Sastry and Kurmi (1970) cited in Gonella and Peterson (1978); Gonella and Peterson (1978) Peterson (1981) Gonella and Peterson (1977, 1978) Dooner and Kermicle (1974) Kermicle (1980) Kermicle ( 1980) Kermicle (1980)

Chromosome location unknown Luteus: Gene associated with chlorophyll production lu Somatic instability is expressed as a yellow seedling variegated for normal green tissue Mutable allele lu-m (“mutable luteus”)

Element Spm? (autonomous)

References McClintock (1946, 1951a, 1961b); Smith (1960)*

Yellow-green chlorophyll: The recessive y g locus mutates to form a darker color chlorophyll

Mutable allele

Element

References McClintock (1948, 1951a) McClintock (1948, 1951a)

Pmp-1817

-W

A dominant mutable allele affected chlorophyll development in the mature plant Instability is expressed as virescent older leaves with dark green stripes. Element at the locus is unknown, but it is not of the E n 4 system (Peterson, 1963, 1964) Variegated white seedlings: White seedling with sectors of pale yellow or of normal green tissue. Element unknown (McClintock , 1946) Variegated light green: Light green seedling with streaks of normal green tissue. Element unknown (McClintock, 1946)

The nomenclature and phenotype given for a particular gene were taken from Neuffer er a / . (1968). The nomenclature of the various mutable alleles may be different from that given in a particular publication-for often even one author uses various designations for the same allele. For example, the mutant a,m-’ (B. McClintock’s nomenclature) has been referred to as a l- m - / or as u-ml in the literature. We chose the latter nomenclature for simplicity. Many of these mutable alleles are discussed in recent review articles on maize transposable elements (Fedoroff, 1983; Doring and Starlinger, 1984). Only the original mutable allele is listed, although many alleles gave rise to both stable and unstable derivatives. Many new unstable alleles are being generated, especially by the mutator system (Mu)described by Robertson (1978). This system induces mutations at a frequency approximately 30 times greater than the usual spontaneous mutation rate for maize, with approximately 35% of the new mutants being unstable (Robertson, 1978). These unstable loci include a, a2, bz, and hundreds of mutable nuclear chloroplast pigment mutants (D. S . Robertson, personal communication). To date, there is not enough information concerning most of these unstable alleles to include them in the table. Orton and Brink (1966) describe 146 independent unstable mutations at the P locus, most of which are due to Mp at the locus. Element refers to the element responsible for the mutable allele in the original unstable mutant. If the regulatory element is listed, the allele is considered to be autonomously controlled. The element listed is designated according to the name given in the publication(s) although several such elements are thought to be functionally equivalent (e.g., Spm = En, Ac = M p ) . The designation of an element at a nonautonomous locus is given by a small r as the first letter. This r stands for “receptor” (e.g., rDr means the receptor element that requires the regulator Dt to be present in the genome in order to produce the instability). Most of the elements are discussed in the text or listed in Table IV. However, a few comments are given as follows: rSpm = I : since Spm and En are functionally equivalent (Peterson, 1965a) then the rSpm (receptor for S p m ) is functionally equivalent to I, the receptor element for the en regulatory element; M u l : An element thought to be associated with a high frequency of mutation (Strommer et al., 1982); E d : barley stripe 1, an element present in a line previously infected with barley stripe mosaic virus; I d j Defective I, where I is the receptor element for the E n 4 two-element system; rSpm-P: A receptor element that responds to the Spm element isolated by R. Phillips; rE2h, rSd, and rBg may be the same receptor element for the two-element Bergamo system ( B g ) (Salamini, 1981); Tz86: Transposon Zea 86. The reference list for some mutable alleles may not be complete but should suffice to initiate an inquiry into the details of that allele. Most references to articles in the Maize Genetics Cooperation News Letter have not been included. Peterson (1978a) lists 72 independently derived En-controlled mutables of the a2 and c loci. Only a few of the better characterized alleles are included in this table. f sh-ml and sh-m2 mutable alleles (both controlled by the Ac-Ds system) are only mentioned in Table 1 of McClintock (1964). It was unclear if these are the same as alleles already listed. mRnj consists of 26 mutable alleles which all carry Mp (Brink and Williams, 1973). Reference was not available to us.

