The Molecular Biology of Chloroplasts and Mitochondria in (Advances in Photosynthesis and Respiration)

The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas

Advances in Photosynthesis

VOLUME 7

Series Editor: GOVINDJEE University of Illinois, Urbana, Illinois, U.S.A.

Consulting Editors: Jan AMESZ, Leiden, The Netherlands Eva-Mari ARO, Turku, Finland James BARBER, London, United Kingdom Robert E. BLANKENSHIP, Tempe, Arizona, U.S.A. Norio MURATA, Okki, Japan Donald R. ORT, Urbana, Illinois, U.S.A.

Advances in Photosynthesis is an ambitious book series seeking to provide a comprehensive and state-of-the-art account of photosynthesis research. Pho­ tosynthesis is the process by which higher plants, algae and certain species of bacteria transform and store solar energy in the form of energy-rich organ­ ic molecules. These compounds are in turn used as the energy source for all growth and reproduction in these organisms. As such, virtually all life on the planet ultimately depends on photosynthetic energy conversion. This series of multiauthored books spans topics from physics to agronomy, from femtosecond reactions to season long production, from the photophysics of reaction centers to the physiology of whole organisms, and from X-ray crys­ tallography of proteins to the morphology of intact plants. The intent of this series of publications is to offer beginning researchers, graduate students, and even research specialists a comprehensive current picture of the remark­ able advances across the full scope of photosynthesis research.

The titles to be published in this series are listed on the backcover of this volume.

The Molecular Biology of

Chloroplasts and

Mitochondria

in Chlamydomonas

Edited by

J.-D. Rochaix M. Goldschmidt-Clermont Departments of Molecular Biology and Plant Biology,

University of Geneva,

Geneva, Switzerland

and

S. Merchant Department of Chemistry and Biochemistry

and Molecular Biology Institute,

University of California-Los Angeles,

Los Angeles, U.S.A.

KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

eBook ISBN: Print ISBN:

0-306-48204-5 0-7923-5174-6

©2004 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow Print ©1998 Kluwer Academic Publishers Dordrecht All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: and Kluwer's eBookstore at:

http://kluweronline.com http://ebooks.kluweronline.com

This book is dedicated to Paul Levine for his pioneering studies on the genetics of photosynthesis in Chlamydomonas and to the memory of Ruth Sager for her seminal contributions to organellar genetics.

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Contents

Preface

xvii

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1–11

Introduction to Chlamydomonas Elizabeth H. Harris

Summary I. Why Chlamydomonas? II. CellArchitecture III. Life Cycle IV. Laboratory strains of Chlamydomonas reinhardtii V. Genetic Analysis VI. Molecular Biology VII. Resources Acknowledgment References

2

Perspectives on Early Research on Photosynthesis in Chlamydomonas Robert K. Togasaki and Stefan J. Surzycki

1

1

3

3

4

6

7

7

8

8

13–23

Summary I. General Background II. The Levine Laboratory in the Early 1960s III. Establishment of Chlamydomonas reinhardtii as a Legitimate Model Organism IV. Development of New Techniques V. Emergence of New Research Targets VI. Old Experiments Becoming Reality Acknowledgment References

3

Organization of the Nuclear Genome Carolyn D. Silflow

Summary I. Introduction and Scope II. General Characteristics of the Nuclear Genome III. Organization of the Genome IV. Characteristics of Chlamydomonas Genes Transcribed by Polymerase II IV. Physical Mapping of the Chlamydomonas Genome VI. Future Prospects Acknowledgments References

4

Nuclear Transformation: Technology and Applications Karen L. Kindle

Summary I. Introduction II. A Brief History of C. reinhardtii Nuclear Transformation

13

13

14

15

18

19

21

21

22

25–40 25

26

26

26

30

36

37

37

37

41–61 42

42

42

III. Selectable Markers IV. Methods for Introducing DNA into the Nuclear Genome of C. reinhardtii V. Reporters and Promoters VI. Characteristics of Transformation Events VII. Insertional Mutagenesis and Gene Tagging VIII. Gene Isolation by Complementation of a Mutant Phenotype IX. Homologous Recombination and Gene Targeting X. The Use of Nuclear Transformation to Study Promoter Function XI. Conclusion Acknowledgments References

5

43

45

46

48

51

54

54

56

58

58

58

Modes and Tempos of Mitochondrial and Chloroplast Genome Evolution in Chlamydomonas: A Comparative Analysis 63–91 Aurora M. Nedelcu and Robert W. Lee

Summary I. Introduction II. Phylogenetic Position of Chlamydomonas III. Monophyletic versus Polyphyletic Origin of Mitochondria and Plastids:

The Chlamydomonas Case IV. Evolution of Mitochondrial and Chloroplast Genome Size in Chlamydomonas V. Evolution of Mitochondrial and Chloroplast Genome Organization in

Chlamydomonas VI. Evolution of Mitochondril and Chloroplast Gene Structure and Organization

in Chlamydomonas VII. Evolution of Mitochondrial and Chloroplast DNA Sequences in

Chlamydomonas VIII. Conclusions Acknowledgments References

6

Uniparental Inheritance of Chloroplast Genomes E. Virginia Armbrust Summary I. Introduction II. Historical Overview of the Uniparental Inheritance of Chloroplast DNA III. Mating-Type Control of Life Cycle Events IV. Protection of Plus Chloroplast DNA V. Zygote Specific Elimination of Minus Chloroplast DNA VI. Regulation of Chloroplast DNA Inheritance VI. Evolution of the Uniparental Inheritance of Organelle Genomes Acknowledgments References

7

Replication, Recombination, and Repair in the Chloroplast Genetic System of Chlamydomonas Barbara B. Sears

Summary I. Introduction II. Replication III. Recombination IV. Repair

63

64

64

65

69

79

82

85

87

87

87

93–113 93

94

95

98

101

103

108

110

110

110

115–138 115

116

116

123

130

viii

V. Perspectives and Conclusions Acknowledgments References

8

Chloroplast Transformation and Reverse Genetics Michel Goldschmidt-Clermont

133

133

133

139–149

Summary I. Introduction II. Delivery of DNA to the Chloroplast III. Selectable Markers and Reporters IV. Fate of Transforming DNA V. Reverse Genetics VI. Conclusion and Perspective Acknowledgments References

9

Chloroplast RNA Stability Jörg Nickelsen

139

140

140

140

142

147

147

148

148

151–163

Summary I. Introduction II. Cell Cycle Dependent Regulation of Chloroplast RNA Stability III. Nuclear Mutants Affected in Chloroplast RNA Stability IV. Towards a Molecular Model of Chloroplast RNA Stabilization/Degradation V. Conclusions and Perspectives Acknowledgments References

10 Chloroplast RNA Synthesis and Processing David B. Stern and Robert G. Drager

Summary I. Transcription of Chloroplast Genes II. Processing of Chloroplast mRNAs Acknowledgments References

151

152

152

154

154

161

161

161

165–183 165

166

171

177

177

11 RNA Splicing in the Chloroplast 183–195 David L. Herrin, Tai-Chih Kuo and Michel Goldschmidt-Clermont

Summary I. Introduction II. Group I Introns III. Group II Introns and Trans-Splicing IV. Perspective Acknowledgments References

183

184

184

190

193

193

193

197–217 12 Regulation of Chloroplast Translation Charles R. Hauser, Nicholas W. Gillham and John E. Boynton

197

Summary 198

I. Introduction II. The Role of Physiological and Environmental Factors in Translational Control 200

ix

III. Current Biochemical and Genetic Approaches to Dissect Mechanisms of

Translational Regulation IV. Cis-acting Sequences Involved in Translation Initiation V. Translational Regulation Involves Interactions between cis-Acting

Sequences and trans-Acting Factors VI. Ribosomes, Membranes and Tethers VII. Translational Regulation of Complex Assembly VIII. How are the Regulatory Proteins Regulated? IX. Is there Hierarchical Control of Chloroplast mRNA Translation? Acknowledgments References

13 Chloroplast Protein Translocation Mireille C. Perret, Karen K. Bernd and Bruce D. Kohorn

219

220

220

222

223

226

228

229

229

233–254

Summary I. Introduction II. Cell and Chloroplast Morphology III. Ultrastructural Organization of Thylakoid Membranes IV. Dynamic Aspects of Thylakoid Membrane Organization V. Biogenesis VI. Conclusion Acknowledgment References

15 Assembly of Photosystem II Jeanne Marie Erickson

205

209

211

212

213

214

214

219–231

Summary I. Introduction II. Chloroplast Import III. Sorting of Proteins Within the Chloroplast IV. Thylakoid Translocation V. Mutations Affecting Translocation VI. Perspectives Acknowledgments References

14 Supramolecular Organization of the Chloroplast and of the Thylakoid Membranes Jacqueline Olive and Francis-André Wollman

202

203

234

234

235

239

246

248

250

251

251

255–285

Summary I. Introduction II. Developmental Biogenesis of Photosystem II III. Assembly of Photosystem II Complexes IV. Assembly of the Extrinsic Membrane Polypeptides of the PS II

Oxygen-Evolving Complex V. Assembly of Manganese: The Catalytic Center of the Oxygen-Evolving

Complex Acknowledgments References

x

255

256

257

260

270

273

277

277

16 Functional Analysis of Photosystem II Stuart V. Ruffle and Richard T. Sayre Summary I. Introduction II. The Photosystem II Complex III. The Chloroplast DNA Encoded Small Polypeptides of Photosystem II IV. The Nucleus Encoded Polypeptides of the Photosystem II Complex V. Perspectives Acknowledgments References

17 Structure and Function of Photosystem I Andrew N. Webber and Scott E. Bingham

Summary I. Introduction II. Structure of Photosystem I III. Nature and Function of Electron Transfer Cofactors IV. Antenna Structure and Function V. Function of Photosystem I Subunits VI. Biogenesis of Photosystem I Acknowledgments References

18 Reexamining the Validity of the Z-Scheme: Is Photosystem I

Required for Oxygenic Photosynthesis in Chlamydomonas? Kevin Redding and Gilles Peltier

Summary I. The Z-Scheme of Oxygenic Photosynthesis and Alternative Schemes II. Electron Transport in the Absence of PS II III. Photosynthesis in the Absence of PS I IV. Putative Electron Transport Pathways Outside of the Z-Scheme V. Thermodynamic Considerations VI. Evolutionary Considerations VII. Conclusions Acknowledgments References

19 Assembly of Light-Harvesting Systems J. Kenneth Hoober, Hyoungshin Park, Gregory R. Wolfe, Yutaka Komine and Laura L. Eggink Summary I. Thylakoid Biogenesis in Chlamydomonas II. Analysis of LHCII Assembly III. Site of Assembly of LHCII During Initial Greening IV. Conclusions Acknowledgments References

xi

287–322

287

288

289

308

311

315

315

315

323–348 324

324

325

328

332

333

341

344

344

349–362

349

350

351

354

357

358

358

359

359

360

363–376 363

364

366

368

371

373

373

20 Pigment Biosynthesis: Chlorophylls, Heme, and Carotenoids Michael P. Timko

377–414

Summary I. Introduction II. Tetrapyrroles and Their Derivatives—An Overview III. Formation of ALA IV. The Pathway from ALA to Protoporphyrin IX V. The Magnesium Branch—Chlorophyll Formation VI. The Iron Branch—Formation of Heme VII. Light and Metabolic Regulation of Chlorophyll Formation VIII. Carotenoids Acknowledgments References

21 Glycerolipids: Composition, Biosynthesis and Function in Chlamydomonas Antoine Trémolières

378

378

378

379

383

388

397

398

401

406

406

415–431

Summary Introduction II. Glycerolipid and Fatty Acid Composition of Chlamydomonas III. Lipid Metabolic Pathway in Chlamydomonas spp. IV. In vivo Modifications of Lipid Composition in Chlamydomonas V. Mutants Affected in Lipid Composition VI. Involvement of Lipids in the Functional Organization and the Biogenesis

of the Photosynthetic Apparatus Acknowledgments References

22 In vivo Measurements of Photosynthetic Activity: Methods Pierre Joliot, Daniel Béal and René Delosme Summary I. Introduction II. Kinetic Analysis of the Fluorescence Yield III. Fluorescence Emission Spectra at Low Temperature IV. Delayed Fluorescence Measurements V. Oxygen Measurements VI. Absorption Spectroscopy VII. Photoacoustic Measurements VIII. Conclusion and Perspectives Appendix A: Estimation of the Signal-to-Noise Ratio in Fluorescence

Measurements Appendix B: Flash Spectrophotometer Acknowledgment References

23 New Digital Imaging Instrument For Measuring Fluorescence and Delayed Luminescence Pierre Bennoun and Daniel Béal

Summary I. Introduction II. Setup for Fluorescence and Delayed Luminescence Video Imaging

xii

415

416

417

422

425

426

428

429

429

433–449 433

434

436

439

439

439

440

443

445

446

446

448

448

451–458 451

452

452

III. Digital Fluorescence Imaging Related to Photosynthetic Electron Transfer IV. Digital Fluorescence Imaging Related to the Permanent Thylakoid

Electrochemical Gradient V. Digital Delayed Luminescence Imaging Related to Light-Induced and

Permanent Thylakoid Electrochemical Gradient Acknowledgments References

24 The Structure, Function and Biogenesis Of Cytochrome Complexes Francis-André Wollman

Summary I. General Traits II. Biochemical and Structural Studies III. Functional Studies IV. The pet Genes V. Biogenesis and Assembly VI. Concluding Remarks Acknowledgments References

25 Assembly and Function of the Chloroplast ATP Synthase Heinrich Strotmann, Noun Shavit and Stefan Leu

Summary I. Introduction II. Structure of

III. Molecular Genetics of IV. Mechanism of V. Regulation of VI. Conclusions References

452

453

455

457

457

459–476 460

460

461

463

466

467

472

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477–500 477

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478 482

487

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26 Molecular Aspects of Components of the Ferredoxin/Thioredoxin 501–514 Systems Jean-Pierre Jacquot, Mariana Stein, Stéphane Lemaire, Paulette Decottignies, Pierre Le Maréchal and Jean-Mark Lancelin Summary I. Introduction II. Ferredoxin Dependent Systems III. Thioredoxin Dependent Systems IV. Conclusion Acknowledgment References

501

502

505

508

511

512

512

515–527

27 Genetic Engineering of Rubisco Robert J. Spreitzer

Summary I. Introduction II. Chloroplast Genetic Screening and Selection

xiii

515

516

518

III. Directed Mutagenesis and Chloroplast Transformation IV. Rubisco Nuclear Mutants V. Conclusion and Perspective Acknowledgments References

28

Acquisition. Acclimation to Changing Carbon Availability

Martin H. Spalding

Summary

I. Introduction II. Photosynthetic Carbon Assimilation III. Induction of the CCM and Related Adaptations to Limiting Acknowledgments References

521

523

524

524

524

529–547 529

530

530

539

544

544

549–567

29 Regulation of Starch Biosynthesis Steven G. Ball

Summary

I. Starch: Structure and Function II. The Starch Pathway III. The Genetics of Starch Biosynthesis IV. A Model Explaining the Biogenesis of the Plant Starch Granule V. Future Prospects Acknowledgments References

30 State Transition and Photoinhibition Nir Keren and Itzhak Ohad

Summary

I. Introduction II. State Transition: The Phenomenon III. Light Stress: Photoinhibition and Recovery IV. Concluding Remarks and Perspectives Acknowledgments References

31 Synthesis of Metalloproteins Involved in Photosynthesis: Plastocyanin and Cytochromes Sabeeha Merchant

Summary

I. Introduction II. Copper-Responsive Synthesis of Plastocyanin and Cytochrome III. Genetic Analysis of Chloroplast Metalloprotein Assembly IV. Conclusions Acknowledgments References

32 Responses to Deficiencies in Macronutrients John P. Davies and Arthur R. Grossman Summary I. Introduction

549

550

554

559

563

564

565

565

569–596 569

570

574

578

590

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597–611 598

598

600

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608

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613–635 614

614

xiv

II. Nutrients in the Environment III. Specific Responses IV. Common Responses V. Model Integrating the Responses to Nutrient Deprivation VI. Regulation of the Responses to Nutrient Deprivation VII. Identification of Mutants Deficient in the Acclimation to Nutrient Deprivation Acknowledgments References

33 Nitrogen Assimilation and its Regulation Emilio Fernández, Aurora Galván and Alberto Quesada

Summary I. Introduction. Pathways for Nitrogen Assimilation in Chlamydomonas II. Assimilation of Ammonium III. Assimilation of Amino Acids IV. Assimilation of Purines V. Assimilation of Nitrate and Nitrite VI. Concluding Remarks Acknowledgments References

34 Mitochondrial Genetics Claire Remacle and René F. Matagne

Summary I. Introduction II. Mitochondrial Genome III. Mitochondria and the Electron Transport Chain IV. Mutations Affecting the Mitochondrial Genome V. Transmission of Mitochondrial Genes in Meiotic Zygotes VI. Transmission of Mitochondrial Genes in Vegetative Zygotes and

Mapping of Mitochondrial Mutations by Recombinational Analysis VII. Mitochondrial Transformation Acknowledgments References

35 Chlororespiration, Sixteen Years Later Pierre Bennoun

Summary I. Introduction II. The Thylakoid Electrochemical Gradient Present in the Dark III. Reduction of Plastoquinone in the Dark IV. Oxidation of Plastoquinol in the Dark V. Conclusion Cautionary Note Acknowledgments References

36 Perspectives Lauren J. Mets and Jean-David Rochaix

Summary I. Introduction II. The Niche of Chlamydomonas in Photosynthesis Research