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PATRICIA NEVERS E T A L .

Plant: Antirrhinum majus Alleles: pullida-recurrens pallida-tubocolorata

(red-and-white variegated flowers) (white flowers with ring of red color at base)

FI Genotype: pal-reclpal-tub Phenotype: Variegated flowers

F2 Expected phenotypes: pal-rec Gametes I pal-rec pal-tub

variegated variegated

pal-tub

variegated white

3 variegated : 1 white

Temperature at which F,grown 25°C 15°C

Observed phenotypes: Total numbers of plants scored Variegated 2017 3199

1493 I589

Phenotypes White

Revertant

Others

499 843

55 672

0 96

Fig. 1. Monoallelic segregation of the pallida-recurrens allele of Antirrhinum mujus. The pallida locus is involved in a late step in anthocyanin synthesis (Harrison and Stickland, 1974). The pallida-recurrens allele is thought to be caused by integration of a transposable element at the pallida locus. Frequent somatic reversion of this allele leads to variegated pigmentation in flowers. The pallida-tubocolorata allele is a stable, recessive allele characterized by white flowers with only very little pigmentation at the base of the corolla. Note that in the Ff, revertants and other new phenotypes are observed that are not expected by normal Mendelian genetics. The incidence of such individuals (and the degree of somatic variegation) is higher at 15°C than at 25°C. The number of variegated progeny is accordingly somewhat lower at 15°C. (Data taken from Harrison and Fincham, 1964.)

Sometimes the segregation ratios indicate that not one nuclear gene but two or more are involved in producing the variegated phenotype. One contributing factor is always a mutable allele that frequently reverts to wild type, presumably due to the presence of a transposable element. Regarding the second locus, two different cases can be distinguished: those where the second factor is required for mutability of the first component, and those where the second factor only modulates mutability of the first. In some instances the second factor is known to be a transposable element itself, while in others this is not clear (see Section V). The most prominent examples where a second independently segregating locus is required for mutability of another one are those of the two-

PLANT TRANSPOSABLE ELEMENTS

133

component systems in Zea mays. Here the second factor is a transposable element capable of autonomous integration, excision, transposition, and other types of activities typical of such elements. This component has been termed the “regulator” (McClintock, 1961a) or autonomous component (Fedoroff, 1983). The element it regulates is defective with respect to all of these activities and can perform none of them of its own accord. This defective element can inhibit expression of a locus simply by virtue of insertion, but it is stable unless a suitable regulatory element is present somewhere in the genome. The regulator can activate in trans the defective component, probably by means of some diffusible protein or RNA “signals.” Accordingly the defective component has been termed the “receptor” (Fincham and Sastry, 1974; Peterson, 1965a) or nonautonomous component (Fedoroff, 1983). Data from a cross exhibiting a segregation ratio typical for two-component transposable element systems are shown in Fig. 2. Information concerning the “signals” produced by a regulatory element is derived primarily from classical genetic experiments. One function that seems to be common to all regulators is a “mutator” function required for excision, transposition, and other activities of both the regulator and related receptor components (McClintock, 1954, 1957, 1961b, 1965a; Fedoroff, 1983). The complex regulatory component of the Spm(En) system is believed to encode one or possibly two other activities in addition to the mutator function: a suppressor, required for complete inhibition of expression at a locus under the control of an element from this system (McClintock, 1954), and possibly an activator for maintaining transcription of the element (McClintock, 1971). A model of how these three functions interact was presented by Nevers and Saedler (1977). The interaction between the two components in a two-component system is highly specific, that is, a given receptor component responds to signals of certain regulatory components but not to others (Peterson, 1978a; Peterson, 1980; Friedemann and Peterson, 1982). This specificity has been exploited by Peterson and his co-workers (Peterson, 1980; Friedemann and Peterson, 1982) to classify several two-component systems in maize, summarized in Table IV together with very recent data on two new systems (Rhoades and Dempsey, 1982, 1983). On the basis of specificity of interaction, the independently isolated regulatory elements En and S p m are considered to be functionally equivalent (Peterson, 1965a), likewise Ac and M p (Barclay and Brink, 1954). It should also be noted that regulatory elements that show the same basic specificity of interaction may not be functionally identical. Thus Ac2 can readily transactivate some Ds elements but not others, and Spfvaries in its ability to mobilize certain receptor components (see Table IV).