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615

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629

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637–659 638

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661–674 661

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662

664

665

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669

671

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675–683 675

676

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680

680

682

682

682

685–703 685

686

687

III. Forefront Problems in Photosynthesis and Organelle Research Acknowledgments References

Index

696 700 700

705

xvi

Preface

The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas is the seventh volume to be published in the series Advances in Photosynthesis of Kluwer Academic Publishers (Series Editor: Govindjee). Volume 1 dealt with The Molecular Biology of Cyanobacteria; Volume 2 with Anoxygenic Photosynthetic Bacteria; Volume 3 with Biophysical Techniques in Photosynthesis; Volume 4 with Photosynthesis and the Environment; and Volume 6 with Lipids in Photosynthesis: Structure, Function and Genetics. The main goal of this book is to provide a comprehensive overview of current research with the green alga Chlamydomonas on chloroplast and mitochondrial biogenesis and function, with special emphasis on the assembly and structure-function relationships of the constituents ofthe photosynthetic apparatus. The editors have encouraged the contributors of this volume to emphasize the particular features of Chlamydomonas that make this unicellular organism uniquely suited for study­ ing photosynthesis and its multiple regulatory mechanisms operating under various environmental and stress conditions. A second, but equally important aim is to show that current research in photosyn­ thesis is multidisciplinary and combines molecular genetics, biochemical, biophysical and physiological approaches. Although Chlamydomonas has also proven to be a powerful system for understanding the structure, function and assembly of flagella, this topic is not covered in the book. Chlamydomonas research would not have reached its present status without the pioneering studies of the late Ruth Sager and of Paul Levine. Organellar genetic analysis of this alga started over 40 years ago when Ruth Sager discovered that during crosses certain traits were transmitted uniparentally to the progeny from the mating-type plus parent, but not from the mating-type minus parent. These uniparental traits were shown later to be specified by the chloroplast genome. Sager also found that, in rare cases, the uniparental traits of both parents could be inherited and that the analysis of their segregation pattern during crosses could be used to construct a genetic map. The potential of using C. reinhardtii for

a genetic dissection of photosynthesis was first recognized by Paul Levine. Together with his coworkers, he initiated a long-range genetic approach which proved to be highly successful. It provided genetic support for the linear Z scheme of photosynthesis and led to the identification of new components of the photosynthetic electron transfer chain such as the Rieske protein ofthe cytochrome complex. During the past 20 years, the powerful techniques of molecular biology and genetics, and the development of methods for efficient nuclear and chloroplast transformation of C. reinhardtii have greatly enhanced the potential of this organism as an experimental system for studying chloroplast biogenesis. This has led to impressive advances in our understanding of the regulation of chloroplast gene expression and it has provided important new insights into the complex cooperative interplay between the chloroplast and nuclear compartments in the assembly of the photosynthetic apparatus. At the same time, the ability to manipulate the chloroplast genome with surgical precision has opened the door for a detailed structure-function analysis of photosynthetic complexes in vitro, and thanks to the refinements and new developments in spectroscopic and fluorescence techniques, also in vivo. We feel strongly that a book on these recent exciting advances in research on photosynthesis in Chlamydomonas is timely and important. The first part of the book provides a general introduction to Chlamydomonas (Chapter 1, Harris), a historical chapter on early research on photo­ synthesis in this organism (Chapter 2, Togasaki and Surzycki) and chapters on nuclear genome organi­ zation (Chapter 3, Silflow), nuclear transformation (Chapter 4, Kindle), mitochondrial and chloroplast genome evolution (Chapter 5, Nedelcu and Lee), chloroplast uniparental inheritance (Chapter 6, Armbrust), chloroplast DNA metabolism (Chapter 7, Sears) and chloroplast transformation and reverse genetics (Chapter 8, Goldschmidt-Clermont). The second part includes several chapters on chloroplast gene expression: RNA stability (Chapter 9, Nickelsen), RNA processing (Chapter 10, Stern and

xvii

Drager), splicing (Chapter 11, Herrin et al.) and translation (Chapter 12, Hauser et al.). Protein targeting in the chloroplast is discussed in Chapter 13 (Perret et al.). The third part includes articles on the biosynthesis and function of thylakoid membranes (Chapter 14, Olive and Wollman), Photosystem II (Chapter 15, Erickson; Chapter 16, Ruffle and Sayre), Photosystem I (Chapter 17, Webber and Bingham; Chapter 18, Redding and Peltier), LHCII (Chapter 19, Hoober et al.), pigments (Chapter 20, Timko), glycerolipids (Chapter 21, Trémolières), the complex (Chapter 24, Wollman), the cytochrome ATP synthase (Chapter 25, Strotmann et al.), ferredoxin and thioredoxin (Chapter 26, Jacquot et al.) and of ribulose 1,5 bisphosphate carboxylaseoxygenase (Chapter 27, Spreitzer). In addition, Chapters 22 (Joliot et al.) and 23 (Bennoun and Beal) describe new and powerful techniques used for measurements of photosynthetic activity in vivo. These techniques are particularly suited for Chlamydomonas. The fourth part includes chapters uptake (Chapter 28, Spalding) and starch on biosynthesis (Chapter 29, Ball). Several articles are devoted to the responses of Chlamydomonas to various stress conditions, such as high light (Chapter 30, Keren and Ohad), copper deficiency (Chapter 31, Merchant) and macronutrient depri­ vation (Chapter 32, Davies and Grossman). Nitrogen assimilation and its regulation is discussed in Chapter 33 (Fernández et al.). Chapter 34 (Remacle and Matagne) describes mitochondrial genetics and

Chapter 35 (Bennoun) discusses the current models of chlororespiration. The last Chapter (36, Mets and Rochaix) offers a perspective on research on photosynthesis with Chlamydomonas. We thank the authors for their invaluable contributions which we hope will make this book very useful for researchers and students interested in photosynthesis and organellar biology in Chlamy­ domonas. The book is also intended for a wide audience, but is specifically designed for advanced undergraduate and graduate students and researchers in the fields of biochemistry, molecular biology, physiology, biophysics, plant biology, phycology and biotechnology. We also hope that this book will stimulate scientists outside the Chlamydomonas research community to use this organism for their studies. We wish to express our gratitude to Larry Orr for his patience with inexperienced editors, generous help and remarkable efficiency, to Govindjee for his continued interest and for his many helpful suggestions, and to Nicolas Roggli and Michael Hippler for their help in designing the cover graphics. Finally, we hope that by showing the extraordinary power and uniqueness of Chlamydomonas as a research tool in photosynthesis and by documenting the fast pace of progress achieved in the past years with this unicellular organism, this book will help promote Chlamydomonas as the ‘green yeast’ among the plant research community. Jean-David Rochaix Michel Goldschmidt-Clermont Sabeeha Merchant

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Chapter 1

Introduction to Chlamydomonas Elizabeth H. Harris

DCMB Group, Department of Botany, Box 91000,

Duke University, Durham, NC 27708-1000, U.S.A.

Summary I. Why Chlamydomonas? II. Cell Architecture III. Life Cycle IV. Laboratory strains of Chlamydomonas reinhardtii V. Genetic Analysis VI. Molecular Biology VII. Resources Acknowledgment References

1 1 3 3 4 6 7 7 8 8

Summary The unicellular green alga Chlamydomonas has found widespread use as a model experimental system for diverse studies in cell and molecular biology. The ability of C. reinhardtii to grow heterotrophically with acetate as its sole carbon source has made this species especially useful for investigation of chloroplast biogenesis and function, since mutants unable to carry out photosynthesis are viable. The simple vegetative and sexual cycles are easily manipulated in the laboratory, making this organism a powerful tool for genetic analysis of photosynthesis as well as many other cellular functions. The usual laboratory strain of C. reinhardtii is the descendant ofan isolate made in Massachusetts in 1945. Several additional strains interfertile with this one have been isolated from nature, all from North America, and are providing a useful source of molecular diversity. More than 300 genetic and molecular loci have now been identified in seventeen linkage groups in the nuclear genome. Maps, references, cultures and other resources for Chlamydomonas research are available from the Chlamydomonas Genetics Center and other collections.

I. Why Chlamydomonas? Sometimes called the ‘green yeast’ (Goodenough, 1992; Rochaix, 1995), this unicellular chlorophyte alga has achieved recognition as a model system for the study of photosynthesis, organelle biogenesis, and many other aspects of cell biology. A simple life cycle that is easily manipulated in the laboratory, minimal nutritional requirements, and rapid growth have all favored selection of this alga as an

experimental organism. Although mutants of a Chlamydomonas species were isolated and crossed as early as 1918 by A. Pascher (see Harris, 1989 for review), the modern age of Chlamydomonas research can be dated from the 1940s, with isolation of the principal laboratory strains of C. reinhardtii and C. moewusii. (Smith, 1946; Lewin, 1949). Conditions were established for laboratory culture and for manipulation of the sexual cycle (Smith and Regnery, 1950; Lewin, 1951; Sager and Granick, 1953, 1954),

J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 1–11. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

2 and the first mutants were selected (Lewin, 1952, 1953, 1954; Sager, 1954, 1955). Nearly fifty years and roughly 5,000 research papers later, we know many intimate details of the life of our favorite subject, but there are secrets still to be revealed. R. K. Togasaki and S. J. Surzycki (Chapter 2) have traced the history of research on photosynthesis in Chlamydomonas. The present chapter introduces the organism and provides suggestions for further reading on other aspects of its biology. Species of the genus Chlamydomonas are distinguished from other unicellular Volvocales by the presence of a cell wall, a pair of apical flagella, and a basal chloroplast surrounding one or more pyrenoids (Fig. 1). H. Ettl (1976) recognized 459 species based on morphological criteria, but the trend in recent years has been toward consolidation into a smaller number (Ettl and Schlösser, 1992; Schlösser, 1994). Molecular phylogenies now suggest that the genus comprises a diverse collection of organisms, some only distantly related (Buchheim et al., 1990, 1996; Chapter 5, Nedelcu and Lee). Some Chlamydomonas species have close affinity to certain colonial Volvocales (Buchheim et al., 1994; Liss et al., 1997). Although the Volvocales are regarded as a side branch from the evolutionary tree leading to land plants (Chapman and Buchheim, 1992), components of the photosynthetic process are highly conserved, and the relevance of research with Chlamydomonas to our understanding of chloroplast function in vascular plants is undisputed. Studies of photosynthesis and chloroplast biogenesis have been greatly facilitated by the ability of some species, notably C. reinhardtii, to grow with acetate as their sole carbon source (Harris, 1989). Mutants unable to carry out photosynthesis are thus viable when supplied with acetate. In nature this metabolism is usually found in organisms living in environments rich in organic compounds, and these so-called ‘acetate flagellates’ can tolerate the low oxygen tension and relatively high levels of associated with these conditions (Pringsheim, 1937; Hutner and Provasoli, 1951). Most laboratory isolates of C. reinhardtii have in fact come originally from nutrient-rich soil in cultivated fields or gardens (Sack et al., 1994). C. moewusii and its close relative, the strain usually known in laboratory research as C. eugametos (Gowans, 1963), are also widely used as experimental subjects, especially for studies of the sexual cycle and to some extent for flagellar biogenesis and

Elizabeth H. Harris

nutritional investigation. Because these species, which are only distantly related to C. reinhardtii (Lemieux et al., 1985; Boudreau and Turmel, 1996; Buchheim et al., 1996), are obligate phototrophs, they have not been employed in many photosynthesis studies. C. monoica, a homothallic species, has been developed by VanWinkle-Swift and colleagues (VanWinkle-Swift and Hahn, 1986; van den Ende, 1995) as a genetic system for study of processes involved in the sexual cycle, including investigations on mechanism of uniparental transmission of the chloroplast genome in crosses (VanWinkle-Swift and Aubert, 1983), but few physiological and molecular studies have been done with this species. Thus most research on chloroplast biogenesis and function has been carried out with C. reinhardtii, and in the present book, ‘Chlamydomonas’ can be

Chapter 1

Introduction to Chlamydomonas

assumed to refer to C. reinhardtii unless otherwise specified.

II. Cell Architecture Cells of C. reinhardtii are spherical to ovate in shape, varying from approximately 8 to as much as in length over the course of the vegetative cell cycle (see Ettl, 1976). A pair of apical flagella extend through a specialized collar region in the cell wall, and are typically 1.5 to 2× the length of the cell body. Within the cell, the flagella terminate in a pair of basal bodies connected by a striated fiber (see Jarvik and Suhan, 1991; Johnson and Rosenbaum, 1992). The basal bodies are connected to the nucleus by centrin fibers (Taillon et al., 1992; Salisbury, 1995), and to a cruciate system of four sets of microtubules, the flagellar roots (Lechtreck and Melkonian, 1991). Two contractile vacuoles are usually seen between the basal bodies and the nucleus (Luykx et al., 1997). For concise reviews of flagellar architecture and biogenesis in Chlamydomonas, see Johnson and Rosenbaum (1993), Johnson (1995), Dutcher (1995a), and Smith and Lefebvre (1997). Bernstein (1995) has reviewed flagellar kinesins, and Porter (1996; also Porter et al., 1996) has discussed dynein structure and genetics. Experimental procedures and reviews relating to the motility system are presented in a series of articles in the volume edited by Dentler and Witman (1995). The nucleus is partially surrounded by a cupshaped chloroplast in C. reinhardtii (Fig. 1), whereas in some other species the chloroplast may be displaced laterally (Ettl, 1976). A prominent pyrenoid is situated within the chloroplast distal to the nucleus. More information on chloroplast structure and the functions of the pyrenoid can be found in Chapters 14 (Olive and Wollman), 28 (Spalding) and 29 (Ball). The eyespot or stigma lies at the anterior of the chloroplast, just within the outer chloroplast envelope and close to the overlying cytoplasmic membrane, and contains carotenoid pigments (Crescitelli et al., 1992; Lawson and Satir, 1994). A unique rhodopsin has been identified as the photoreceptor for both phototaxis and photophobic responses (Derguini et al., 1991; Kroeger and Hegemann, 1994; Deininger et al., 1995), and the nature of the rhodopsin-mediated photoresponse is becoming understood (Pazour et al. 1995; Holland et al., 1996, 1997; Nonnengaesser et al., 1996). Witman (1993) has summarized earlier

3 investigations of phototaxis in Chlamydomonas. For general reviews of the cell biology of algal photoresponses, see Kreimer (1994) and Hegemann (1997). The evolution of algal visual proteins is reviewed by Walne and Gualtieri (1994). Mitochondria may appear in sections as small oval or elongate bodies (Fig. 1), or under some growth conditions can form an interconnecting, branching network throughout the cell (see Harris, 1989). Mitochondrial DNA and genetics are covered in this volume in Chapter 5 (Nedelcu and Lee) and Chapter 34 (Matagne), respectively. Respiratory physiology, including the cytochrome and alternative oxidase pathways, has been discussed by Weger and colleagues (Weger et al., 1990; Derzaph and Weger, 1996; Weger, 1996), and Eriksson et al. (1996) have published an improved method for isolation of physiologically active mitochondria. Geraghty and Spalding (1996) have discussed mitochondrial responses to changing concentrations. Cells of C. reinhardtii are surrounded by a complex cell wall composed of glycoproteins with high hydroxyproline content (Goodenough and Heuser, 1985; Matsuda, 1988; Adair and Snell, 1990; Woessner and Goodenough, 1994). Genes encoding some of the constituent proteins have been cloned and sequenced (Waffenschmidt et al., 1993; Kurvari, 1997). Mutants with absent or defective cell walls have been isolated (Harris, 1989; Voigt et al., 1997), and have proved useful as recipients for trans­ formation and in other experimental applications where the wall would otherwise present a barrier to manipulation.

III. Life Cycle Vegetative cells of C. reinhardtii are haploid. Growing logarithmically on a 12:12 light-dark cycle, they divide synchronously during the dark period in (usually) two mitotic divisions in rapid succession, releasing four daughter cells from a single sporangial wall after secretion of a specific lytic enzyme (Schlösser et al., 1976; Spessert and Waffenschmidt, 1990). Under optimal laboratory conditions in sufficient light, stationary phase is reached at about 1 cells/ml. Gene regulation and expression to through the cell cycle have been investigated by several laboratories (Savard et al., 1996; Voigt et al., 1996; Lechtreck and Silflow, 1997), and mutants blocked at specific points in the cycle have been

4 isolated (Howell and Naliboff, 1973; Harper et al., 1995; Wu et al., 1997). A number of biological processes in Chlamydomonas exhibit circadian rhythms (e.g. Fujiwara et al., 1996; Hwang et al., 1996; Jacobshagen et al., 1996), and Goto and Johnson (1995) have proposed that the cell division cycle itself is controlled by a circadian clock. In nature, the sexual cycle is a response to adverse conditions. In the laboratory, gametogenesis is controlled by nitrogen deprivation and a blue lightresponsive signal transduction pathway (Matsuda et al., 1992; Beck and Haring, 1996; Gloeckner and Beck, 1997; Pan et al., 1997). The light requirement has been obviated by mutation to constitutive expression of one or more regulatory genes in some laboratory strains (Gloeckner and Beck, 1995). is genetically determined Mating type by a complex locus on linkage group VI (Ferris and Goodenough, 1994, 1997) and gametes express mating type-specific glycoproteins (agglutinins) on their flagellar surfaces (Goodenough et al., 1995). Flagellar pairing initiates a cascade of events, including lysis of the gametic cell walls by a specific enzyme distinct from the vegetative cell lysin (Matsuda, 1988; Buchanan et al., 1989), contact of mating type-specific structures at the cell apices (Ferris et al., 1996; Wilson et al., 1997), and fusion of the gametic cytoplasms. For review of the extensive literature on signal transduction in mating, see Musgrave (1993) and Quarmby (1994). A transient quadriflagellate stage is followed by resorption of all flagella, and nuclear and chloroplast fusion follow within a few hours. Within the first 24 hours after mating, a hard, impermeable wall is secreted (Woessner and Goodenough, 1989, 1992), creating a durable zygospore that is resistant to desiccation or other environmental insults. After a maturation period of four to seven days, or longer, restoration of nitrogen, moisture and light induce the germination process. Over the next 18 to 24 hours, meiosis takes place, sometimes followed by a mitotic division, and the zygospore wall bursts to release the four or eight haploid progeny. Separation of these meiotic products to allow each to form a clone is the basis of Chlamydomonas genetics by tetrad analysis (Harris, 1989; Dutcher, 1995b). Nuclear genes are inherited in a Mendelian fashion and segregate 2:2 among the tetrad products. Chloroplast genes are inherited uniparentally from parent in most zygotes (>95% under the usual the laboratory conditions), whereas mitochondrial DNA

Elizabeth H. Harris in C. reinhardtii is transmitted uniparentally from parent. The mechanism by which the the inheritance oforganelle DNA is controlled was hotly debated for years, and is still not fully understood (Chapter 6, Armbrust). This book covers many aspects of metabolism in Chlamydomonas, including synthesis of lipids (Chapter 21, Trémolières), pigments (Chapter 20, Timko), and starch (Chapter 29, Ball). Nitrogen metabolism is discussed by Fernández et al. (Chapter 33), and micronutrient metabolism by Davies and Grossman (Chapter 32). For additional background on Chlamydomonas physiology, see Harris (1989).