A. A two-component system Plant: Mirubilis julapu L. Alleles: Y (dominant allele; red flowers) Y“ (white flowers when V is not present in the genome; white flowers with red spots when V is present in the genome) V (regulatory component whose activity is required for mutability of Y “ ) y (regulatory component mutated, inactive, or missing)

F, Genotype: Phenotype:

Gametes

I

YP vu Red flowers

F2 Expected phenotypes: Y, v Y, u P,v red red red red

red red red red

P>u

red red variegated variegated

red red variegated white

12 red : 3 variegated : I white

Observed phenotypes: 38 red : 7 variegated : 3 white (0.3 < p < 0.5)

B. A mutable allele plus an independently segregating modifier Plant: Alleles:

Antirrhinum majus pallida-recurrens (red-and-white variegated flowers) pallida-tubocolorata (white flowers with ring of red color at base) “Stabilizer” (factor that reduces mutability of pal-rec) StlSr (low mutability of pal-rec) Stlst (medium mutability of pal-rec) stlst (high mutability of pal-rec)

FI Genotype: Phenotype:

pal-reclpal-tub, Stlsr

Medium variegated flowers

F2 Expected phenotypes: pal-rec

Gametes pal-rec, St pal-rec, sr pal-tub,

pal-rec

pal-tub St

si

pal-tub st

low variegated

medium variegated

low variegated

medium variegated

medium variegated

highly variegated

medium variegated

highly variegated

low variegated

medium variegated

white

white

medium variegated

highly variegated

white

white

st

pal-tub, st

6 medium variegated : 3 highly variegated : 3 low variegated : 4 white

Observed phenotypes: 99 medium variegated : 54 highly variegated : 73 low Variegated : 65 white 214 medium variegated : 152 highly variegated :90 low variegated : 136 white

PLANT TRANSPOSABLE ELEMENTS

135

Evidence for two-component systems in which the second component seems to be a regulatory element comparable to those of maize has also been obtained for Oryza sutiva, Mirabilis jalapa, Elymus junceus, and Imputiens balsaminu L. (see Table I for references). Other cases in which segregation ratios indicate that two loci contribute to the variegated phenotype are those in which a modifier of variegation is involved. In these cases one locus apparently carries an autonomous transposable element capable of executing all the activities required to produce a variegated phenotype. But a second locus can influence the activities of the transposable element so that a new kind of variegation pattern occurs. However, this modifier is not absolutely required for variegation. Sometimes it has a stabilizing effect on the transposable element in question (Demerec, 1931; Imai, 1931; Rhoades, 1938; Harrison and Fincham, 1968; Harrison and Carpenter, 1973, 1980; Gerats et al., 1982), so that fewer reversion events occur. Sometimes the modifier enhances the instability of a transposable element so that the degree of variegation and/or germinal reversion is increased (McClintock, 1956c, 1957, 1958, 1965a; Punnett, 1935; Ashman, 1960, 1965; Mulder etal., 1981). Still other modifiers can alter the time in development at which reversion events occur (Punnett, 1935; Sand, 1976; Reddy and Peterson, 1983). For example, in the presence of a modifier, fine spotting, indicative of delayed events, may occur instead of the coarse spotting characteristic of earlier reversion events (Reddy and Peterson, 1983; Sand, 1976). In all cases involving modifiers, plants that are heterozygous for both loci segregate progeny with two different kinds of variegation pattern, as shown in Fig.