IV. Laboratory strains of Chlamydomonas reinhardtii The standard laboratory strains of C. reinhardtii derive from a zygospore isolated by G. M. Smith from a soil sample collected near Amherst, Massachusetts, in 1945 (Hoshaw, 1965). Smith gave his strains to several of the early investigators, with the result that three principal lineages exist today, all apparently tracing their ancestry to the same sample (Table 1; see Harris, 1989, for further discussion). Most of the early nonphotosynthetic mutants were isolated in the strain used by R. P. Levine, W. T. Ebersold and coworkers, usually known as 137C (Smith’s original designation for his isolate, now usually applied specifically to the Levine line). These strains lack nitrate reductase activity and thus require a reduced nitrogen source for growth. Comparison of the strains used by R. A. Lewin, Levine and others suggests that this deficiency was present in this stock as early as 1949. Ruth Sager also obtained strains from Smith in the early 1950s. Her wild type isolate, 21 gr, was selected as a clone that remained green in the dark, in contrast to other strains, collectively designated y mutants, that fail to accumulate chlorophyll in the dark and maintain only a rudimentary chloroplast structure under these conditions (Sager and Palade, 1954). Beginning in the late 1960s, I. Ohad, J. K. Hoober, and others carried out extensive studies of rediffer­ entiation of chloroplast structure on transfer of y mutants into light (Chapter 2, Togasaki and Surzycki; Chapter 19, Hoober et al.). In contrast to the Levine 137C strain, Sager’s 21 gr and the original y1 strains used by her and by Ohad are able to grow on nitrate

Chapter 1

Introduction to Chlamydomonas 5

Elizabeth H. Harris

6 as their sole nitrogen source. The C8 and C9 strains ofthe Japanese culture collection (IAM) are probably strains from Sager, deposited by Y. Tsubo. This fundamental physiological difference between Sager’s and Levine’s strains produced speculation that these strains might have independent origins (Harris, 1989). Molecular analysis, particularly of insertion of the Gulliver transposon (Ferris, 1989) and ofpolymorphisms in chloroplast DNA (Harris et al., 1991), argues, however, for a common origin, followed by loss early on ofnitrate reductase activity in the Levine line, through mutations in the structural gene for nitrate reductase (Nit1) and in an unlinked regulatory gene (Nit2) (Chapter 33, Fernández et al.). Spreitzer and Mets (1981) crossed the Sager 21 gr strain with a Levine 13 7C isolate to create the hybrid strain 2137. Many nonphotosynthetic mutants have been isolated in this background. A third line of C. reinhardtii deriving from Smith’s original isolate made its way to the British (CCAP), German (SAG) and American (Indiana University, subsequently UTEX) algal collections (Table 1; Harris, 1989). These strains have been less frequently used than the aforementioned strains for studies of chloroplast function. There are several strains extant in the major culture collections that are listed as independent isolates of C. reinhardtii, but, based on molecular criteria, they appear to be incorrectly labeled as cultures of one of the Smith strains (Table 1). Ferris (1989) found that insertion patterns for the transposon Gulliver were remarkably similar when four cultures from the SAG collection, ostensibly of independent origin, were compared with the usual laboratory isolates, and chloroplast DNA from these strains shows restriction digest patterns identical to the Smith strains (Harris et al., 1991). In contrast, several strains of undisputed inde­ pendent origin (Table 1) are cross-fertile with laboratory strains of C. reinhardtii, and in recent years have been exploited as sources of molecular diversity. The oldest ofthese strains is the one isolated from South Deerfield, Massachusetts, and identified by Smith as C. reinhardtii but subsequently redescribed by Hoshaw and Ettl (1966) as the type specimen of the new species C. smithii. Hoshaw and Ettl identified it as mating type minus, based on tests against strains 89 and 90 of the Indiana collection. These strains were subsequently found to have been labeled with their mating types reversed (Harris, 1989; Starr and Zeikus, 1993), and the C. smithii

strain from Massachusetts is in fact The strain identified by Hoshaw and Ettl as the opposite mating type of C. smithii was isolated from Santa Cruz CA, and appears to be only slightly interfertile, if at all, with C. reinhardtii. Based on sequence analysis of the ITS-2 region of the nuclear ribosomal repeats, Coleman and Mai (1997) have suggested that this strain bears closer affinity to C. culleus, a species also found on the U.S. west coast, than to C. reinhardtii. Several other more recent ‘wild’ Chlamydomonas strains (Gross et al., 1988; Spanier et al., 1992; Sack et al., 1994; E. H. Harris, unpublished) are fully interfertile with laboratory strains of C. reinhardtii (Table 1). These strains show molecular polymorphisms in both nuclear and chloroplast DNA, and variations in incidence and insertion sites of the Gulliver and TOC transposons (Ferris, 1989; Harris et al., 1991 and unpublished; Sack et al., 1994; Chapter 3, Silflow). It is perhaps worth noting that all the authentic interfertile strains of C. reinhardtii isolated to date have been found in North America east of the Rocky Mountains.

V. Genetic Analysis The first genetic maps published for the nuclear genome of C. reinhardtii (Levine and Goodenough, 1970) comprised non-motile (pf, ‘paralyzed flagella’), non-photosynthetic (ac, ‘acetate-requiring’), anti­ biotic resistant (e.g. streptomycin, cycloheximide), and auxotrophic mutants (requiring arginine, nicotinamide, thiamine or p-aminobenzoic acid). Although the specific mutations are now understood in much greater detail, and some additional mutant types have been identified, these remain the major broad categories. More than 200 genetic loci have been defined based on mutation, and an additional 170 molecular loci have been identified (Ranum et al., 1988; Chapter 3, Silflow,). Levine and his colleagues used mutants to dissect the path of photosynthetic electron transport (Levine, 1969; Chapter2,Togasaki and Surzycki). Flagellarstructure and biogenesis have likewise been elucidated primarily through mutant studies (Curry and Rosenbaum, 1993; Dutcher, 1995a), as have the pathways of nitrogen assimilation (Chapter 33, Fernández et al.). Specific mutations affecting chloroplast structure and function are discussed in detail throughout the present book. Seventeen nuclear linkage groups are currently

Chapter 1

Introduction to Chlamydomonas

recognized (Harris, 1993; Chapter 3, Silflow). Linkage groups XII and XIII as defined by Levine and Goodenough (1970) have been consolidated (Dutcher et al., 1991), as have XVI and XVII (Harris, 1989; Dutcher et al., 1991). Chromosome cytology in C. reinhardtii is poor (see Harris, 1989, for review), and electrophoretic separation of chromosomes has not been fully satisfactory (Hails et al., 1993). Most of the chromosomes co-migrate and are resolved into four bands on pulsed-field gels. Sixteen linkage groups have now been localized to these bands by blot analysis, permitting unmapped DNA probes to be rapidly assigned to a subset of co-migrating chromosomes (A. Day, personal communictation). Linkage group XIX, or UNI (for the uniflagellate mutation uni1), was defined by Ramanis and Luck (1986), who reported genetic evidence suggesting that it might be circular rather than linear, as well as novel effects of temperature during zygospore maturation on recombination frequency. As first described, this linkage group contained only markers relating to motility, but subsequently mutations with no apparent flagellar association have been found linked to other loci on this group (Dutcher et al., 1992). Hall et al. (1989) reported that molecular probes for the UNI linkage group hybridized to basal bodies and concluded that the UNI linkage group corresponds to basal body DNA. This report was challenged by K. A. Johnson and Rosenbaum (1990), who did not find immunologically detectable DNA associated with basal bodies; by D. E. Johnson and Dutcher (1991), who found that DNA sequences from this linkage group were present in the same copy number per cell as other nuclear linkage groups in both haploid and diploid strains, and in mutants lacking basal bodies; and by Holmes et al. (1993) who obtained a linear genetic map in multifactor crosses. Hall and Luck (1995) subsequently confirmed the linearity of the genetic map and the stoichiometry reported by Johnson and Dutcher, but based on in situ hybridization data, they believe that DNA of this linkage group does occupy a unique position in interphase cells specifically at the anterior edge of the nucleus proximal to the basal body complex.

VI. Molecular Biology The past ten years have seen a virtual revolution in the style of Chlamydomonas research, and the types

7 of studies that are feasible, entirely because of advances in techniques for molecular analysis and genetic engineering. Successful introduction of exogenous DNA into Chlamydomonas chloroplasts by biolistic bombardment (Boynton et al., 1988) was soon followed by application of this procedure to nuclear genes (Debuchy et al., 1989; Kindle et al., 1989), and then by a simple procedure based on agitation of cells in the presence of DNA and glass beads (Kindle, 1990) that made transformation feasible in any laboratory. High efficiency trans­ formation by electroporation has recently been reported by Shimogawara et al. (1998). Kindle (Chapter 4) and Goldschmidt-Clermont (Chapter 8) have reviewed transformation technology for nuclear and chloroplast genes, respectively in this volume. Whereas DNA introduced into the chloroplast genome usually integrates at homologous sites, in the nuclear genome integration is more often by non­ homologous recombination. Tam and Lefebvre (1993) turned this seeming difficulty into advantage by using transformation as a means of generating tagged mutants by insertional mutagenesis, and their technique has now been used by many laboratories to identify a diverse collection of genes (see Kindle, Chapter 4, for details). Expression of foreign genes in Chlamydomonas has also been a stumbling block, owing perhaps to a combination of codon bias, gene silencing, and perhaps other factors, but this obstacle too now is being surmounted (Hall et al., 1993; Sizova et al., 1996; Stevens et al., 1996;Cerutti et al., 1997a,b). With solution ofthese problems specific to Chlamydomonas, application of general techniques of molecular biology to algal research becomes straightforward. The results will be apparent throughout this volume.

VII. Resources Historical background, descriptions of mutants, and many methods are available in The Chlamydomonas Sourcebook (Harris, 1989). The Chlamydomonas Genetics Center maintains a collection of wild type and mutant strains of C. reinhardtii, C. moewusii and C. eugametos, as well as bacterial cultures carrying plasmids with inserts of Chlamydomonas DNA (Chlamydomonas Genetics Center, c/o Dr. Elizabeth H. Harris, DCMB Box 91000, Duke University, Durham NC 27708-1000, U.S.A.; e-mail chlamy@ duke.edu; phone 919-613-8164; fax 919-613-8177).

8 The Center also maintains a web page (http:// www.botany.duke.edu/DCMB/chlamy.htm) with methods files, announcements, and links to pages of many laboratories engaged in Chlamydomonas research, and a database that is part of the Plant Genome project of the U.S. National Agricultural Library (http://probe.nalusda.gov:8300/cgi-bin/ browse/chlamydb). Cultures of other species of Chlamydomonas can be obtained from several major algal collections, i n c l u d i n g UTEX (University of Texas Algal Collection, Department of Botany, Austin TX 78713­ 7640, U.S.A.; see also Starr and Zeikus, 1993), CCAP (Culture Centre of Algae and Protozoa, Freshwater Biology Association, The Ferry House, Ambleside, Cumbria LA22 OLP, U.K.), SAG (Sammlung von Algenkulturen, Pflanzenphysio­ logisches Institut, Universität Göttingen, Nikolaus­ berger Weg 18, D-3400 Göttingen, Germany; see also Schlösser, 1994), and IAM (Institute of Applied Microbiology, The University of Tokyo, 1-1-1 Yayoi, Bunkyou-ku, Tokyo 113, Japan). A few Chlamy­ domonas cultures are also maintained by the American Type Culture Collection (12301 Parklawn Drive, Rockville MD 20852, U.S.A.). Several specialized collections also have significant numbers of Chlamydomonas accessions: Peterhof Chlamy­ domonas Collection (Genetics Department, St. Petersburg State University, St. Petersburg V 164, 199164, Russia); Culture Collection of Autotrophic Organisms (Institute of Botany, Czech Academy of Sciences, Dukelska 145, CS-379 82 Trebon, Czech Republic); Culture Collection of Microalgae (K. A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, 35 Botanicheskaya Street, Moscow, 127276, Russia), and the University of Toronto Culture Collection (Department of Botany, University of Toronto, Toronto, Ontario M5S 3B2, Canada).

Acknowledgment The Chlamydomonas Genetics Center is supported by NSF Grant DBI-9319941.

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9 Goto K and Johnson CH (1995) Is the cell division cycle gated by a circadian clock? The case of Chlamydomonas reinhardtii. J Cell Biol 129: 1061–1069. Gowans CS (1963) The conspccificity of Chlamydomonas eugametos and Chlamydomonas moewusii: An experimental approach. Phycologia 3: 37–44 Gross CH, Ranum LPW and Lefebvre PA (1988) Extensive restriction fragment length polymorphisms in a new isolate of Chlamydomonas reinhardtii. Curr Genet 13: 503–508 Hails T. Jobling M and Day A (1993) Large arrays of tandemly repeated DNA sequences in the green alga Chlamydomonas reinhardtii. Chromosoma 102: 500–507 Hall JL and Luck D (1995) Basal body-associated DNA: In situ studies in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 92: 5129–5133 Hall JL, Ramanis Z and Luck DJL (1989) Basal body/centriolar DNA: Molecular genetic studies in Chlamydomonas. Cell 59: 121–132 Hall LM, Taylor KB and Jones DD (1993) Expression of a foreign gene in Chlamydomonas reinhardtii. Gene 124: 75–81 Harper J, Wu L, Sakuanrungsirikul S and John P (1995) Isolation and partial characterization of conditional cell division cycle mutants in Chlamydomonas. Protoplasma 186: 149–162 Harris EH (1989) The Chlamydomonas Sourcebook. A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego Harris EH (1993) Chlamydomonas reinhardtii. In: O’Brien SJ (ed) Genetic Maps. Locus Maps of Complex Genomes, 6th edition, pp 2.157–2.169. Cold Spring Harbor Laboratory, Cold Spring Harbor Harris EH, Boynton JE, Gillham NW, Burkhart BD and Newman SM (1991) Chloroplast genome organization in Chlamy­ domonas. Arch Protistenk 139: 183–192 Hegemann P (1997) Vision in microalgae. Planta 203:265–274 Holland EM, Braun FJ, Nonnengaesser C, Harz H and Hegemann P (1996) Nature of rhodopsin-triggered photocurrents in Chlamydomonas .1. Kinetics and influence of divalent ions. Biophys J 70: 924–931 Holland EM, Harz H, Uhl R and Hegemann P (1997) Control of phobic behavioral responses by rhodopsin-induced photo­ currents in Chlamydomonas. Biophys J 73: 1395–1401. Holmes JA, Johnson DE and Dutcher SK (1993) Linkage group XIX of Chlamydomonas reinhardtii has a linear map. Genetics 133: 865–874 Hoshaw RW (1965) Mating types of Chlamydomonas from the collection of Gilbert M. Smith. J Phycol 1: 194–196 Hoshaw RW and Ettl H (1966) Chlamydomonas smithii sp. nov.—a Chlamydomonad interfertile with Chlamydomonas reinhardtii. J Phycol 2: 93–96 Howell SH and Naliboff JA (1973) Conditional mutants in Chlamydomonas reinhardtii blocked in the vegetative cell cycle. I. An analysis of cell cycle block points. J Cell Biol 57: 760–772 Hutner SH and Provasoli L (1951) The phytoflagellates. In: Lwoff A (ed) Biochemistry and Physiology of Protozoa, Vol 1, pp 27–128. Academic Press, New York Hwang S, Kawazoe R and Herrin DL (1996) Transcription of tufA and other chloroplast-encoded genes is controlled by a circadian clock in Chlamydomonas. Proc Natl Acad Sci USA 93: 996–1000 Jacobshagen S, Kindle KL and Johnson CH (1996) Transcription

10 of CABII is regulated by the biological clock in Chlamydomonas reinhardtii. Plant Mol Biol 31: 1173–1184 J a r v i k JW and Suhan JP (1991) The role of the flagellar transition region: Inferences from the analysis of a Chlamydomonas mutant with defective transition region structures. J Cell Sci 99: 731–740 Johnson DE and Dutcher SK (1991) Molecular studies of linkage group XIX of Chlamydomonas reinhardtii: Evidence against a basal body location. J Cell Biol 113: 339–346 Johnson KA (1995) Keeping the beat: Form meets function in the Chlamydomonas flagellum. BioEssays 17: 847–854 Johnson KA and Rosenbaum JL (1990) The basal bodies of Chlamydomonas reinhardtii do not contain immunologically detectable DNA. Cell 62: 339–346 Johnson KA and Rosenbaum JL (1992) Replication of basal bodies and centrioles. Curr Opin Cell Biol 4: 80–85 Johnson KA and Rosenbaum JL(1993) Flagellar regeneration in Chlamydomonas: A model system for studying organelle assembly. Trends Cell Biol 3: 156–161 Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 87: 1228–1232 Kindle KL, Schnell RA, Fernández E and Lefebvre PA (1989) Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol 109: 2589–2601 Kreimer G (1994) Cell biology of phototaxis in flagellate algae. Int Rev Cytol 148: 229–310 Kroeger P and Hegemann P (1994) Photophobic responses and phototaxis in Chlamydomonas arc triggered by a single rhodopsin photoreceptor. FEBS Lett 341: 5–9 Kurvari V (1997) Cell wall biogenesis in Chlamydomonas: Molecular characterization ofa novel protein whose expression is up-regulated during matrix formation. Mol Gen Genet 256: 572–580 Lawson MA and Satir P (1994) Characterization of the eyespot regions of ‘blind’ Chlamydomonas mutants after restoration of photophobic responses. J Euk Microbiol 41: 593–601 Lechtreck KF and Melkonian M (1991) An update on fibrous flagellar roots in green algae. Protoplasma 164: 38–44 Lechtreck KF and Silflow CD (1997) SF-assemblin in Chlamydomonas: Sequence conservation and localization during the cell cycle. Cell Motil Cytoskeleton 36: 190–201 Lemieux B, Tunnel M and Lemieux C (1985) Chloroplast DNA variation in Chlamydomonas and its potential application to the systematics of this genus. BioSystems 18: 293–298 Levine RP (1969) The analysis of photosynthesis using mutant strains of algae and higher plants. Annu Rev Plant Physiol 20: 523–540 L e v i n e RP and Goodenough UW (1970) The genetics of p h o t o s y n t h e s i s and the chloroplast in Chlamydomonas reinhardi. Annu Rev Genet 4: 397–408 Lewin RA (1949) Genetics of Chlamydomonas—paving the way. Biol Bull 97: 243–244 Lewin RA (1951) Isolation of sexual strains of Chlamydomonas. J Gen Microbiol 5: 926–929 Lewin RA (1952) Ultraviolet induced mutations in Chlamy­ domonas moewusii Gerloff. J Gen Microbiol 6: 233–248 L e w i n RA (1953) The genetics of Chlamydomonas moewusii Gerloff. J Genet 51: 543–560 Lewin RA (1954) Mutants of Chlamydomonas moewusii with

Elizabeth H. Harris impaired motility. J Gen Microbiol 11: 358–363 Liss M, Kirk DL, Beyser K and Fabry S (1997) Intron sequences provide a tool for high-resolution phylogenetic analysis of volvocine algae. Curr Genet 31: 214–227 Luykx P, Hoppenrath M, Robinson DG (1997) Structure and behavior of contractile vacuoles in Chlamydomonas reinhardtii. Protoplasma 198: 73–84. Matsuda Y (1988) The Chlamydomonas cell walls and their degrading enzymes. Japan J Phycol 36: 246–264 Matsuda Y, Shimada T and Sakamoto Y (1992) Ammonium ions control gametic differentiation and dedifferentiation in Chlamydomonas reinhardtii. Plant Cell Physiol 33: 909–914 Musgrave A (1993) Mating in Chlamydomonas. Prog Phycol Res 9: 193–237 Nonnengaesser C, Holland EM, Harz H and Hegemann P (1996) The nature of rhodopsin-triggered photocurrents in Chlamy­ domonas .2. Influence of m o n o v a l e n t i o n s . Biophys J 70: 932– 938 Pan JM, Having MA and Beck CF (1997) Characterization of blue light signal transduction chains that control development and maintenance of sexual competence in Chlamydomonas reinhardtii. Plant Physiol 1 1 5 : 1241–1249 Pazour GJ, Sineshchekov OA and Witman GB( 1995) Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii. J Cell Biol 131: 427–440. Porter ME (1996) Axonemal dyneins: Assembly, organization, and regulation. Curr Opin Cell Biol 8: 10–17 Porter ME, Knott JA, Myster SH and Farlow SJ (1996) The dynein gene family in Chlamydomonas reinhardtii. Genetics 144: 569–585 Pringsheim EG (1937) Beiträge zur Physiologie saprotropher Algen und Flagellaten. 3. Mitteilung: Die Stellung der Azetatflagellaten in einem physiologischen Ernährungssystem. Planta 27: 61–72 Quarmby LM (1994) Signal transduction in the sexual life of Chlamydomonas. Plant Mol Biol 26: 1271–1287 Ramanis Z and Luck DJL( 1986) Loci affecting flagellar assembly and function map to an unusual linkage group in Chlamy­ domonas reinhardtii. Proc Natl Acad Sci USA 83: 423–426 Ranum LPW, Thompson MD, Schloss JA, Lefebvre PA and Silflow CD (1988) Mapping flagellar genes in Chlamydomonas using restriction fragment length polymorphisms. Genetics 120: 109–122 Rochaix J - D ( 1 9 9 5 ) Chlamydomonas reinhardtii as the photosynthetic yeast. Annu Rev Genet 29: 209–230 Sack L, Zeyl C, Bell G, Sharbel T, Reboud X, Bernhardt T and K o e l e w y n H ( 1 9 9 4 ) I s o l a t i o n of four new strains of Chlamydomonas reinhardtii (Chlorophyta) from soil samples. J Phycol 30: 770–773 Sager R (1954) Mendelian and non-Mendelian inheritance of streptomycin resistance in Chlamydomonas reinhardi. Proc Natl Acad Sci USA 40: 356–363 Sager R (1955) Inheritance in the green alga Chlamydomonas reinhardi. Genetics 40: 476–489 Sager R and Granick S (1953) N u t r i t i o n a l studies with Chlamydomonas reinhardi. Ann New York Acad Sci 56: 831– 838 Sager R and Granick S (1954) Nutritional control of sexuality in Chlamydomonas reinhardi. J Gen Physiol 37: 729–742 Sager R and Palade GE (1954) Chloroplast structure in green and yellow strains of Chlamydomonas. Exp Cell Res 7: 584–588