Fig. 2. Examples of diallelic segregation ratios obtained with mutable alleles. (A) Segregation of the factors of a two-component system in Mirabilis jalapa. The Y locus is involved in pigment synthesis in this plant. The Y" allele is a mutable allele that may harbor an element comparable to a receptor component of Zea mays. The V allele is an independently segregating factor required for mutability of Y" and is thus comparable to a maize regulatory component. In all plants a wild-type R allele is present in the genome, which permits red pigments to be synthesized. Note that P is stable when the V component is missing or inactive. (Data taken from Spitters er a / . , 1975.) (B) Segregation of a mutable allele and an independently segregating modifier of this allele in Antirrhinum majus. The pallida-recurrens allele is an unstable allele of the pallida locus, which is involved in anthocyanin synthesis. The pallidatubocolorara allele is a stable recessive mutation causing pigmentation in the flower to be restricted to the base of the corolla. "Stabilizer" is a semidominant locus, perhaps a transposable element (see text), that reduces instability of the pal-rec locus so that less somatic variegation is observed. The observed values differ slightly from the expected ones, possibly due to difficulties in scoring. Scoring may be complicated by the fact that pal-reclpal-rec plants have twice as many flakes as pal-reclpal-rub ones (Harrison and Fincham, 1968). Note that the pal-rec allele is by itself capable of inducing variegation, regardless of whether or not the stabilizer is present in the genome. (Data taken from Harrison and Fincham, 1968.)

TABLE IV Two-Component Systems of Transposable Elements in Maize"

Responsive allele:

I a-m(r)

rSpm a-ml

Ds a-Ds

Ds *b

Ds bz2-m

rdt a-dt

rFcu r-cu

rFcu r#IO

ruq a-ruq

rBg o2-m(r)

rMrh a-mrh

rMut bz-mut

+

-

-

+

+

-

+

Data are taken from Friedemann and Peterson (1982), and from Rhoades and Dempsey (1983). * indicates Ds in standard position on chromosome 9, which generates variegation by inciting chromosome breakage. A + symbol indicates that a receptor element is capable of responding to signals from the regulator, usually manifested by a variegated phenotype. A - symbol means that there is no response or the response has not been tested. Where neither nor - is present, no test has been performed. (-) means that the response requires unusually high doses of the regulator element (see text).

+

PLANT TRANSPOSABLE ELEMENTS

137

2. However, it should be emphasized that definite conclusions concerning the presence of a modifier can be drawn only if a clear-cut diallelic segregation ratio can be demonstrated. In other cases where progeny with different degrees of variegation are observed but no Mendelian segregation ratios, other explanations are possible. Here one could imagine that multiple modifiers are responsible (see Section V) or that alterations have occurred in or around the transposable element causing mutability. Origin of Receptor Elements There is considerable evidence that some receptor elements originate from regulatory components. Thus in a number of cases it was found that when the autonomous regulatory element En(Spm) transposes away from a locus, part of it seems to be left behind. This residual element behaves like a receptor element in that it responds to transactivating signals from a regulatory element elsewhere in the genome (McClintock, 1962; Peterson, 1961, 1968; Reddy and Peterson, 1976). In other cases alterations within an autonomous element inserted at a particular locus seem to give rise to nonautonomous derivatives without transposition of the element away from the locus. For example, derivatives of the bz-m2 mutant, which harbors A c at the bz locus, have been found to behave as if a Ds element were present at the bz locus (McClintock, 1955, 1956b, 1962). The Ds element at the wx locus in the mutant wx-m9 also arose in the same manner (McClintock, 1963). Thus in these cases one can expect that the structure of the receptor component resembles that of the corresponding regulators. Support for this idea on the basis of molecular data will be presented in Section 111. C. REVERSION EVENTS

Since most of the mutable alleles analyzed so far are alleles of the nuclear genome, we began our considerations on how to identify transposable element-induced mutations be examining segregation patterns typical for nuclear mutations. We shall now direct our attention to a prominent feature of most mutable alleles, their reversion to a phenotypically wild-type condition in somatic and/or germinal tissue. As early as 1910 Correns interpreted the greedwhite variegation of some mutants of Mirabifisjulapa as being due to a transition from the homozygous recessive state to a dominant heterozygous state in the colored parts of the plant. Depending on whether this transition occurs early or later in development, larger or smaller sectors of colored tissue arise. Although this hypothesis has not yet been verified by molecular data for Mimbilis jalupa, supporting evidence is available for other unstable mu-

138

PATRICIA NEVERS E T A L .