Chapter 1

Introduction to Chlamydomonas

Salisbury JL (1995) Centrin, centrosomes, and mitotic spindle poles. Curr Opin Cell Biol 7: 39–45 Savard F, Richard C and Guertin M (1996) The Chlamydomonas reinhardtii LI818 gene represents a distant relative of the cabI/ II genes that is regulated during the cell cycle and in response to i l l u m i n a t i o n . Plant Mol Biol 32: 461–473 Schlösser UG (1994) SAG—Sammlung von Algenkulturen at the University of Göttingen. Bot Acta 107: 111–186 Schlösser UG, Sachs H and Robinson DG (1976) Isolation of protoplasts by means of a ‘species-specific’ autolysine in Chlamydomonas. Protoplasma 88: 51–64. Shimogawara K, Fujiwara S, Grossman A and Usuda H (1998) High efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics, in press Sizova I A , Lapina TV, Frolova ON, Alexandrova NN, Akopiants KE and Danilenko VN (1996) Stable nuclear transformation of Chlamydomonas reinhardtii with a Streptomyces rimosus gene as the selective marker. Gene 181: 13–18 Smith GM (1946) The nature of sexuality in Chlamydomonas. Amer J Bot 33: 625–630 Smith EF and Lefebvre PA (1997) The role of central apparatus components in flagellar motility and microtubule assembly. Cell Motil Cytoskeletonj 38: 1–8 Smith GM and Regnery DC (1950) Inheritance of sexuality in Chlamydomonas reinhardii. Proc Natl Acad Sci USA 36: 246– 248 Spanier JG, Graham JE and Jarvik JW (1992) Isolation and preliminary characterisation of three Chlamydomonas strains interfertile with Chlamydomonas reinhardtii (Chlorophyta). J Phycol 28: 822–828 Spessert R and Waffenschmidt S (1990) Studies on the vegetative autolysin during the vegetative life cycle in Chlamydomonas reinhardtii. Eur J Cell Biol 51: 17–22 Spreitzer RJ and Mets L (1981) Photosynthesis-deficient mutants of Chlamydomonas reinhardii with associated light-sensitive phenotypes. Plant Physiol 67: 565–569 Starr RC and Zeikus JA (1993) UTEX—the culture collection of algae at the University of Texas at Austin. J Phycol 29 suppl: 1–106 Stevens DR, Rochaix JD and Purton S (1996) The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Mol Gen Genet 251: 23–30 Taillon BE, Adler SA, Suhan JP and Jarvik JW(1992) Mutational analysis of centrin: An EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas. J Cell Biol 119: 1613–1624 Tam LW and Lefebvre PA (1995) Insertional mutagenesis and isolation of tagged genes in Chlamydomonas. Methods Cell Biol 47: 519–523 van den Ende H (1994) Vegetative and gametic development in the green alga Chlamydomonas. Adv Bot Res 20: 125–161 van den Ende H (1995) Sexual development in the homothallic

11 green alga Chlamydomonas monoica Strehlow. Sexual Plant Reprod 8: 139–142 Van W i n k l e - S w i f t KP and Aubert B ( 1 9 8 3 ) Uniparental inheritance in a homothallic alga. Nature 303: 167–169 VanWinkle-Swift KP and Hahn JH (1986) The search for matingtype-limited genes in the homothallic alga Chlamydomonas monoica. Genetics 1 1 3 : 601–619 Voigt J, Hinkelmann B, Liebich I and Mix M (1996) Alteration of the cell surface during the vegetative cell cycle of the unicellular green alga Chlamydomonas reinhardtii. Plant Cell Physiol 37: 726–733 Voigt J, Hinkelmann B and Harris EH (1997) Production of cell wall polypeptides by different cell wall mutants of the unicellular green alga Chlamydomonas reinhardtii. Microbiol Res 152: 189–198 Waffenschmidt S, Woessner JP, Beer K and Goodenough UW (1993) Isodityrosine cross-linking mediates insolubilization of cell walls in Chlamydomonas. Plant Cell 5: 809–820 Walne PL and Gualtieri P (1994) Algal visual proteins: An evolutionary point of view. Crit Rev Plant Sci 13: 185–197 Weger HG (1996) Interactions between respiration and inorganic phosphate uptake in phosphate-limited cells of Chlamydomonas reinhardtii. Physiol Plant 97: 635–642 Weger HG, Chadderton AR, Lin M, Guy RD and Turpin DH (1990) Cytochrome and alternative pathway respiration during transient ammonium assimilation by N-limited Chlamy­ domonas reinhardtii. Plant Physiol 94: 1131–1136 Wilson NF, Foglesong MJ, Snell WJ (1997) The Chlamydomonas mating type plus fertilization tubule, a prototypic cell fusion organelle: Isolation, characterization, and in vitro adhesion to mating type minus gametes. J Cell Biol 137: 1537–1553 Witman GB (1993) Chlamydomonas phototaxis. Trends Cell Biol 3: 403–408 Woessner JP and Goodenough UW (1989) Molecular charac­ terization of a zygote wall protein: An extensin-like molecule in Chlamydomonas reinhardtii. Plant Cell 1: 901–911 Woessner JP and Goodenough UW (1992) Zygote and vegetative cell wall proteins in Chlamydomonas reinhardtii share a common epitope, (SerPro)x. Plant Sci 83: 65–76. Woessner JP and Goodenough UW (1994) Volvocine cell walls and t h e i r c o n s t i t u e n t g l y c o p r o t e i n s : An e v o l u t i o n a r y perspective. Protoplasma 181: 245–258 Woessner JP, Molendijk AJ, Van Egmond P, Klis FM, Goodenough UW and Haring MA (1994) Domain conservation in several volvocalean cell wall proteins. Plant Mol Biol 26: 947–960 Wu LP, Hepler PK, John PCL (1997) The met 1 mutation in Chlamydomonas reinhardtii causes arrest at mitotic metaphase with persisting p34cdc2-like H1 histone kinase activity that can promote mitosis when injected into higher-plant cells. Protoplasma 199: 135–150

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Chapter 2

Perspectives on Early Research on Photosynthesis in Chlamydomonas Robert K. Togasaki and Stefan J. Surzycki Department of Biology, Indiana University, Bloomington, Indiana 47405, U.S.A.

Summary I. General Background II. The Levine Laboratory in the Early 1960s III. Establishment of Chlamydomonas reinhardtii as a Legitimate Model Organism IV. Development of New Techniques V. Emergence of New Research Targets VI. Old Experiments Becoming Reality Acknowledgment References

13 13 14 15 18 19 21 21 22

Summary Personal perspectives of the authors on research conducted in the laboratory of R. P. Levine at the Biological Laboratories, Harvard University in the latter half of the 1960s to the early 1970s is described. The chapter summarizes the state ofresearch in photosynthesis and chloroplast biology in the early 1960s. The authors recall experiments and events that led to the establishment of Chlamydomonas reinhardtii as a model organism for the study of photosynthesis and the molecular biology of chloroplasts. These reminiscences include personal anecdotes that try to convey the excitement, and elation and disappointments, that were experienced during these pioneering times in Chlamydomonas research. I. General Background As of 1964, the field of photosynthesis had several dominant topics. Most important among them were photosynthetic electron transport, photophos­ phorylation, and photosynthetic carbon assimilation. The precise measurement of photosynthetic activities in micro algae, pioneered by Warburg, led to the discovery of the red drop phenomenon by Emerson and Lewis (Emerson and Lewis, 1943). This was followed by the discovery of the enhancement phenomenon by Emerson’s group (Emerson et al., 1957), and Hill and Bendall’s proposal of two photosystems, the Z scheme, for photoelectron transport (Hill and Bendall, 1960). Subsequently the

Z scheme was elegantly corroborated by Duysen’s work on red algae (Duysens et al., 1961). Thus, the foundation for today’s generally accepted model of photosynthetic electron transport in photosynthetic eukaryotes and cyanobacteria was well established by the mid 1960’s. Furthermore, electron transport, using water as the electron donor and as the electron acceptor, was demonstrated in vitro by work in San Pietro’s laboratory at Johns Hopkins (San Pietro and Lang, 1957). Photophosphorylation in isolated chloroplast fragments was achieved by Arno’s group at Berkeley (Arnon et al., 1954). In addition, in the 1960s, work on the chemiosmotic basis of ATP synthesis by thylakoid membranes was initiated by

J. -D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 13–23. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

14 Jagendorf and coworkers in their pioneering paper ( H i n d and Jagendorf, 1963). In the area of photosynthetic carbon metabolism, the reductive photosynthetic carbon cycle was well established by mid-1950 (Bassham, 1957), but analysis of photosynthesis was just beginning with Kortschak’s paper (Kortschak et al., 1965). This was to be followed later by the elucidation ofthe carbon assimilation pathway by Hatch’s and Slack’s group (see Edwards and Walker, 1983). At this time, research with intact organisms was conducted mainly with cells of the unicellular green algae Chlorella, Scenedesmus, the cyanobacterium Anacystis and Euglena. The measurements made were either physical measurements, such as or gas exchange, or the fluorescence and biochemical analysis of fixation products. At the subcellular level, thylakoid membranes were isolated from plant leaves, mainly spinach and peas, for the analysis of photosynthetic electron transport and photophosphorylation. Chloroplasts with intact envelopes were also isolated successfully from these leaves and used extensively in studies of photo­ synthetic carbon metabolism (for review, see Edwards and Walker, 1983). Many plants and unicellular organisms were analyzed by the application of both biophysical and biochemical techniques, with a focus on the components of the photosynthetic apparatus and their function. The discovery of one component of the photosynthetic machinery in one organism was quickly extended to other systems in a quest to find all common components of the photosynthetic apparatus. For example, the discovery of plastocyanin in Chlorella was extended rapidly to other plant systems (Katoh et al., 1961) and its position in the electron transport chain was incorporated into most models for eukaryotic and cyanobacterial photo­ synthesis. In general, the emphasis was on refinement and exploitation of biophysical and biochemical research tools, better spectrophotometers, fluor­ imeters, and more specific chemical inhibitors. The idea that the experimental organism itself can offer a powerful research advantage remained dormant. Among these early studies, however, Duysens’ study of photosynthetic electron transport in Abbreviations: DCMU – 3-(3,4-dichlorophenyl) 1,1 -dimethyl­ urea; DC IP –2,6-dichloroindophenol; and – components of chloroplast ATP synthase; PMS – phenazine methosulfate; PS I – Photosystem I; PS II – Photosystem II; Rubisco – ribulose1,5-biphosphate carboxylase/oxygenase; TAP – trisacetate-phosphate

Robert K. Togasaki and Stefan J. Surzycki Porphyridium cruentum stands out as an example of superb exploitation of a biological trait, unique to a given experimental material (Duysens et al., 1961). In this red alga, light absorbed by an accessory pigment, phycoerythrin (563 nm), drives PS II while light absorbed by chlorophyll a (680 nm) drives PS I activities exclusively. Thus, Duysens could clearly demonstrate photoreduction and photooxidation of cytochrome f by PS II and PS I, respectively. This was an exceptional case; the more common approach was to use ever more powerful biochemical and biophysical tools on the usual experimental organisms. In an alternative approach, the alteration of the genetic constitution and hence the phenotype of a photosynthetic organism became a major research tool. This approach, pioneered by R. P. Levine with Chlamydomonas reinhardtii and N. Bishop with Scenedesmus obliquus (see Bishop, 1973), used the paradigm ofTatum and Beadle’s work of elucidating the order of enzymatic reactions in biochemical pathways through the study of mutants. It was reasoned that the best way to discover all of the components of the photosynthetic apparatus, as well as the mode and place of their action, would be to create mutants that affectphotosynthesis. One should marvel on the farsightedness of this approach at that time, realizing that it is still in use thirty years after its introduction into photosynthetic research. The analysis of photosynthetic mutants, using established experimental procedures began to yield new information by the early 1960s. At that time, the S. obliquus system was already yielding much new information whereas research on photosynthesis with C. reinhardtii was only beginning. However, the potential of C. reinhardtii as a model organism to study photosynthesis appeared much greater than for S. obliquus because of the possibility of genetic analysis. This possibility led one of us (R. Togasaki) to choose Levine’s laboratory over Bishop’s for postdoctoral studies.

II.The Levine Laboratory in the Early 1960s R. P. Levine began to use Chlamydomonas for his research by focusing on the isolation and charac­ terization of amino acid auxotrophs, as well as by studying genetic recombination mechanisms (Levine and Ebersold, 1958). By the early 1960s, the main focus of the laboratory shifted to the genetics and

Chapter 2 Perspective on Early Research biochemistry of photosynthesis. It is our under­ standing that the very low yield of amino acid auxotrophs and the high yield of acetate auxotrophs during early mutant screening was one factor for this change. The other was Levine’s association with Bob Smillie at Brookhaven National Laboratory and their collaborative work on photosynthesis (Levine and Smillie, 1962). In 1962, while Levine was away in France, N. Gillham, G. Hudock and G. Russell in Levine’s laboratory carried out a large scale isolation of acetate auxotrophs (Hudock, personal commun­ ication). They characterized some of these mutants as being deficient in photosynthesis by using an autoradiography screen developed earlier by Levine (Levine, 1960). Thus, by the fall of 1964, several papers on the photosynthetic electron transport system and photophosphorylation in Chlamydomonas had been published (for review, see Harris, 1988). In the fall of 1964, after Levine returned from France, three new postdoctoral fellows joined his laboratory, R. K. Togasaki, S.J. Surzycki and J.P. Hastings. Robert Togasaki arrived from the laboratory ofM. Gibbs where he hadjust finished his dissertation work on photosynthetic carbon metabolism in micro algae. He intended to study potential photosynthetic carbon cycle mutants in C. reinhardtii. Stefan Surzycki came from Gajewski’s laboratory in Warsaw where he was studying the mechanism of meiotic gene conversion in Ascobolus immersus. He, in turn, wanted to study the molecular basis of recombination using Chlamydomonas as an experimental system. Philip Hastings came from the Whitehouse laboratory at Cambridge University, England where he worked on the formulation of the molecular theory ofgenetic recombination. During their stay, they witnessed and participated in the evolution and development of approaches to study the function (photosynthesis) and structure (molecular biology) of the chloroplast in Chlamydomonas.

III. Establishment of Chlamydomonas reinhardtii as a Legitimate Model Organism During the 1960s, C. reinhardtii had to earn its citizenship in the community of photosynthetic research model organisms. The green algae Chlorella and Scenedesmus, the cyanobactenum Anacystis, as well as Euglena were dominant model organisms for biochemical, biophysical and physiological analysis of photosynthesis at the level of intact cells. Spinach

15 and peas were, in turn, favored material for in vitro studies using isolated, intact chloroplasts or thylakoid membranes. A researcher using C. reinhardtii had to face the dual task of establishing reliable experi­ mental procedures for this organism, and of demonstrating the unique advantage ofthis organism for the study of photosynthetic and molecular processes. By 1964, much of the biochemical and biophysical characterization of the photosynthetic apparatus had been accomplished, including the determination of the sequence ofevents from the photophysics of light absorption to the biochemical events of carbon assimilation. The notable exception was the oxygen evolving mechanism in PS II. Thus, much of early photosynthesis research activity in Chlamydomonas involved the isolation and characterization ofmutants deficient in photosynthesis. The aim of this research was to localize the site of the biochemical lesion in the photosynthetic apparatus caused by these mutations. Mutants lacking PS II and PS I activities, or photophosphorylation activity, and those lacking a component of photosynthetic electron transport, such as plastocyanin or cytochrome f, were discovered and characterized (see Chapter 31, Merchant). Figure 1 presents a diagram of the electron transport system and shows the positions of components affected in mutants isolated in Levine’s laboratory. Table 1 lists properties ofthese mutants. The study of these Chlamydomonas mutants confirmed, at first, the findings of published biochemical research. In addition, their genetic mapping provided the genetic basis for understanding the biochemical data and the organization of genes encoding photosynthetic components (Hastings et al., 1965). However, very soon, the mutant approach yielded new and unexpected results. The first major demonstration of an advantage of the Chlamydomonas system came with the unequiv­ ocal demonstration of the positions of cytochrome f and plastocyanin in the electron transport chain (Gorman and Levine, 1966b). At that time, the copperchelating agent, salicylaldoxime, was shown to inhibit photoreduction of cytochrome f. Since plastocyanin is a copper-containing protein, the results implied that plastocyanin is located before cytochrome f in the electron transport chain (Fork and Urbach, 1965). Gorman and Levine used two mutants in their study, one lacking cytochrome f activity (ac-206), and the other, missing plastocyanin (ac-208). In the absence of plastocyanin (ac-208), cytochrome f was

16

photoreduced by PS II but not photooxidized by PS I. The absence of cytochrome f in ac-206 did not prevent photooxidation of the exogenously added reductant (ascorbate and DCIP) by PS I, while the absence of plastocyanin did. These data showed that cytochrome f preceded plastocyanin in the electron transport chain. The Gorman and Levine experiments demonstrated the usefulness of mutants in photo­

Robert K. Togasaki and Stefan J. Surzycki

synthetic research and illustrated the danger of artifacts that can arise from the use of chemical inhibitors in in vitro experiments. Gorman and Levine’s conclusions were later confirmed for spinach chloroplasts (Kimimura and Katoh, 1972). Moreover, this research was also an early example of data from Chlamydomonas being confirmed by later bio­ chemical work and not the other way around. Robert

Chapter 2 Perspective on Early Research Togasaki recalls the following episode. A very prominent researcher in the photosynthetic electron transport field was visiting the Levine laboratory during the time when work with mutants ac-208 and ac-206 was in progress. He was shown the latest data on the relative positions of cytochrome f and plastocyanin in the electron transport chain. His comment was, ‘Well, is that how you want to order them? But, most other people think otherwise!’ This was symptomatic of the time when the power of the genetic approach was not fully appreciated by established researchers in the field. Another important mutant isolated during this time was ac-21. This mutation identified a new essential component, called M, in the photosynthetic electron transport chain located between PS II and cytochrome f (Levine and Smillie, 1962; Gorman and Levine 1966a; Levine 1968). Many years later it was found that M is the Rieske iron-sulfur protein (Bendall et al., 1986). One advantage ofworking in a new field is thatjust about anything you do or find is novel. Eventually many of these findings pioneered new fields of research. Some of the early doctoral dissertations from Levine’s laboratory centered on the isolation and characterization of a single mutant affecting a key component ofthe photosynthetic apparatus. These were: The isolation and characterization of the first mutant defective in PS I function, ac-80 (Givan and Levine, 1967). A. Givan showed that ac-80 did not display the absorbance change associated with P700. The mutant could not reduce with electrons from either water or an artificial donor (the dye DCIP and ascorbate), but it had considerable Hill activity. The cells could not carry out cyclic photophosphorylation but were capable still ofnoncyclic photophosphorylation coupled to the reduction of ferricyanide. These experiments allowed them to conclude that one coupling site is situated between the two photosystems. Analysis of a 520 nm light-induced absorbance change (Chua and Levine, 1969). N. -H. Chua studied the 520 nm light-induced absorbance change in wild type and in mutants deficient in electron transport to analyze the function of quinones in the electron transport system.