tants discussed in Section III,G. In these cases the two types of tissue in the variegated plant definitely differ genotypically. However, when molecular data are not available, the strongest indication that variegation involves mutational change is when variegated plants produce some wildtype or nearly wild-type offspring that later breed true. This can be attributed to reversion events occurring in germinal tissue, which results in wild-type gametes. If reversions to wild type occur frequently in germinal tissue, distorted Mendelian ratios will be found in crosses involving a mutable allele. Wildtype or nearly wild-type plants may occur where they are not at all expected, or an excess of wild-type individuals may be observed (see Fig. 1). Somatic and germinal reversion may not always involve excision of the element responsible for mutability at a given locus (McClintock, 1953, 1956c; Doodeman et al., 1984a). Other possibilities will be discussed in Section 111. One question for which there is no universal answer is whether the reversion events that occur in somatic tissue are equivalent to those that take place in germinal tissue. In a few cases that have been analyzed at a molecular level, reversion was shown to involve excision of the transposable element, and this could be demonstrated in both somatic tissue and germinal revertants (see Section 111,G). The fact that the frequencies of somatic and germinal reversion events are often strongly correlated also lends support to the concept of equivalence. The stabilizing effect of higher temperatures (25°C) on both somatic and germinal reversion of the mutable pul-rec mutation of Antirrhinum majus (Harrison and Fincham, 1964; see Fig. 2) provides further evidence for a positive correlation of this kind. There are, however, some striking exceptions such as the perlutea-uuriubilis allele in Antirrhinum (Stubbe et al., 1981) and the mutable alleles of Callistephus chinensis (Hondelman, 1959), where no positive correlation can be observed. In still other cases either somatic or germinal reversion may be missing completely. For example, some mutants of Antirrhinum have been reported in which frequent reversion in germinal tissue obviously occurs, leading to a large number of revertants among the progeny of the mutant, but there is no incidence of somatic reversion (see Table 11). Reversion in germinal tissue of the A . mujus mutant perlutea-variabilis occurs only in the presence of a specific modifier (Stubbe et al., 1981). The modifier of the mutable u locus in Nicotiana has the same effect (Sand, 1976). Germinal reversion but no somatic reversion is also observed in innumerable inbred lines with a tendency toward “sporting” or toward producing socalled “rogues.” In these lines wild-type or off-type individuals appear

PLANT TRANSPOSABLE ELEMENTS

139

(Burr and Burr, 1981a). very frequently, sometimes at the rate of 1 x This subject has been reviewed by other authors (Dermen, 1960; Jensen, 1965; Pearson, 1969) and interpreted by Burr and Burr (1981a) as follows: “Rogue” or “sporting” lines are often commercial lines containing mutations that have been maintained for many generations by strong artificial selective pressure. It is conceivable that a large number of these mutations originated by insertions of transposable elements and are thus basically unstable. They revert frequently to wild type, and the entire population would eventually revert to wild type in the absence of counterselective pressure. Cases in which somatic variegation but no germinal reversion is observed have already been discussed (Section 1,B). Since reversion does not take place in germinal tissue, it is questionable whether the somatic events observed are due to genetic alterations. Thus transposable elements may not be involved in these cases. D. REVERSE VARIEGATION

In considering reversion events we have so far discussed mainly those examples in which reversion occurs from a homozygous recessive state to a heterozygous one, manifested, for example, by pigmented spots on a colorless or nearly colorless background. The reverse phenotype, that is, colorless spots on a pigmented background, has also been observed (McClintock, 1968; Peterson and Weber, 1969; Peterson, 1980; Reddy and Peterson, 1984; Cornu, 1977; Harrison and Carpenter, 1977, 1979, 1980; Doodeman et al., 1984b; Gerats et al., 1984b). This phenomenon can be interpreted in terms of transposable elements as well. Several different theoretical explanations for the reverse variegated phenotype are conceivable. In the maize mutants a-m2 (McClintock, 1968) and a-m(Au) (Peterson, 1980) elements belonging to the Sprn(En) system are known to be integrated at the a locus, yet expression of the locus is apparently unimpaired, and kernels of these mutants are basically pigmented. Here one could imagine that the element is located in the control region of the locus in such a way that transcription originates at a promoter within the element and continues into the locus (see Nevers and Saedler, 1977). Expression of the locus would therefore depend on active transcription of the element. If for some reason transcription should no longer be initiated at the promoter in the eIement, as during an inactive phase, then expression of the a locus would also cease. Thus occasional inactivation of transcription might explain the white sectors sometimes observed with such mutants. Another case involves revertants of the transposable element-induced mutants anl-sl+ and a d - s l p - + in Petunia hybrida (Bianchi et al., 1978;