17 Effects ofmanganese on photosynthetic activities and chloroplast structure of C. reinhardtii (Teichler-Zallen and Levine, 1969). D. TeichlerZallen found that Mn deficiency led to severe reduction of PS II activity but did not affect PS I activity. The extent of thylakoid membrane stacking decreased in parallel to the reduction of PS II activity. This established a significant role for Mn in the structure and function ofthylakoid membranes. Isolation and characterization ofthe first mutant lacking phosphoribulokinase activity with near wild type levels of several other Calvin cycle enzymes, F-60 (Moll and Levine, 1970). B. Moll provided the first clear genetic evidence to support the Calvin cycle. Interestingly, in spite of extensive searching, this mutant remained the only photosynthetic carbon cycle mutant until the isolation of Rubisco mutants (Spreitzer and Mets, 1980; Chapter 27, Spreitzer). The phosphoribulokinase mutation eventually was used in many research projects including studies of fermentation and photohydrogen evolution and the hydrogenase system by Gibbs and coworkers (for review, see Togasaki and Whitmarsh, 1983). Isolation and characterization of the first mutant deficient in photophosphorylation that lacked coupling factor, F-54 (Sato et al., 1971). V. Sato provided the first genetic evidence, linking the absence of thylakoid membrane surface particles, the lack of ATPase, and photophosphorylation activity to a single mutation. Analysis of the structure of chloroplast DNA using electron microscopy (Rochaix, 1972). This work was one of the first attempts to create a physical map of organelle DNA. Excitation energy transfer and chlorophyll orientation was studied in intact cells of Chlamydomonas (Whitmarsh and Levine, 1974). Fluorescence polarization for intact cells was measured with horizontally polarized exciting light, in the presence and absence of DCMU to test the validity of the Förster mechanism of excitation energy transfer.

18 The pioneering work on chloroplast genetics and biology of Chlamydomonas was not restricted to the Levine’s laboratory at this time. Ruth Sager and Nicholas Gillham performed independently an extensive genetic analysis of uniparental inheritance in Chlamydomonas (Sager, 1960; Gillham, 1965). Laurie Mets and Laurie Bogorad began the analysis of the chloroplast translational apparatus, studying erythromycin binding by plastid ribosomes (Mets and Bogorad, 1971).

IV. Development of New Techniques Since C. reinhardtii was a new model organism, most of the experimental techniques had to be modified or developed de novo. A few examples given below will demonstrate the effort ofthis task in the early period of working with this alga in Levine’s laboratory. In 1960, R. P. Levine developed an effective screening method to identify mutants deficient in photosynthesis (Levine, 1960a). Mutagenized cells were plated on acetate supplemented agar plates, and allowed to develop colonies that, in turn, were replicated to minimal media plates to screen for acetate auxotrophs. Acetate auxotrophic colonies were replica plated and allowed to carry out assimilation in the light. The incorporation was stopped by exposure to acid, and colonies were incorporation by autoradiography. analyzed for This double screen, involving both acetate auxotrophy and autoradiography, was labor intensive and lacked a positive selection for mutants. However, this test was the least biased mutant isolation procedure developed to date. It is no wonder, therefore, that this test yielded many different photosynthetic mutants affecting electron transport, photophosphorylation and carbon metabolism. All of the early acetate requiring mutants isolated in Levine’s laboratory were obtained using this procedure. In the summer of 1966, P. Bennoun visited the Levine laboratory and developed a rapid screen for the isolation of photosynthetic mutants. This procedure was based on the fluorescent light output ofindividual colonies (Bennoun, 1967). Mutagenized cells were plated on acetate containing plates (TAP), allowed to form colonies, irradiated with blue light and photographed through a red filter using a red and infrared sensitive Polaroid film. Colonies of the mutants with defects localized after PS II gave high

Robert K. Togasaki and Stefan J. Surzycki fluorescence and could be easily differentiated from wild type colonies with normal fluorescence. While a positive selection for mutants deficient in photosynthesis was still absent in this procedure, the simplicity and rapidity ofthis detection method more than made up for it. The development of this elegant procedure literally began with a big bang! In the very first experiment, the absence of a heat filter between the Zeiss microscope lamp and the primary blue glass filter caused the expensive glass filter to overheat and shatter to pieces. The new method permitted Bennoun, Chua, and Togasaki, to isolate and partially characterize a large number of photosynthesis negative mutants in short time. A similar method was published by Garnier’s group (Garnier, 1967). This non-invasive screening method for photosynthetic mutants was eventually adopted for plants, in particular maize, by D. Miles (Miles and Daniel, 1973). The Bennoun screening procedure is an elegant example of a hybrid between the genetic and microbiological advantage of the Chlamydomonas system with the powerful biophysical tool of fluorescence analysis (Chapter 23, Bennoun and Béal). It was extended later to a method capable of analyzing fluorescence kinetics ofindividual colonies that became a powerful tool in the isolation of photosynthetic electron transport and photophos­ phorylation mutants, and is still used today to isolate mutants and study the phenotypes of strains carrying site-directed mutations. While a well established procedure for assaying photophosphorylation by spinach thylakoid mem­ branes existed by then, the spinach procedure did not work with Chlamydomonas cells. Trying to adapt the method developed for spinach to Chlamydomonas, D. Gorman found that grinding cells in mortar with sand was the only effective means of cell disruption for obtaining chloroplast fragments capable of noncyclic phosphorylation (Gorman and Levine, 1965). This method made it possible to isolate and characterize the first photophosphorylation negative mutant (Sato et al., 1971). It is said that a noted expert in the photophosphorylation field was visiting the Levine laboratory when this work was in progress. He saw how thylakoid fragments with photophos­ phorylation activity were isolated from Chlamy­ domonas by grinding the cells with sand. He was bemused, and set out to demonstrate how it should really be done. After a period of frustration, he also began using sand grinding for his experiments. This procedure was in use for more than 10 years until

Chapter 2

Perspective on Early Research

Brand, in San Pietro’s laboratory, developed a more convenient and rapid preparation procedure (Brand et al, 1975). When Togasaki joined the Levine laboratory, fixation was measured in 25 ml flasks using 2 ml of cell suspension. One flask was used for one time point to measure the rate of photosynthetic carbon fixation after stopping the reaction by injecting acid into the flask. A number of flasks were required for a single kinetic experiment. Togasaki modified this method by using the newly available automatic pipettor to withdraw samples. This reduced sample size to 0.1 ml or less, and made the kinetic analysis of fixation more rapid, more reliable and less expensive. In the process, he also developed a convenient method to control the gas composition fixation (Togasaki and Botos, 1972). during The Levine laboratory also developed a new liquid growth medium for culturing photosynthetic mutants that remains in use presently. Sueoka’s high salt medium, commonly used at this time, formed a precipitate after autoclaving and the liquid medium appeared cloudy. To analyze the growth rate of the cells, the change in optical density at 750 nm was used usually as an index of cell growth. The precipitate in the liquid medium interfered with these measure­ ments. Furthermore, to maintain the pH of the medium at pH 7.0, Sueoka’s medium used phosphate buffer, the pK of which is 6.83. As acetate was consumed by growing Chlamydomonas cells, the pH of the medium increased to the point where the phosphate buffer ceased to have buffering capacity. Gorman and Levine devised Tris Acetate Phosphate (TAP) medium that solved both problems. The TAP medium does not form a precipitate during autoclaving and the correct pH is maintained. In the course ofstudies ofchloroplast development and biogenesis, synchronously grown cultures of Chlamydomonas were required. Existing methods for synchronizing algae were time consuming and resulted in very poor synchrony of cell division. To remedy this, Surzycki developed a new method of synchronizing cell division in Chlamydomonas using a specific light and temperature regime (Surzycki, 1967).

V. Emergence of New Research Targets During the 1960s, the focus of the entire laboratory was largely on the isolation of new mutants and the

19 analysis of components of the photosynthetic apparatus affected in these mutants. Thus, the study of one of these photosynthetic mutants, ac-20, yielded quite unexpected and surprising results that led some of us to the study of chloroplast biogenesis. At the beginning of 1965, Levine asked R. Togasaki to analyze a number of acetate requiring mutants that still retained the ability to fix in the light at wildtype levels. The basic idea was that these mutants were normal in their photocarboxylation capacities but were defective at some later step in the photosynthetic carbon cycle. Determining these steps was a very interesting problem. This kind of study could possibly uncover unknown steps in photo­ synthesis. The photo-assimilation of in such mutants should result in the accumulation of radioactive intermediates that would indicate the site of the genetic lesion leading to their unusual phenotype. To tackle this problem, R. Togasaki developed a high resolution (for that time) column chroma­ tography method for the analysis of radioactive products of short term photosynthesis. Using these columns he compared the product profile of a short term labeling of the mutant cells to that of wild-type cells. R. Togasaki analyzed seven mutants which were provided by Levine from his mutant collection. All ofthese mutants, except ac-20, yielded a product profile indistinguishable from wild-type cells. In contrast, ac-20 produced the same level of radioactive products of light-independent fixation as wild type cells, but much reduced products fixation. Moreover, as of light-dependent R. Togasaki learned later, this mutant was the only mutant in the group that fixed at a much lower rate than wild type. Upon further analysis, he found that the ac-20 cells had greatly reduced ribulose-1,5bisphosphate carboxylase (Rubisco) activity. At first, it was presumed, that the genetic lesion in these cells resulted from defective Rubisco function (Levine and Togasaki, 1965). However, ac-20 cells did not die on minimal medium, and much to our surprise, had a much higher Rubisco activity when grown on minimal medium than when grown on acetate supplemented medium. With background training in pure biochemical analysis of photo­ synthetic carbon metabolism, R. Togasaki was not ready to handle this new phenomena, namely the regulation of enzyme biosynthesis. To learn more about regulation of enzyme biosynthesis he attended a Gordon conference on the regulation of enzyme

20 synthesis in the summer of 1965 and discovered that 90% of the meeting was focused on the regulation of the lac operon. Armed with a new perspective, he began to analyze the effect of growth conditions on Rubisco activity in ac-20 cells. The conclusion of this research was, that in ac-20 cells, cultured in acetate in either the light or dark, there is a block in the biosynthesis of Rubisco. This block is removed during the incubation of the cells in the absence of acetate in either light or dark conditions. Because Rubisco activity in this mutant was light-dependent, the removal of acetate, when cells were grown in the dark, would not result in synthesis of the enzyme. The appearance of Rubisco activity had a distinct time lag suggesting that de novo enzyme synthesis occurred at this time rather than induction of activity (Togasaki and Levine, 1970). Clearly a new approach was needed to solve the problem of induction of Rubisco activity in the mutant cells of ac-20. Around this time, Ursula Goodenough began to examine the structure of photosynthetic Chlamy­ domonas mutants by electron microscopy (Goode­ nough and Levine, 1969). Analyzing the structure of the chloroplast in light- or dark-grown cells of ac-20 she discovered that, in the presence of acetate, in the light or dark, this mutant had a very small number of chloroplast ribosomes which increased upon removal of acetate. Thus, she identified the chloroplast ribosomes as the rate limiting block in the biosynthesis of Rubisco in these cells. She further demonstrated the participation of chloroplast ribosomes in Rubisco biosynthesis and went on to characterize this regulatory phenomena in more detail (Goodenough and Levine, 1970). This research added a cell biology perspective to the laboratory’s research. This was one of the early examples of regulation of biosynthesis of the photosynthetic apparatus. Subsequent work in Levine’s laboratory extended this approach to the analysis of the site of coding and synthesis of many components of the chloroplast photosynthetic, transcriptional andtranslational apparatus (Surzycki, 1969; Surzycki et al., 1970; Armstrong et al. 1971; Beck and Levine, 1974). It is intriguing looking back today, that some ofthe ‘anomalies’ we found at the time, became the starting point for very fruitful later investigations. For example, in their analysis of the ac-208 mutant, Gorman and Levine found that these cells easily acquired some suppressor mutations that could restore much ofthe photosynthetic activity. This observation

Robert K. Togasaki and Stefan J. Surzycki eventually led to the work of Wood on regulation of plastocyanin and cytochrome (now called biosynthesis (Wood, 1978). His work was followed by an elegant study of copper involvement in the regulation of plastocyanin (Merchant and Bogorad, 1986). Another observation was that in order to have ac-208 cells with a ‘non-suppressed’ phenotype, the cells ofthis mutant must always be maintained in the logarithmic growth phase. We had no idea what caused this phenomenon. In hindsight, the organism was trying to tell us that there is an interesting and complex regulatory problem awaiting investigation. Goodenough provided the combined analysis of thylakoid membrane structure and photosynthetic electron transport activities on ac-31,a mutant devoid of thylakoid membrane stacking (Goodenough et al., 1969). This paper, on the effect of membrane architecture on photosynthetic efficiency, was another seminal work for the field of excitation energy distribution. When S. Surzycki and P. Hastingsjoinedthe Levine laboratory in 1964, genetics and molecular biology of the organelle, in this case the chloroplast, was in its infancy. A quote from a 1970 paper reflects the new direction in the Levine laboratory that began to emerge at this time. ‘In the early 1960s, the presence of DNA and RNA in the chloroplast was beginning to be documented. At this time, several models were published that depicted a highly autonomous chloroplast carrying the synthesis of most of its components, and largely independent of the cytoplasm, except for the supply of small metabolites. Such models were of some puzzlement to students of Chlamydomonas, for during the same period many genes controlling chloroplast functions were being mapped to what appeared to be classical nuclear linkage groups.’ (Surzycki et al., 1970). Genetic data and specific chemical tools were combined to study chloroplast structure and function at a molecular level. Characterized mutants and antibiotics that specifically inhibit chloroplastic transcription (rifampicin) ortranslation(spectinomycin), were used to ask more molecular questions, namely the sites of transcription and translation for some chloroplast components. The work of Surzycki and Amstrong (Surzycki et. al., 1970; Amstrong et al., 1971) established the similarity between the effect of spectinomycin and the ac-20 mutation which reduced the level of 70S ribosomes in the presence of acetate. Using an

Chapter 2 Perspective on Early Research inhibitor of chloroplast translation together with rifampicin, an inhibitor of transcription in chloro­ plasts, they determined the site of synthesis and transcription of many components of the photo­ synthetic and genetic apparatus ofthe chloroplast. In a variation of this approach they used transcriptional and translational inhibitors in synchronized cultures of wild-type cells. This permitted the analysis of transcription and translation in normal, photo­ synthetically active cells, that were in the same physiological state at a given time. This refinement permitted the analysis of the chloroplastic trans­ cription and translation requirements, as well as cytoplasmic translational requirements for several chloroplast components. The work of P. Hastings, J.-D. Rochaix and Surzycki on the regulation of expression and on the structure of the ribosomal gene cluster was also carried out in the Levine laboratory (Surzycki and Hastings, 1968; Surzycki and Rochaix, 1971). This work was one of the first examples of the application of molecular biology methods to the field of chloroplast biogenesis in Chlamydomonas. Thus, the molecular analysis of chloroplast biogenesis was well on its way.

VI. Old Experiments Becoming Reality During the 1960s, the investigation of chloroplasts and mitochondria took place separately in cell free preparations. However, in intact organisms, these organelles coexist and function in close proximity within the same cell. Since chloroplast or mitochon­ drial functions can be genetically perturbed in Chlamydomonas, this alga should provide a powerful tool for the analysis of interactions between these organelles. A glimpse of this possibility emerged in 1967, and recent findings appear to support it. In 1967, Robert Togasaki attempted to duplicate a fixation protocol designed for spinach chloroplasts using intact cells of Chlamydomonas. He reasoned that if DCIP/ascorbate can supply electrons to PS I and cyclic photophosphorylation can supply ATP, the cell should be able to photoassimilate in the absence of PS II contribution (namely in the presence of 0.1 mM fixationby spinach chloroplasts DCMU). Since was favored in a nitrogen atmosphere, his experiments were also carried out under nitrogen. The results showed a small but significant light-dependent

21 fixation by the cells in the presence of 0.1 mM DCMU. However, in the absence of ascorbate and even of DCIP, control cells fixed Further experiments showed that DCMU inhibition of fixation was complete only for cells kept in aerobic conditions. There was always a significant rate of fixation in the presence of DCMU when the nitrogen atmosphere was maintained. Furthermore, addition of DCIP and especially PMS increased this anaerobic, DCMU-independent, fixation substantially (Levine, 1969). Togasaki concluded that there must be an electron donor pool within Chlamydomonas cells, and both atmospheric oxygen, and PS I are competing for it. In air, oxygen out competes PS I due to its more positive redox potential. However, in the absence of oxygen, PS I serves as an alternate electron acceptor. This PS II independent fixation under nitrogen suggests the diversion of electrons from mitochondria to the chloroplast. Thus, we can imagine a flow of electrons between chloroplasts and mitochondria. If this prediction is true then mutants defective in oxidative electron transport should fix even under aerobic conditions in the presence of DCMU. Recent data of G. Peltier, K. Redding and P. Bennoun reveal that a small amount ofPS II-dependent electron flow occurs in the absence of PS I, but that the reducing power is somehow transported and fed into the mitochondrial electron transport chain thus revealing the existence ofelectron traffic between the two organelles (Chapter 18, Redding and Peltier; Chapter 35, Bennoun). In summary, during the latter half of the 1960s to the early 1970s, we were exploring the research potential of Chlamydomonas and witnessed the emergence of many areas of research. It was a very productive period, and one of the major driving forces was R. P. Levine’s abundant curiosity and drive, coupled with his ability to assimilate new thinking and data, from both within and outside of his laboratory. He provided us with freedom to pursue our ideas and with an environment which stimulated productive interactions. It was an exciting time.