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Doodeman et al., 1984b). Plants heterozygous for the original mutable allele and an anZ-+l+ revertant allele have red flowers with many white spots. Such spots are not observed in heterozygotes carrying the mutable allelle and a true wild-type allele. Moreover, the white spots on the flowers of the hybrid revertant contain colored dots. As discussed by Doodeman et al., (1984a), this suggests that the element is retained at the locus in phenotypic revertants and continues to be unstable. Rearrangements in the sequence of either the element or the target site may be responsible for switching from the mutant to the revertant condition and back again, although other explanations are conceivable. A revertant line derived from the unstable pal-rec mutant of A. majus has been isolated that also has red flowers with many pale-colored and some white spots. In this case a completely different interpretation has been advanced (Harrison and Carpenter, 1979). It is based on the assumption that the element responsible for the pal-rec mutation is excised in the process of reversion and that this element and/or others continue to transpose in the revertant. The pale spots are thought to arise by insertion of a transposable element at a locus involved in anthocyanin synthesis, generating a phenotypically distinguishable heterozygous condition. To explain the occurrence of white spots either a one-step or a two-step process can be postulated. In the first instance one could imagine that an element integrates in a single chromosome homologue and generates a dominant mutation leading to acyanic tissue. This is plausible since dominant mutations of this kind are known, for example, the c-Z and c2-Zdfmutations in Zea mays and the Eluta mutation in A. majus, which are thought to result in accumulation of an inhibitor of pigmentation. On the other hand, if insertion of a transposable element leads to a recessive mutation in one chromosome homologue, then a two-step process is required to generate a white spot. In this case some form of gene conversion or mitotic crossing over would be necessary to reproduce the recessive mutation in the second chromosome homologue. Both processes would require a high frequency of transposition or possibly a preference for integration at loci involved in anthocyanin synthesis. The novel aspect of the interpretation of reverse variegation in A. majus presented by Harrison and Carpenter (1979) is that spotting is due to transposition and integration of transposable elements. Accordingly this phenomenon was termed “resurgence of instability.” That reverse variegation in A. majus arises by the same mechanism that generates red spots on white flowers in a pal-rec line, that is, by transposition, is supported by the fact that both kinds of spotting are affected by the same factors (Harrison and Carpenter, 1979). In both cases the frequency of spotting is reduced at higher temperatures (25°C) and under the influence of a stabi-

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lizing gene elsewhere in the genome. It is possible that “resurgence of instability” is due to specific mobilization of elements related to the one responsible for the pal-rec mutation. Molecular analyses of the elements responsible for the original pal-rec mutation and for a new niu-rec allele derived from the unstable pal-rec revertant are being conducted at present (H. Sommer, personal communication) and should help to clarify the matter.

E. VARIEGATION ASSOCIATED WITH CHROMOSOMAL ANOMALIES

So far we have discussed two characteristic aspects of transposable element-induced mutations in the nuclear genome: nearly Mendelian segregation of the variegated trait and reversion of the mutable allele in somatic and germinal tissue. An unusual set of variegated mutants is known which are definitely of nuclear origin and exhibit germinal reversion but nonMendelian segregation ratios. These are mutants in which defined chromosomal alterations such as breaks and translocations occur that can be observed cytologically. In maize, for example, the transposable element Ds (Dissociation) induces chromosomal breaks that can be observed directly (McClintock, 1945, 1946, 1947, 1948, 1951a). These breaks only occur when an Ac regulatory element is present in the genome (McClintock, 1947; Barklay and Brink, 1954). In a variegated plant of Nicotiana, mutant tissue exhibits a conspicuous block of heterochromatin at a defined position in the genome that is missing in the genome of revertant tissue (Burns and Gerstel, 1967). Many other examples of chromosomal anomalies associated with a variegated phenotype are summarized by Kirk and Tilney-Bassett (1978). It is not certain whether transposable elements are involved in all of these instances, but the case of Ds suggests that this may be true in some instances. F. STABLE A N D UNSTABLE ALLELIC VARIANTS OF MUTABLE ALLELES

None of the criteria discussed so far for recognizing transposable elements is absolutely foolproof. If segregation ratios and evidence for somatic and germinal reversion are ambivalent, a third characteristic of transposable elements can be considered: their ability to generate new derivatives with heritably altered phenotypes. Some of these derivatives may have a stable recessive or intermediate phenotype while others may exhibit an altered pattern of variegation with respect to frequency and timing of somatic reversion. Cases in which new alleles have been derived from a mutable one are shown in Tables I and I1 (Table 111 does not

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include this information although many mutable alleles of Zea mays have produced allelic variants). When a derivative with an altered phenotype is isolated, the first question to be answered is whether or not this is due to a mutation. It is a wellestablished fact, for example, that environmental factors such as temperature and light can strongly influence the degree of variegation observed, irrespective of any mutational change in the organism (Winton and Haldane, 1935; Rhoades, 1941; FabergC and Beale, 1942; Sand, 1957; Harrison and Fincham, 1964; Harrison and Carpenter, 1973, 1979; Bianchi et al.. 1978). The obvious indication that a hereditary alteration is involved is when a true-breeding line exhibiting the altered phenotype can be established. This, of course, depends in turn upon the stability of the allele responsible for the altered phenotype. If the new allele is itself highly unstable, it may be difficult if not impossible to establish a truebreeding line. Assuming that only one or two factors are responsible for the altered phenotype, another question to be answered is where the mutation has taken place, at the locus controlled by a transposable element or at an independent modifying locus? This can be determined by examining segregation ratios in appropriate crosses. In the two-element systems of Zea mays, for example, mutations within or adjacent to either the receptor or the regulator component can lead to an altered phenotype. Mutational alterations of independently segregating Ac and Spm regulator elements are known which cause a reduction in mutability, manifested by kernels with fewer andor smaller spots of revertant tissue (McClintock, 1956c, l957,1961a, 1963,1964, 1965b). Such weak “states” of the regulator may alternate with stronger “states” of the element (McClintock, 1959; 1961a,b, 1963,1965b). Still other allelic derivatives of En(Spm) and A c are known that exhibit cyclic changes in activity (McClintock, 1957, 1958, 1959, 1961b, 1962, 1964, 1971; Peterson, 1966). In addition to mutations in or around an independently segregating locus, alterations can occur at the locus immediately controlled by a transposable element. For example, colorless, intermediate, and various different variegated alleles of loci apparently controlled by autonomous elements have been reported for Callistephus chinensis (Hondelman, 1959), Anfirrhinurn majus (Fincham and Herrison, 1967), and Petunia hybrida (Bianchi et al., 1978; Gerats et al., 1984a,b; Farcy and Cornu, 1979). Likewise, many new derivatives of En-controlled a and a2 loci in Zea mays have been found (Peterson, 1960, 1961, 1968, 1970b, 1976a,b, 1980; Fowler and Peterson, 1974, 1978; Reddy and Peterson, 1976). Many of the latter seem to have arisen when En(Spm) transposed away from the locus.