Acknowledgment The authors wish to express their thanks to Judith A. Surzycki for reading, discussion and editorial advice in the preparation of this manuscript.

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References Armstrong JJ, Surzycki SJ, Moll, B and Levine RP (1971). Genetic transcription and translation specifying chloroplasts components in Chlamydomonas reinhardi. Biochemistry 10: 692–701 Arnon DL, Whatley FR and Allen, MB. (1954). Photosynthesis by isolated chloroplasts. II. Photosynthetic phosphorylation, the conversion of light into phosphate bond energy. J Am Chem Soc 76: 6324–6329 Beck DP and Levine RP (1974). Synthesis of chloroplast membrane polypeptides during synchronous growth of Chlamydomonas reinhardi.. J. Cell Biol 63: 759–772 Bendall DS, Sanguansermsri M, Girard-Bascou J and Bennoun P (1986) Mutations of Chlamydomonas reinhardtii affecting the cytochrome complex. FEBS Lett 203: 31–35 Bennoun P and Levine RP (1967 ). Detecting mutants that have impaired photosynthesis by their increased level of fluorescence. Plant Physiol 42: 1284–1287 Bassham JA and Calvin M (1957) The path of carbon in photosynthesis. Prentice Hall, Englewood Cliffs Bishop NI (1967) Analysis of photosynthesis in green algae through mutation studies. In: Giese AC (ed) Photophysiology, Vol 8, pp 65–96. Academic Press, New York Brand JJ, Curtis VA, Togasaki RK and San Pietro A (1975) Partial reaction of photosynthesis in briefly sonicated Chlamydomonas. II Photophosphorylation activities. Plant Physiol 55: 187–191 Chua N and Levine RP (1969) The photosynthetic electron transport chain of Chlamydomonas reinhardi. VIII. The 520 nm light induced absorbance change in the wild type and mutant strains. Plant Physiol 44: 1–6 Duysens LNM, Amesz J and Kamp BM (1961) Two photo­ chemical systems in photosynthesis. Nature 190: 157–161 Edwards G and Walker DA (1983) C3, C4: Mechanisms and cellular and environmental regulation of photosynthesis. University of California Press, Berkeley Emerson R and Lewis CM (1943) The dependence of the quantum yield of Chlorella photosynthesis on wave length of light. Am J Bot 30: 126–139 Emerson R, Chalmers RV and Cederstand C (1957) Some factors influencing the long-wave limit of photosynthesis. Proc Natl Acad Sci USA 43: 133–143 Fork DC and Urbach W (1965) Evidence for the localization of plastocyanin in the electron transport chain of photosynthesis. Proc Natl Acad Sci USA 53: 1307–1315 Garnier J (1967) Une methode de detection, par photographie, de souches d’Algues vertes emettant in vivo une fluorescence anormale. CR Acad Sci, Ser D 265: 874–877 Gillham NW (1965) Linkage and recombination between non­ chromosomal mutations in Chlamydomonas reinhardi. Proc Natl Acad Sci USA 54: 1560—1567 Goodenough U W and Levine RP (1969) chloroplast ultrastructure in mutant strains of Chlamydomonas reinhardtii lacking components of the photosynthetic apparatus. Plant Physiol 44: 990–1000 Goodenough UW and Levine RP (1970) Chloroplast structure a mutant strain of Chlamydomonas and function in reinhardtii. III. Chloroplast ribosomes and membrane organization. J Cell Biol 44: 547–562

Robert K. Togasaki and Stefan J. Surzycki Goodenough UW, Armstrong JJ and Levine RP (1969) Photosynthetic properties of a mutant strain of Chlamydomonas reinhardtii devoid of chloroplast membrane stacking. Plant Physiol. 44: 1001–1012 Gorman DS and Levine RP (1965) Cytochrome f and plastocyanin: Their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 54: 1665–1669 Gorman DS and Levine RP (1966a) Photosynthetic electron transport chain of Chlamydomonas reinhardtti. III. Lightinduced absorbance changes in chloroplast fragments of the wild type and mutant strains. Plant Physiol 41. 1293–1300 Gorman DS and Levine RP (1966b) Photosynthetic electron transport chain of Chlamydomonas reinhardtti. VI. Electron transport in mutant strains lacking either cytochrome 553 or plastocyanin. Plant Physiol 41: 1648–1656 Harris EH (1988) The Chlamydomonas Sourcebook. A Comprehensive Guide to Biology and Laboratory Use. Academic Press, New York Hastings PJ, Levine EE, Cosbey E, Hudock MO, Gillham NW, Surzycki SJ, Loppes R and Levine RP (1965) The linkage groups of Chlamydomonas reinhardi. Microb Genet Bull 23: 17–19 Hill R and Bendall F (1960) Function of the two cytochrome components in chloroplasts: A working hypothesis. Nature 186: 155–156 Hind G and Jagendorf A (1963) Separation of light and dark stages in photophosphorylation. Proc Natl Acad Sci USA 49: 715–722 Katoh S, Suga I, Shiratori I and Takamiya A. (1961) Distribution of plastocyanin in plants, with special reference to its local­ ization in Chloroplasts. Arch Biochem Biophys 94: 136–141 Kimimura M and Katoh S (1972) Studies on electron transport associated with Photosystem I.I. Functional site of plastocyanin: Inhibitory effects of HgCl 2 on electron transport and plastocyanin in chloroplasts. Biochim. Biophys. Acta, 283: 279–292 Kortschak HP, Hartt CE and Burr GO (1965) Carbon dioxide fixation in sugarcane leaves. Plant Physiol 40: 209–213 Levine RP (1960a) A screening technique for photosynthetic mutants in unicellular algae. Nature 188: 339–340 Levine RP (1960b) Genetic control of photosynthesis in Chlamydomonas reinhardtii Proc Natl Acad Sci USA 46: 972–977 Levine RP (1968) Genetic Dissection of photosynthesis in Chlamydomonas reinhardtii. Science 162: 768–771 Levine RP (1969a) A light-induced absorbance change at 564 nm in wild-type and mutant strains of Chlamydomonas reinhardi. In: MetzneR H (ed) Proc. International Congress of Photosynthesis Research. Progress in Photosynthesis II, pp 971–977. International Union of Biological Sciences, Tuebingen Levine RP (1969b) The analysis of photosynthesis using mutant strains of algae and higher plants. Annual Rev Plant Physiol 20: 523–543 Levine RP and Ebersold WYE (1958) Gene recombination in Chlamydomonas reinhardi. Cold Spring Harbor Symp Quant Biol. 23: 101–109 Levine RP and Goodenough UW (1970) The genetics of photosynthesis and of the chloroplast in Chlamydomonas reinhardi. Annual Rev Genetics 4: 397–408

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Levine RP and Smillie RM (1962) The pathway of triphospho­ pyridine nucleotide photoreduction in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 48: 417–421 Levine RP and Togasaki RK (1965) A mutant strain of Chlamydomonas reinhardi lacking ribulose diphosphate carboxylase activity. Proc Natl Acad Sci USA 53: 987–990 Merchant S and Bogorad L (1986) Regulation by copper of the expression of plastocyanin and cytochrome c552 in Chlamydomonas reinhardti. Mol Cell Biol 6: 462–469 Miles CD and Daniel DJ (1973) A rapid screening technique for photosynthetic mutants of higher plants. Plant Science Letters 11: 237–240 Mets LG and Bogorad L (1971) Mendelian and uniparental alterations in erythromycin binding by plastids ribosomes. Science 174: 707–709 Moll B and Levine RP (1970) Characterization ofa photosynthetic mutant strain of Chlamydomonas reinhardti deficient in phosphoribulokinase activity. Plant Physiol 46: 576–580 Rochaix JD (1972) Cyclization of chloroplast DNA fragments of Chlamydomonas reinhardi. Nature New Biol 238: 76–78 San Pietro A. and Lang HM (1957) Photosynthetic pyridine nucleotide reductase. 1 Partial purification and properties of the enzyme from spinach. J Biol Chem 231: 211–227 Sato VL, Levine RP and J. Neumann J (1971) Photosynthetic phosphorylation in Chlamydomonas reinhardti. Effects of a mutation altering an ATP synthesizing enzyme. Biochim BiophysActa 253: 437–448 Sager R (1960) Genetic Systems in Chlamydomonas. Science 132: 1459–1465 Spreitzer RJ and Mets LJ (1980) Non-mendelian mutation affecting ribulose-1,5-bisphosphate carboxylase structure and activity. Nature 285: 114–115 Surzycki SJ (1969) Genetic functions of the chloroplast of Chlamydomonas reinhardi: Effects of rifampin on chloroplast DNA-dependent RNA polymerase. Proc Natl Acad Sci USA 63: 1327–1334

23 Surzycki SJ (1971) Synchronously grown culture of Chlamy­ domonas reinhardi. Methods Enzymol 23: 67—73 Surzycki SJ and Hastings PJ (1968) Control of chloroplast RNA synthesis in Chlamydomonas reinhardi. Nature 220: 786–787 Surzycki SJ and Rochaix JD (1971) Transcriptional mapping of ribosomal RNA genes of the chloroplast and nucleus of Chlamydomonas reinhardi. J Mol Biol 62: 89–109 Surzycki SJ, Goodenough UW, Levine RP and Armstrong JJ (1970) Nuclear and chloroplast control of chloroplast structure and function in Chlamydomonas reinhardti. Symposia Soc Exp Biol 24: 13–37 Teichler-Zallen, D. and R. P. Levine. (1969). The effect of manganese on chloroplasts structure and photosynthetic ability of Chlamydomonas reinhardi. Plant Physiol 44:701–710 Togasaki RK and Botos CR (1971) Enhanced dark fixation by preilluminated algae: A tool for analysis of photosynthetic mechanisms in vivo. In: Forti G, Avron M and Melandri A (eds) Proceedings IInd International Congress on Photo­ synthesis Research, Vol 3, pp 1759–1772. Dr. W. Junk N.V. Publishers, The Hague Togasaki RK and Levine RP (1970) Chloroplast structure and function in ac-20, a mutant strain of Chlamydomonas reinhardi. I. CO2. fixation and riublose-l,5-diphosphate carboxylase synthesis. J Cell Biol 44: 531–539 Togasaki RK and Whitmarsh J. (1986) Multidisciplinary research in photosynthesis: a case history based on the green alga Chlamydomonas. Photosynthesis Research 10: 415–422 Whitmarsh J and Levine RP (1974) Excitation energy transfer and chlorophyll orientation in the green alga Chlamydomonas reinhardi. Biochim Biophys Acta 368: 199–213 Wood PM (1978) Interchangeable copper and iron proteins in algal photosynthesis. Studies on plastocyanin and cytochrome c552 in Chlamydomonas. Eur J Biochem 87: 9–19

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Chapter 3 Organization of the Nuclear Genome Carolyn D. Silflow Department of Genetics and Cell Biology, Department of Plant Biology, Plant Molecular Genetics Institute, University of Minnesota, 1445 Gortner Ave., St. Paul, MN 55108, U.S.A.

Summary I. Introduction and Scope II. General Characteristics of the Nuclear Genome Organization of the Genome A. Interspersed Repeated Sequences 1. Transposable Elements 2. Transcribed Repetitive DNA Sequences B. Tandemly Repeated Sequences 1. Large Tandem Repeats 2. Simple Sequence Repeats 3. Telomere Repeat Sequences 4. Ribosomal DNA C. Low Copy Number Sequences (Gene Families) D. The Mating-Type Locus IV. Characteristics of Chlamydomonas Genes Transcribed by Polymerase II A. Promoters B. Codon Bias C. Introns D. Translation Start Codon and Stop Codon Sequence Context E. 3´ Noncoding Sequences IV. Physical Mapping of the Chlamydomonas Genome VI. Future Prospects Acknowledgments References

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Summary The unicellular green alga Chlamydomonas reinhardtii is an outstanding system for investigation of numerous cellular processes including photosynthesis and other metabolic pathways, biogenesis of organelles, assembly and motility of flagella, gametogenesis and mating, phototaxis and circadian rhythms. Genetic studies have generated mutations at more than 200 nuclear loci whose products function in these processes. Recent advances in molecular genetic techniques including transformation, expression of selectable marker genes, insertional mutagenesis, and genetic rescue methods have facilitated the isolation of genes identified by mutation. The nuclear genome has a sequence complexity approximately 20–30 times that of Escherichia coli, a GC content of 62%, and a large proportion of unique sequences. Among the classes of repeated sequences in the genome are several families of transposable elements which have proven useful for gene tagging, and the interspersed simple sequence repeat DNA sequences in the mating-type locus are highly rearranged between the genomes. DNA sequences obtained from approximately 200 genes and cDNAs have led to a general and picture of gene structure that includes features such as biased codon usage, a high frequency of small introns, J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 25–40. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

Carolyn D. Silflow

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and a putative polyadenylation signal that differs from the consensus in other eukaryotes. Genes located in the mating-type locus lack some of these features. The construction of a molecular map of the genome has utilized polymorphic interfertile strains to align molecular markers with the genetic map. The molecular map, together with genomic libraries in YAC (yeast artificial chromosome) and cosmid vectors, presents opportunities for further physical mapping of the genome.

I. Introduction and Scope This review will focus on the nuclear genome of Chlamydomonas reinhardtii, a species that provides distinct experimental advantages for the investigation ofnumerous biological processes. Outside the scope of this article are several other Chlamydomonas species whose phylogenetic relationships to C. reinhardtii are relatively distant. Also outside the present scope are the genomes of Volvox and other related multicellular species. The fascinating questions that remain regarding the evolutionary relationships among the genomes of this group of algal species have been discussed (Schmitt et al., 1992; Liss et al., 1997). Many aspects of the C. reinhardtii genome were reviewed previously by Harris (1989) and by Rochaix (1995). This review will summarize work that has provided insight into the organization of the nuclear genome and the structure of Chlamydomonas genes. Information gained from these studies may be particularly relevant for further development of molecular genetic tools for this system.

II. General Characteristics of the Nuclear Genome Estimates of the DNA content ofthe haploid genome (Harris, 1989). The range from genetic map consists of 17 linkage groups on which nearly 200 molecular markers have been mapped (Dutcher et al., 1991; Harris, 1993). Chromatin made from isolated nuclei contains polynucleosomes, the complement ofhistone proteins typical of eukaryotes, and a nucleosomal DNA repeat length of 189 bp (Morris et al., 1990). One unusual feature of the genome is its high GC content of 62% (Harris, 1989). In contrast to DNA from higher plants, relatively little methylation of cytosine residues in Abbreviations: PCR – polymerase chain reaction; RFLP – restriction fragment length polymorphism; YAC – yeast artificial chromosome

the CpG dinucleotide was detected in Chlamy­ domonas nuclear DNA (Day et al., 1988). Con­ clusions from early studies of genome organization differed as to the content of repetitive DNA in the nuclear genome, ranging from estimates of almost none to 30% (reviewed by Harris, 1989). Studies in the past dozen years have characterized numerous classes of repetitive sequences.

III. Organization of the Genome

A. Interspersed Repeated Sequences 1. Transposable Elements The Chlamydomonas genome contains examples of each of two groups of transposons as classified by the mechanism oftransposition (Finnegan, 1989). Class I elements or retrotransposons are flanked by long terminal repeats (LTRs) at each end and transpose via an RNA intermediate using the activity ofreverse transcriptase. Class II elements have inverted terminal repeats at each end and transpose via a DNA intermediate. The first Chlamydomonas sequence with charac­ teristics of a transposable element was discovered as an insertion in the gene encoding the oxygen-evolving enhancer 1 (OEE1) protein (Day et al., 1988). The TOC1 element is a 5.7 kb sequence found in variable copy number (2 to more than 30) in different strains. The element is dispersed in the genome, as shown by its hybridization to several chromosomes when used as a probe for in situ hybridization experiments (Hall and Luck, 1995). The element contains LTRs as found in Class I elements; however, the repeats are arranged in an unusual manner with part of the leftend LTR found at the right end of the element (Day et al., 1988). The 4.6 kbp internal region of TOC1 was found to share certain characteristics with nonviral retrotransposons including several copies of a 76 bp repeat sequence and the absence of large open reading frames encoding retroviral proteins (Day and Rochaix, 1991 a). Most TOC1 elements appear to

Chapter 3

The Nuclear Genome

be relatively recent amplification products of a single progenitor element (Day and Rochaix, 1991b). A possible retrotransposition intermediate ofthe TOC1 element was represented by a nearly full-length sense transcript (Day and Rochaix, 1991c). Comparison of two members of the TOC1 family led to the discovery of a 1.2 kb sequence inserted into a TOC1 element (Day, 1995). This novel element, termed TOC2, was shown to contain short 14-bp imperfect inverted terminal repeats and was found in ten or more copies in the DNA of several Chlamy­ domonas strains, some of which contain only one or two copies of TOC1. These results suggest that TOC2 sequences represent an independent family of Class II transposable elements. The Gulliver element, which shares several features with Class II elements in other systems, was discovered by Ferris (1989) who conducted a genomic genomic region walk in the mating-type plus on linkage group VI. This 12 kb element was shown to contain a 15-bp imperfect inverted repeat at its termini and to create an 8-bp target site duplication upon insertion. Twelve copies of the element were found dispersed in the genome in a laboratory strain, but the element was absent in the field isolate S1-D2 (Gross et al., 1988). Analysis of several independent transposition events indicated that the site of insertion was often genetically linked to the donor site (Ferris, 1989). The Gulliver element was used by Schnell and Lefebvre (1993) in a transposon-tagging strategy to isolate the Nit2 gene. They found that two of 14 spontaneous nit2 mutations were caused by insertion of a Gulliver element. Additional transposable element families have been discovered through analysis ofspontaneous mutations in several Chlamydomonas genes. The Tcr1 and Tcr2 elements were found to be associated with mutations in the Nit2- gene (Schnell and Lefebvre, 1993) and the Tcr1 element also caused a fus1 mutation (Ferris et al., 1996). Both the Tcr1and Tcr3 elements appear to be Class II transposons. Analysis of the fus1-1 mutation caused by insertion of a 9.4 kb Tcr1 element showed that it created an 8-bp target site duplication and contained terminal 140-bp perfect inverted repeats (Ferris et al., 1996). The Tcr3 element was associated with two additional fus1 mutations (Ferris et al., 1996) as well as a nit8 mutation (S.-C. Wang and P. Lefebvre, personal communication). The Tcr3 insertions contained 56 or 58-bp imperfect inverted terminal repeat sequences and created a 2-bp target site duplication upon insertion. Estimates of copy

27 number for the Tcr3 element in several Chlamy­ domonas strains ranged from two to approximately twenty copies per genome (S.-C. Wang and P. Lefebvre, personal communication). An element termed Pioneer 1 was isolated after selection for spontaneous mutations in the NIT1 gene in the field isolate strain JG224 (Graham et al., 1995). This 2.8 kb element appeared to be associated with a 2-bp duplication of the target sequence, but it did not contain other features oftransposable elements such as terminal repeats. The Pioneer element was present in low copy number in several field isolate strains of Chlamydomonas but was not present in laboratory strains 137c and 21gr.