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Under the influence of an appropriate regulator component (McClintock, 1955) mutations can also occur in or around a receptor element integrated at a given locus (McClintock, 1955, 1957, 1958, 1965a, 1968; Peterson, 1961, 1970a; Reddy and Peterson, 1984). McClintock (1955) referred to these variants as “states of a locus.” The best documented examples of this kind involve derivatives of mutable alleles of the a locus in maize. The receptor elements in some of these derivatives no longer respond to mutator signals of the regulator, and kernels of these derivatives have a stable, nonvariegated phenotype even when an active regulator is present in the genome. Pigmentation in such stable derivatives is homogeneous and can vary from null to an almost wild-type level. Other derivatives exhibit altered patterns of somatic variegation. A unique allelic variant of an Spm-I-controlled locus is a2-ml. It contains a receptor element of the Spm system that responds to the suppressor of Spm but not to the mutator function (McClintock, 1957, 1958, 1961a,b). Kernels of this derivative are almost fully pigmented in the absence of Spm and colorless in its presence. Still other derivatives of the a locus contain elements that are unstable only in particular parts of the plant. For example, in the Enbearing mutant a-m(crown) variegation is observed only in the crown of the kernels, while instability is restricted to the base of the kernel in am(flow) (Peterson, 1965b, 1966). In these derivatives En-(Spm) seems to be programmed to respond to tissue-specific signals. It is also conceivable that mutations in the immediate vicinity of a transposable element lead to an altered phenotype. Deletions of a flanking sequence, for example, might affect excision of the transposable element or its influence on gene expression. One such case involving the maize element MuZ has been verified by DNA sequence analysis (L. Taylor and V. Walbot, personal communication; see Section 1,H). Similarly, mutation of an external promoter might affect transcription across the element and alter transcription from sequences within the element itself. It has been suggested that a deletion may have fused the bz locus to the control region of the sh locus in the maize mutant bz-m4, and that this is responsible for the altered timing and tissue specificity of bz expression observed in the mutant (Gerats et a f . ,1983b). However, it is now known that the bzm4 mutant contains a deletion that extends to a position 500 bp upstream from the translational start signal of the s h locus (U. Courage, personal communication; Weck et al., 1984).To accommodate this finding with the fusion hypothesis one would have to assume that the sh locus is subject to control by a DNA sequence that is further upstream than the promoter region of the sh locus. Finally the exact position of a transposable element in the genome may have a decisive effect on the kind of variegation pattern produced. Peter-

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son (1976a, 1977) has proposed that varying insertional sites of a transposable element at a given locus may be responsible for various derivatives of mutable alleles in maize. Likewise Brink and Williams (1973) concluded that different modes of integration of Mp(Ac) at the r locus in maize are responsible for the wide array of derivatives with different patterns of somatic variegation observed. The frequency of germinal reversion of the maize od-m(r) allele controlled by the receptor element of the Bg system also seems to be dependent on the exact position of the regulatory component on the same chromosome: the farther away the regulator is located, the lower the frequency of reversion (F. Salamini, personal communication). Molecular analysis of derivatives of mutable systems will be required to distinguish between the alternative explanations for “states of a locus” listed above. Examples of such investigations are presented in Section II1,H. G. TRANSPOSITION: THE MOST CHARACTERISTIC PROPERTY OF

TRANSPOSABLE ELEMENTS

1 . General Remarks The property of transposable elements which is unique is at the same time the one most difficult to demonstrate unequivocally, and that is their ability to transpose. Several lines of classical genetic evidence outlined below suggest that transposition in plants occurs by excising an element from one position and reinserting it at another. Based on the assumption that somatic reversion also occurs by means of excision and that excising is inevitably accompanied by transposition, some authors even go so far as to equate each spot or stripe on a variegated plant with a transposition event. While this may hold true in some cases, caution is warranted in making any generalizations about transposition in plants. First, the means available for studying transposition are relatively crude, involving populations of elements, cells and plants in in uiuo studies and populations of molecules in in uitro experiments. Ideally one would like to be able to follow the movements of one uniquely marked element in a single cell. Second, there is some evidence that transposition may occur by more than one mechanism in a single organism. In prokaryotes, for example, one mode of transposition seems to be a process in which a copy of the element moves (Grindley and Sherratt, 1978; Shapiro, 1979; Arthur and Sherratt, 1979), a mode sometimes referred to as replicative transposition. In this case excision and transposition seem to be independent events. However, preliminary results indicate that transposition in prokaryotes may sometimes proceed via an alternate route, namely via excision and reintegration (Berg, 1983; Bresler et al., 1983; Harayama et al., 1984;

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N. Kleckner, personal communication). By the same token it is possible that more than one mechanism of transposition also operates in plants. Finally, as discussed in Section III,G, phenomena such as colored spots on white tissue that appear phenotypically to have arisen by excision of a transposable element may in fact have some other molecular basis. It follows that reversion events of this kind may have nothing to do with transposition. Since transposition as detected by genetic means is a rare event, one must develop a strategy for picking out the few organisms in which transposition has occurred among perhaps thousands of others. It is in this aspect of selection that most of the cases of transposition reported so far differ. Several examples will be outlined below. 2 . Transposition of a Receptor Element To follow the movements of a transposable element it must possess some property by which it can be identified and mapped wherever it is in the genome. In the case of a receptor element this is difficult since these elem