2. Transcribed Repetitive DNA Sequences A starting point for studying repetitive DNA sequences transcribed by RNA polymerase II is the isolation of cDNA clones that hybridize to repeated sequences in nuclear DNA. Three clones isolated in this way were found to hybridize to multiple transcripts in polyadenylated RNA and to different classes of short (.5 kb) interspersed repetitive DNA sequences (Day and Rochaix, 1989). Because the cDNA clones in this study had small inserts of less than 400 bp, the repetitive elements were likely to be located in 3´ noncoding sequences of the corres­ ponding transcripts. The hybridizing DNA fragments were polymorphic between a C. reinhardtii laboratory strain and two interfertile strains, C. smithii and the field isolate S1-D2 (Gross et al., 1988), providing multiple RFLP markers for a single hybridization probe. Other examples of highly repeated sequences within cloned genes have been noted (e.g. Debuchy et al., 1989)

B. Tandemly Repeated Sequences 1. Large Tandem Repeats Evidence for large arrays of tandemly repeated sequences in the Chlamydomonas nuclear genome was found by analyzing discrete fragments of high molecular weight DNA (50–500 kb) resulting from digestion of DNA with enzymes that cut nuclear DNA with relatively high frequency (Hails et al., 1993). One class of repeat (represented by the clone pTANC1.5) was found associated with three large DNA fragments ranging in size from 200 to 700 kb and representing approximately 1% of the nuclear

Carolyn D. Silflow

28 genome by mass. Within these large arrays, four different repeat units of 1.5 to 2.5 kb were observed by digestion with BamH I, an enzyme that cleaves once within each repeat. The presence of the four repeat units varied among different C. reinhardtii strains; some interfertile strains including C. smithii and various field isolates were deficient in all four units, indicating that the sequence is an unstable or rapidly evolving component of the nuclear genome. Analysis of tetrad progeny from crosses of these polymorphic strains indicated that the large arrays represented at least five different loci and that the repeat units were not extensively interspersed with each other. Ferris and Goodenough (1994) in a genomic walk through the MT locus found a region of 1.1 kb repeat units that also were found in other genomic locations.

2. Simple Sequence Repeats The simple sequence repeat or microsatellite has been found in the genomes of numerous eukaryotic organisms including Chlamydomonas (Morris et al., 1986). Because the number of repeated motifs in each element may be highly variable, these elements can serve as a source of polymorphic DNA markers. An estimated reiteration frequency of 25 elements per 100 kb ofChlamydomonas DNA (based on genomic DNA hybridization) was found to be approximately 200-fold and 3.4-fold higher than the frequency estimates obtained for maize and mouse DNA, respectively (Morris et al., 1986). Based on repeats in a library of the frequency of cloned genomic DNA fragments, Kang and Fawley (1997) estimated a frequency of 5–6 elements per 100 kb of Chlamydomonas DNA. They found that elements is the presence and frequency of highly variable among different Chlamydomonas species. This group also demonstrated that the polymerase chain reaction (PCR) could be used to amplify small genomic fragments surrounding elements which proved to be specific polymorphic in size among different isolates of C. reinhardtii.

3. Telomere Repeat Sequences As is true for most eukaryotic species studied to date, telomeres in C. reinhardtii are composed of short tandem repeat sequences. Petracek et al. (1990) identified the telomere sequences from Chlamy-

domonas by hybridization with telomere repeats from Arabidopsis thaliana. DNA fragments containing these repeats were sensitive to Bal-31 exonuclease digestion, indicating that they were located at the ends of chromosomes. Analysis of cloned DNA containing the putative telomere sequences revealed which was a repeat sequence of estimated to be present in tracts at least 300 bp in length in genomic DNA. The telomere-associated sequences adjacent to the telomere repeats from three different chromosome ends were conserved, with 78% sequence identity over a distance of approximately 100 bp. Although the Chlamydomonas telomere repeat sequence is quite divergent from that found in Sacharomyces cerevisiae it is apparently recognized as a telomere in S. cerevisiae. After ligation of Chlamydomonas DNA fragments to a linearized S. cerevisiae plasmid and transformation of the ligated DNA into S. cerevisiae cells, Hails et al. (1995) recovered a transformant containing a linear plasmid in which Chlamydomonas telomere repeats were capped by telomere repeats of S. cerevisiae.

4. Ribosomal DNA Hybridization of cytosolic ribosomal RNA to DNA fractionated by equilibrium density centrifugation showed that the nuclear ribosomal DNA bands with a slightly lower density than the major band of nuclear DNA, indicating that it has a lower GC content than the majority of nuclear DNA (Harris, 1989), Approximately 400 copies of the ribosomal RNA genes were estimated to be present in the genome, based on saturation hybridization experi­ ments (Howell, 1972). The genomic repeat unit encoding the ribosomal RNAs was cloned and characterized by Marco and Rochaix (1980). They found that the repeat unit is approximately 8.0 kb in length and that it contains genes encoding 18 S and 25 S rRNAs, two internal transcribed spacer regions between the genes as well as a gene encoding 5.8 S ribosomal RNA in the smaller spacer. The DNA sequences and conserved secondary structure of the spacer regions of ribosomal RNA genes have been useful for phylogenetic studies ofgreen algal species (Mai and Coleman, 1997). Cytoplasmic 5S RNA hybridized with the bulk of nuclear DNA in density gradient fractions, indicating that the 5S genes are not a part of the ribosomal DNA repeat unit (Marco and Rochaix, 1980).

Chapter 3

The Nuclear Genome

C. Low Copy Number Sequences (Gene Families)

The minimal number of each of the four genes encoding core histones in the C. reinhardtii genome was estimated at 15, whereas histone H1 genes were estimated to be present in only one or two copies (Fabry et al., 1995). Seven different genomic regions containing core histone genes were examined, revealing that the four core histone genes usually are clustered, with a histone H3, H4 gene pair separated by about 1 kb from a histone H2A, H2B gene pair. DNA sequences from five different histone gene clusters (16 histone genes) have been reported (Fabry et al., 1995; Walther et al., 1995). These data show that each gene pair is separated by an intergenic region of about 300 bp and the genes are transcribed divergently. A single H1 histone gene was found separated by 1.6 kb from a H2A, H2B gene pair (Fabry et al., 1995). Several conserved promoter elements were found in the Chlamydomonas histone genes including a TATA-box and three other motifs. Walther and Hall (1995) showed that at least two histone H4 genes are likely to be located on linkage group XIX and that the other histone genes are probably dispersed on other chromosomes. Several small gene families have been described in Chlamydomonas. The genes encoding lightharvesting complex (LHC) II proteins (CabII genes) have been reported to exist as a small gene family composed of 3–7 members (Imbault et al., 1988) which are dispersed throughout the genome (P. Kathir, C. Silflow, unpublished). Similarly, several genomic fragments hybridized to the L1818 gene which encodes a protein distantly related to other LHC proteins (Savard et al., 1996). The genes encoding dynein heavy chains (Dhc) were analyzed by Porter et al. (1996) who used a PCR-based strategy to clone 11 Dhc genes, eight of which mapped to positions on five different linkage groups, with only two of the genes being closely linked. Pairs of closely linked but differentially regulated genes encode carbonic anhydrase (Fujiwara et al., 1990), Rubisco small subunit (Goldschmidt-Clermont and Rahire, 1986), and a cysteine-rich protein which may be a component of the zygote cell wall (Matters and Goodenough, 1992). In the latter case, evidence for expression of only one gene was obtained, suggesting that one of the genes may be a pseudogene (Matters and Goodenough, 1992). Two genes encoding identical proteins are separated by 12 cM on linkage

29 group XII; two genes encoding identical proteins are located on linkage groups III and IV (Ranum et al., 1988; James et al., 1993). Although the genomes of Chlamydomonas and the higher plant Arabidopsis thaliana are of similar size, Arabidopsis contains six genes encoding (Kopczak et al., and nine genes encoding 1992;Snustad et al., 1992). In some instances, genes with related function have been shown to be located in close physical proximity. For example, five genes involved in nitrate assimilation are linked within a region of 32 kb (Quesada et al., 1993; Chapter 33, Fernández et al.). The Rsp4 and Rsp6 genes encoding related protein components of the flagellar radial spoke heads are closely linked, with the 3´ end of the Rsp4 gene being only 435 bp from the 5´ end of the Rsp6 gene (Curry et al., 1992). Several genes involved in mating are located in the MT locus, (Goodenough et al., 1995).

D. The Mating-Type Locus The most detailed characterization ofthe organization of a genomic region has come from the results of an extraordinary genomic walk through the matingtype locus on linkage group VI that covered a distance of 1.1 Mb (Ferris and Goodenough, 1994; reviewed by Goodenough et al., 1995). This region, which is notable for suppression of recombination for numerous genes that map to the MT locus, was found to contain three domains. The centromereproximal C domain (525 kbp) and the telomereproximal T domain (109 kbp) are colinear in the genomes. In contrast, comparison of the and strain with the central R domain of 190 kbp in the strain showed that this region R domain in the contains major translocations, inversions, deletions and duplications, most likely leading to suppressed recombination. Several genes have now been cloned from this region including Ezy1 which is found in multiple copies in the C domain and which encodes a protein that participates in uniparental inheritance of the chloroplast genome (Armbrust et al., 1993; Chapter 6, Armbrust). The Fus1 gene, which is locus, encodes found only in the R domain of the, a glycoprotein expressed on mating structures (Ferris et al., 1996). The Mid gene, which is located locus, encodes a putative in the R domain ofthe transcription factor that controls the program of gametic differentiation (Ferris and Goodenough, 1997). Three additional genes which reside in the

Carolyn D. Silflow

30 C and T domains but do not play a role in mating functions have been localized on cloned fragments from the walk (Ferris, 1995).

IV. Characteristics of Chlamydomonas Genes Transcribed by Polymerase II

A. Promoters The expression of the four tubulin genes and other genes encoding flagellar proteins is induced coordinately following deflagellation of Chlamy­ domonas cells (Brunke et al., 1982; Schloss et al., 1984), and this induction is partly due to increased rates of transcription of these genes (Keller et al., 1984). The tubulin gene promoters have been studied extensively to identify cis-acting elements necessary for this induction. A TATA box followed by a GCrich region has been found approximately 30 bp upstream of the transcription start site in numerous Chlamydomonas genes including the tubulin genes (reviewed in Davies and Grossman, 1994). Although the GC-rich sequence was essential for transcription gene constructs injected into Xenopus of oocyte nuclei (Bandziulis and Rosenbaum, 1988), its role in transcription in Chlamydomonas cells is unclear. When this sequence was changed to an ATpromoter driving rich sequence in a expression of the arylsulfatase reporter gene, no effect was observed on the constitutive activity ofthe promoter nor on the induction of transcription following deflagellation (Davies and Grossman, 1994). Similarly, a study in which the TATA box and upstream sequences were deleted from the gene promoter construct showed that the TATA box was not required for basal levels of expression (Periz and Keller, 1997). Results from these studies indicate that basal transcription from the tubulin promoters is controlled by sequences within 35 bp upstream of the transcription start site and that the TATA box and the GC-rich regions may not be essential for basal level activity of the promoters, although these elements may be involved in selecting the site of transcription initiation. Comparison of promoter regions for the four tubulin genes identified several copies of a 10-bp motif termed a ‘tub’box (Brunke et al., 1984). Davies and Grossman (1994) examined the role of the tub boxes in induction of promoter gene after deflagellation activity for the and concluded that inclusion of at least two groups of

tub box elements was essential for the induction (i.e. four tub boxes). Studies with the gene promoter showed that two different regions of the promoter, one of which contains two tub boxes, are essential for maximal induction and suggested that promoter elements in addition to tub boxes are important for the response (Periz and Keller, 1997). The analysis of several other promoters has provided insight into gene regulation as well as useful tools for driving the expression of reporter genes. For example, the Cyc6 gene encoding cytochrome is transcribed only under conditions of copper deficiency. Mutational analysis ofthe Cyc6 promoter showed that two different sequences within 127 bp upstream of the transcription start site were sufficient for copper-responsive expression (Quinn and Merchant, 1995). Analysis of the Hsp70 gene promoter showed that the elements responsible for light induction and for heat shock response reside in different regions of a sequence 140 upstream of the transcription start site (Kropat et al., 1995). A 2.5 kb region upstream of the CabII-1 gene was able to confer light-regulated expression when fused to a reporter gene (Blankenship and Kindle, 1992). In addition, a 4 kb region upstream from the CabII-1 gene was able to drive the expression of reporter genes with a circadian rhythm similar to that seen for the expression of the CabII gene family, indicating that the circadian clock regulates transcription ofthis gene (Jacobshagen et al., 1996). A promoter region from the Nit1 gene encoding nitrate reductase conferred ammonium-repressible expression on different reporter genes (Ohresser et al., 1997; Zhang and Lefebvre, 1997). High-level expression of reporter genes has been facilitated by fusion with the promoter of the RbcS2 gene (Kozminski et al., 1993; Nelson et al., 1994; Stevens et al., 1996; Cerutti et al., 1997).

B. Codon Bias A feature of most Chlamydomonas genes is the pronounced codon bias which favors codons with C or G in the third position and reflects the high GC content of the genome. The bias is, however, more than a reflection of the high GC content. For 89 Chlamydomonas sequences, LeDizet and Piperno (1995a) calculated the ‘standardized synonymous codon bias’ or B value which may vary from 0 (no codon bias) to 1 (only one codon used per amino acid)(Long and Gillespie, 1991). They found that the

Chapter 3 The Nuclear Genome B values ranged from 0.316 to 0.78, with a median of 0.617. It is possible that the codon bias is at least partly responsible for the low levels of expression observed when heterologous genes are introduced by transformation into Chlamydomonas. Indeed, a bacterial phleomycin-resistance gene (ble), which has a codon bias similar to that seen in most Chlamydomonas genes, was shown to be expressed when transformed into Chlamydomonas cells (Stevens et al., 1996). Striking exceptions to the common codon bias were found in two genes located in the mating-type (MT) locus. The Fus 1 gene shows no codon preference (B value = .05) and all regions of the gene contain a reduced GC content (coding region = 47.7% GC) (Ferris et al., 1996). Another gene, Mid, also was found to have a very low codon bias (B value = 0.161) and a coding region GC content of 50% (Ferris and Goodenough, 1997). These authors suggest that the dramatic rearrange­ ments that have occurred in the mating-type locus R domain (Section III.D) have led to the changes in nucleotide composition and changes in codon bias that are apparent in genes located in this region. In addition to the lack of codon bias observed in these endogenous genes, further evidence that codon bias is not essential for gene expression in Chlamy­ domonas comes from studies in which a bacterial dominant selectable marker gene which lacks the Chlamydomonas codon bias was able to confer spectinomycin resistance to transformed Chlamy­ domonas cells (Cerutti et al., 1997). Two databases that maintain codon usage tables for C. reinhardtii and other organisms are TransTerm (Dalphin et al., 1996) and CUTG (Nakamura et al., 1996).

C. Introns Based on DNA sequence data for genes and/or cDNAs, one general feature ofChlamydomonas genes that has emerged is that many small introns are often present at high frequency (Table 1). On average, four introns are located within each kilobase of coding sequence and the average intron size is 219 bp. At least two introns are located in untranslatedregions of genes (Mitchell and Kang, 1993; Sugase et al., 1996). Within an average gene, the total sequence contained in introns is nearly equal to the total coding sequence (Table 1). The frequency of introns in Chlamydomonas genes may provide a large target for mutations that affect intron splicing, as suggested by the finding that three different ida4 alleles contain

31

alterations in splice site sequences (LeDizet and Piperno, 1995b). Although most Chlamydomonas genes apparently do contain introns, some intronless genes have been noted including several core histone genes (Fabry et al., 1995). In addition, introns were shown to be unnecessary for the normal expression of the Rsp3 gene, as minigenes consisting of 5´ and 3´ genomic regions fused to a Rsp3 cDNA were able to rescue the mutant phenotype when transformed into pf14 mutant cells (Diener et al., 1993). The possible role of intron splicing in regulating expression of other genes in Chlamydomonas has not been examined rigorously. However, in studies with Volvox carteri, a related alga that also has a high frequency of introns, the presence of at least one intron in cDNA constructs from the nitA gene was shown to enhance the transformation frequency of nitA mutants by ten-fold over intron-less constructs. These results suggest a role for splicing in regulating the expression of the nitA gene which normally contains 10 introns (Gruber et al., 1996). Nucleotide sequences at the 5´ and 3´ splice junctions of Chlamydomonas introns conform in general to the eukaryotic consensus for these sequences (Table 2; Lee et al., 1991; Ledizet and Piperno, 1995b). A consensus sequence (NCTAG) for the putative branch site located 15–51 bp upstream of the 3´ splice site in 56 Chlamydomonas nuclear introns was also observed (Lee et al., 1991). The presence of these consensus sequences suggests that the splicing machinery in Chlamydomonas cells is similar to that in other

32

eukaryotes. Some evidence supporting this con­ clusion has come from experiments reported by Jarvik et al. (1996) who developed a ‘CD-Tagging’ method for inserting small sequences encoding epitopes into various locations in genes of interest. The method involves insertion of engineered miniexons into existing introns to produces genes encoding epitope-tagged proteins that can be localized in cells using specific antibodies. In these experiments, engineered constructs based on intron splice site sequences of Chlamydomonas were able to function in Drosophila cells. Information on the positioning of introns within Chlamydomonas genes has been used in the debate over the origins of spliceosome-dependent introns. Some evidence showing conserved intron positions within ancient genes such as glyceraldehyde-3phosphate dehydrogenase (Kersanach et al., 1994) have supported the hypothesis that introns arose early in the evolution of genes (Dorit et al., 1990). However, most introns in Chlamydomonas genes occur at unique positions when compared with homologous genes in animals, higher plants, fungi and other protists (Dibb and Newman, 1989; Lee et al., 1991; Kopczak et al, 1992; Snustad et al., 1992;

Carolyn D. Silflow

Sugase et al., 1996). These data lend support to the hypothesis that multiple intron insertion events occurred late in the evolution of eukaryotic genes (reviewed by Palmer and Logsdon, 1991). The presence of conserved exon sequences adjacent to introns would not be expected unless these sequences were involved in some way in the acquisition or splicing of introns. Dibb and Newman (1989) proposed that the sequence (A/C) AG(A/G) is a proto­ splice site, where introns are inserted between the conserved G and (A/G) nucleotides. The consensus sequence is clearly present in exon positions adjacent to Chlamydomonas introns (Table 2). Intron sequences have been exploited as tools to evaluate phylogenetic relationships among groups of related algae in the order Volvocales (Liss et al., 1997). Several studies have noted that intron positions are often conserved between genes in C. reinhardtii and Volvox carteri, implying intron insertion into common ancestral genes (Harper and Mages, 1988; Mages et al., 1988; Dietmaier et al., 1995; Fabry et al., 1995). However, DNA sequences in introns have diverged much more rapidly than those in protein coding regions. The rapidly evolving intron sequences provided the resolution necessary to describe the

Chapter 3

The Nuclear Genome

relationships among several strains of Volvox and among several interfertile strains ofChlamydomonas which had been difficult to resolve using rDNA or protein coding sequences (Liss et al., 1997). These authors estimated that the substitution rate in intron sequences is approximately ten-fold higher than the synonymous substitution rate in protein-coding sequences.

D. Translation Start Codon and Stop Codon Sequence Context Because the sequence context surrounding start codons and stop codons affects the efficiency of translation, the TransTerm database was created to compile sequence context data for numerous species including C. reinhardtii (Dalphin et al., 1996). Comparisons of 133 sequences showed, for example, a consensus of (A/C) A (A/C) (A/C) A T G (G/C) for the context ofthe start codon (Table 3). Similarly, the consensus of (G/C) TAA (G/A) for the context ofthe stop codon was obtained from analysis of 149 cloned sequences (Table 3). The stop codon TAA was found in 70% of genes; TGA and TAG stop codons were each found in 15% of the genes surveyed.

33

E. 3´ Noncoding Sequences The 3´ noncoding regions of Chlamydomonas genes are often several hundred bp in length. The most common putative polyadenylation signal is TGTAA, located 10–20 bp upstream of the polyadenylation site in approximately 90% ofChlamydomonas cDNA sequences reported to date. Variations ofthis sequence including TGTAG and TGTTA in the appropriate location have been reported for a few cDNA clones. Two genes found in the mating-type locus, Fus1 and Mid, have among other unusual characteristics (see Sections III.D, IV.B), an absence of the canonical polyadenylation signal, leading to transcripts with a variety of polyadenylation sites (Ferris et al., 1996; Ferris and Goodenough, 1997). The histone genes (including the histone H1 gene) do not contain the canonical Chlamydomonas polyadenylation signal at the 3´ end but do contain a 27-bp palindromic sequence (Fabry et al., 1995; Walther and Hall, 1995). Similar palindromes are involved in regulating the stability of histone mRNA molecules in metazoans and were observed in the histone genes of the related alga Volvox, but not in those of land plants.

34

Carolyn D. Silflow

Chapter 3 The Nuclear Genome

35

36 IV. Physical Mapping of the Chlamydomonas Genome The genetic map of Chlamydomonas consists of 17 linkage groups (Dutcher et al., 1991; Harris, 1993). Although progress has been made in developing techniques for visualizing Chlamydomonas chromo­ somes and for in situ hybridization to specific chromosomes, it has not been possible to verify chromosome number using this approach (Hall and Luck, 1995). In addition, only partial resolution of the chromosomes has been achieved using pulsed field gel electrophoresis (Hall et al., 1989; A. Day, personal communication). The availability of Chlamydomonas strains interfertile with C. rein­ hardtii but divergent in genomic DNA sequence has facilitated the mapping ofrestriction fragment length polymorphisms (RFLP) (Ranum et al., 1988). The progeny of a cross between C. reinhardtii strains containing various genetic markers and the interfertile C. smithii strain were analyzed to align molecular markers with the genetic map. Further mapping has utilized a wild isolate S1-D2 which exhibits RFLPs with C. reinhardtii more frequently than does the C. smithii strain (Gross et al. 1988; Porter et al., 1996). A molecular map currently under construction has analyzed the segregation of markers among 136 random progeny of a cross between C. reinhardtii and the S1-D2 strain (Fig. 1; P. Kathir, P. Lefebvre, and C. Silflow, unpublished data). Currently, this map contains approximately 170 molecular markers including RFLP markers and sequence tagged site (STS) markers that utilize specific primers for the polymerase chain reaction (PCR) to amplify genomic fragments that vary in length between the two polymorphic strains (Silflow et al. 1995). The molecular markers, which include random small Pst I genomic fragments, random cDNA clones, and specific cloned genes or cDNAs contributed by Chlamydomonas researchers, are distributed among all 17 known linkage groups. For most ofthe linkage groups, it has been possible to orient the groups of molecular markers with the genetic map by utilizing cloned genes known to correspond to mutations for which genetic map location was determined previously or by using a C. reinhardtii parent strain containing multiple mutations whose genetic map locations were known (Ranum et al., 1988). In addition to the groups of linked markers shown in Fig. 1, we have identified two groups of markers which do not appear to be linked to known linkage

Carolyn D. Silflow groups. Further analysis will be required to determine whether these groups ofmarkers represent previously unidentified chromosomes or whether they map to a known linkage group but sufficiently distant from the current set of markers that linkage has not been detected. The Chlamydomonas molecular map has been used to determine whether cloned genes/cDNAs correspond to known genetic loci (e.g. Porter et al., 1996). It has proven useful for mapping mutations generated by insertional mutagenesis by using DNA fragments flanking the inserted plasmid. Mapped probes also have been used as the starting points to ‘walk’to nearby genes of interest (e.g. Walther et al., 1994). A large set of molecular markers will facilitate the ordering of cloned genomic fragments into ‘contigs’ of overlapping fragments during the construction ofa physical map ofthe Chlamydomonas genome (see below). By comparing the genetic map containing approximately (Harris, 1993) with the genome complexity (see above), a rough estimate for the physical distance of a recombination unit of 100– 160 kb per cM is obtained. The results ofVashishtha et al. (1996) suggest that this may be an overestimate. They found that severalYAC clones containing inserts of approximately 130 kb encompassed intervals defined by one or two recombination events among 15–20 tetrads tested. The physical distance corres­ ponding to a recombination unit can be much smaller than this estimate, as shown by Debuchy et al. (1989) who found that a genomic fragment of 7.8 kb containing the argininosuccinate lyase gene was able to rescue the phenotype of three mutations, arg2, arg 7-3, and arg7, which had been ordered previously in genetic experiments (Matagne, 1978). The genetic distance over this interval was calculated to be 1.0 to 1.6 map units. Physical distances could be larger in regions of suppressed recombination such as telomeric and centromeric regions. In addition to the molecular maps described above, other necessary tools have been prepared to facilitate gene cloning in Chlamydomonas. Hall and co­ workers have generated a genomic library in a yeast artificial chromosome (YAC) vector which contains approximately four genome equivalents in clones with an average size of 125 kb (Infante et al., 1995; Vashishtha et al., 1996). In another project, an ‘indexed’ genomic library ofcosmid clones has been prepared, with the clones plated individually in wells so that pools of clones can be analyzed in gene cloning experiments (Zhang et al., 1994, Chapter 4,

Chapter 3

The Nuclear Genome

Kindle). An indexed library of genomic fragments also has been constructed using a bacterial artificial chromosome (BAC) vector (P. Lefebvre, personal communication).

VI. Future Prospects To take full advantage of Chlamydomonas as a genetic system, a major goal of future research efforts in many areas will be the cloning of genes identified by mutation. The insertional mutagenesis technique is a powerful new tool that relies on random integration of plasmids containing selectable marker genes into the nuclear genome (Tam and Lefebvre, 1993; Gumpel et al., 1995; Pazour et al., 1995; Davies et al., 1996; Smith and Lefebvre, 1996). This method generally causes gene disruption or deletion, producing null mutations. Mutations in genes essential for viability are not expected to be recovered, nor are mutations with conditional phenotypes. Cloning genes by transposon tagging is another potentially fruitful approach (Day et al., 1988; Schnell and Lefebvre, 1993; Ferris et al., 1996), but more research is needed to characterize the classes of transposons present in the genome and to define the conditions that may influence transposition. For certain nuclear genes that have homologs in E. coli, cloning has been accomplished by functional complementation of the appropriate E. coli mutant with sequences from a Chlamydomonas cDNA library (Matters and Beale, 1994, 1995; Yildiz et al., 1996). For mutations that result in selectable phenotypes, gene cloning may be accomplished by transformation of mutant cells with genomic libraries (Purton and Rochaix, 1994; Zhang et al., 1994). However, the size of the genome and the current transformation efficiency preclude this method in cases where a strong selection method is not available. The phenotypes of many interesting mutations are subtle and require labor-intensive screening methods. The cloning of the affected genes in such mutants will be facilitated by the development of better methods for genome walking from nearby molecular markers. To facilitate these approaches, an important goal is the further development of the physical map in which cloned genomic fragments have been ordered in ‘contigs’ that cover large regions of the genome. A puzzling aspect of molecular genetic studies with Chlamydomonas has been the inability of these cells to express foreign genes including many reporter

37 genes that are expressed routinely in other eukaryotic systems. Speculation about the molecular basis of the block to expression has included unusual aspects of Chlamydomonas genes including the codon bias, intron sequences, and the polyadenylation signal. Some success in solving the problem of lack of expression has been reported (e.g. Stevens et al., 1996; Cerutti et al., 1997). The latter group found evidence that the lack of stable expression of the bacterial spectinomycin resistance gene may have been due to gene silencing or transcript instability. Further investigation into the basic mechanisms involved in control of gene expression in Chlamy­ domonas are important for gaining insight into this problem and for developing the strengths of Chlamydomonas as an experimental system.

Acknowledgments Work in the author’s laboratory was funded by the National Institutes of Health GM51995. I thank O. Sodeinde, S. Merchant and P. Lefebvre for commun­ icating unpublished results and P. Lefebvre for comments on the manuscript.

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39 Mai JC and Coleman A W (1997) The internal transcribed spacer 2 exhibits a common secondary structure in green algae and flowering plants. J Mol Evol 44:258–271 Marco Y and Rochaix J-D (1980) Organization of the nuclear ribosomal DNA of Chlamydomonas reinhardtii, Mol Gen Genet 177:715–723 Matagne RF (1978) Fine structure of the arg-7 cistron in Chlamydomonas reinhardtii. Molec Gen Genet 160:95–99 Matters GL and Beale SI (1994) Structure and light-regulated expression of the gsa gene encoding the chlorophyll biosynthetic enzyme, glutamate 1-semialdehyde aminotransferase, in Chlamydomonas reinhardtii. Plant Mol Biol 24:617–629 Matters GL and Beale SI (1995) Structure and expression of the Chlamydomonas reinhardtii alad gene encoding the chlorophyll biosynthetic enzyme, acid dehydrase (porphobilinogen synthase). Plant Mol Biol 27:607–611 Matters GL and Goodenough UW (1992) A gene/pseudogene tandem duplication encodes a cystein-rich protein expressed during zygote development in Chlamydomonas reinhardtii. Molec Gen Genet 232:81–88 Mitchell DR and Kang Y (1993) Reversion analysis of dynein intermediate chain function. J Cell Sci 105:1069–1078 Morris J, Kushner SR and Ivarie R (1986) The simple repeat poly(dT-dG)-poly(dC-dA) common to eukaryotes is absent from eubacteria and archaebacteria and rare in protozoans. Mol Biol Evol 3:343–355. Morris RL, Keller LR, Zweidler A and Rizzo PJ (1990) Analysis of Chlamydomonas reinhardtii histones and chromatin. J Protozool 37:117–123. Nakamura Y, Wada K, Wada Y, Doi H, Kanaya S, Gojobori T and Ikemura T (1996) Codon usage tabulated from the international DNA sequence databases. Nucl Acids Res 24:214– 215. Nelson JAE, Savereide PB and Lefebvre PA (1994) The CRY1 gene in Chlamydomonas reinhardtii: Structure and use as a dominant selectable marker for nuclear transformation. Mol Cell Biol 14:4011–4019 Ohresser M, Matagne RF and Loppes R (1997) Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii. Curr Genet 31:264– 271 Palmer JD and Logsdon JM (1991) The recent origins of introns. Curr Opin Genet Develop 1:470–77 PazourGJ, Sineschekov OA and Witman GB (1995) Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii. J Cell Biol 131:427–440 Periz G and Keller LR (1997) DNA elements regulating gene induction during regeneration of eukaryotic flagella. Mol Cell Biol 17:3858–3866 Petracek ME, Lefebvre PA, Silflow CD and Berman J (1990) Chlamydomonas telomere sequences are A+T-rich but contain three consecutive G-C base pairs. Proc Natl Acad Sci USA 87:8222–8226 Porter ME, Knott JA, Myster SH and Farlow SJ (1996) The dynein gene family in Chlamydomonas reinhardtii. Genetics 144:569–585 Purton S and Rochaix J-D (1994) Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library. Plant Mol Biol 24:533–537 Quesada A, Galvan A, Schnell RA, Lefebvre PA and Fernandez E (1993) Five nitrate assimilation-related loci are clustered in

40 Chlamydomonas reinhardtii. Mol Gen Genet 240:387–394 Quinn JM and Merchant S (1995) Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators. Plant Cell 7:623–638 Ranum LPW, Thompson MD, Schloss JA, Lefebvre PA, and Silflow CD (1988) Mapping flagellar genes in Chlamydomonas using restriction fragment length polymorphisms. Genetics 120:19–122 Rochaix J-D (1995) Chlamydomonas reinhardtii as the photosynthetic yeast. Annu Rev Genet 29:209–230 Savard F, Richard C and Guertin M (1996) The Chlamydomonas reinhardtii L1818 gene represents a distant relative of the Cab1/II genes that is regulated during the cell cycle and in response to illumination. Plant Mol Biol 32:461–473 Schloss JA, Silflow CD and Rosenbaum JL (1984) mRNA abundance changes during flagellar regeneration in Chlamy­ domonas reinhardtii. Mol Cell Biol 4:424–434 Schmitt R, Fabry S and Kirk DL (1992) In search ofthe molecular origins of cellular differentiation in Volvox and its relatives. Int Rev Cytol 139:189–265 S c h n e l l RA and Lefebvre PA (1993) Isolation of the Chlamydomonas regulatory gene NIT2 by transposon tagging. Genetics 134:737–747 Senapathy P, Shapiro MB and Harris NL (1990) Splice junctions, branch point sites, and exons: Sequence statistics, identification, and applications to genome project. Methods Enzymol 183:252–278 Silflow CD, Kathir P and Lefebvre PA (1995) Molecular mapping of genes for flagellar proteins in Chlamydomonas. Methods in Cell Biol 47:525–530 Smith EF and Lefebvre PA (1996) PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella. J Cell Biol 132:359–370

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Chapter 4

Nuclear Transformation: Technology and Applications Karen L. Kindle

Plant Science Center, Biotechnology Program, Biotechnology Building,

Cornell University, Ithaca, NY 14853

Summary I. Introduction II. A Brief History of C. reinhardtii Nuclear Transformation III. Selectable Markers A. Complementation of C. reinhardtii Mutations B. Drug Resistance Markers IV. Methods for Introducing DNA into the Nuclear Genome of C. reinhardtii A. Particle Bombardment B. Glass Beads, Silicon Whiskers C. Electroporation V. Reporters and Promoters A. Arylsulfatase as a Reporter B. Other Reporters C. Constitutive Promoters D. Regulated Promoters VI. Characteristics of Transformation Events A. Nature and Stability of Introduced DNA B. Insertion Events C. Cotransformation and Expression of C. reinhardtii Genes D. Expression of Foreign Genes/Silencing VII. Insertional Mutagenesis and Gene Tagging A. Development and Application of the Approach B. Technical Considerations C. Isolation of the Gene Responsible for the Mutant Phenotype D. Perspectives VIII. Gene Isolation by Complementation of a Mutant Phenotype IX. Homologous Recombination and Gene Targeting X. The Use of Nuclear Transformation to Study Promoter Function XI. Conclusion Acknowledgments References

J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 41–61. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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Karen L. Kindle

Summary Nuclear transformation is now simple and efficient, and it has revolutionized the kinds of questions that can be addressed using Chlamydomonas reinhardtii as a model system. The highest rates of transformation are obtained using C. reinhardtii genes that complement auxotrophic mutations as selectable markers. Cotransformation is efficient, so effects of mutations engineered in vitro can readily be tested by reintroducing the altered genes into an appropriate mutant strain together with a selectable marker. Because nearly all nuclear transformation events result from apparently random integration of the introduced plasmid into chromosomal DNA, it is possible to generate large numbers of insertional mutants. The subsequent isolation of the disrupted genes by virtue of the molecular tag provides a very powerful means for cloning genes with known mutant phenotypes. Alternatively, since nuclear transformation is so efficient, genes with good selectable phenotypes can be isolated by complementing appropriate recessive mutations with pools of DNA from an indexed genomic library. Recently, expression of eubacterial genes that confer antibiotic resistance to C. reinhardtii has been reported; these genes should be useful as dominant selectable markers in any genetic background. However, transformants are recovered with these bacterial genes at only 1 % the rate obtained by transformation with C. reinhardtii genes, and in about half the cases the introduced genes are silenced under nonselective conditions, sometimes a problem with C. reinhardtii genes as well. Although an active homologous recombination system allows efficient recombination between simultaneously introduced plasmids, the rate of gene-targeted homologous integration events is very low. Further understanding of the factors that limit the expression of reintroduced genes and the rate of gene-targeting could lead to substantial improvements in the capability of manipulating the nuclear genome and of generating or phenocopying mutations corresponding to cloned genes.

I. Introduction Although a subset of chloroplast and mitochondrial proteins is encoded by organellar genes, the biogenesis and functions of these organelles are to a large extent dependent on structural and regulatory proteins encoded in the nucleus. The functions of these nucleus-encoded polypeptides and how they are correctly targeted to the appropriate subcellular compartment are some of the most intriguing questions in organelle ontogeny. The capability of introducing DNA into the nuclear genome is essential for a variety of ‘reverse’ genetic approaches to investigate organellar biogenesis, structure, and function. For example, site-directed mutations can be engineered into a cloned gene in vitro and tested in vivo by transforming the gene into an appropriate mutant strain. With a sufficiently high transformation efficiency, it is possible to isolate a gene by its ability to complement a particular mutant phenotype. Nuclear transformation can also be used as a mutagen, either in the course of random insertion events, or Abbreviations: Ars – arylsulfatase; CabII-1 – chlorophyll a/b binding protein gene; nos – nopaline synthetase gene; nptII – neomycin phosphotransferase gene; ocs – octopine synthetase gene; PEG – polyethylene glycol; Rubisco – ribulose bisphosphate carboxylase/oxygenase; RFLP – restriction fragment length polymorphism; SV40 – simian virus 40; Tn5, 9 – transposons 5 and 9

potentially, by generating gene-targeted disruptions. In the last decade, simple, efficient, and reliable methods for transforming the nucleus of Chlamy­ domonas reinhardtii have been developed. For those who have watched the field since the mid 1980s, it is gratifying to see this technique evolve from frustratingly elusive to so simple that it has been incorporated into many introductory laboratory courses. This review will briefly describe the history of nuclear transformation, then discuss the selectable markers and other molecular tools that are available. Alternative methods for introducing DNA into the nucleus will be presented, and the molecular events that appear to occur during transformation will be reviewed. Finally, some of the limitations and perspectives for the future will be discussed, along with a brief summary of results on promoter structure and function that have been deduced from transformation experiments.

II. A Brief History of C. reinhardtii Nuclear Transformation Many individuals and laboratories have contributed to the development of nuclear transformation technology, a process that is ongoing, since certain technical challenges remain (see below). The first

Chapter 4 Nuclear Transformation reports of nuclear transformation utilized ‘foreign’ (non-C. reinhardtii) genes. Thus, the yeast ARG4 gene was shown to complement the arg7 mutation in C. reinhardtii (Rochaix and vanDillewijn, 1982). Unfortunately, the transformation rate ( cell) was not much higher than the frequency of reversion. Although transformants apparently contained integrated copies ofthe introduced plasmid DNA, genetic crosses to demonstrate that the phenotype was due to the introduced gene were not performed, and the hybridization pattern changed when transformed strains were maintained under nonselective conditions. Putative replication origins were identified by cloning C. reinhardtii restriction fragments into the ARG4 transformation vector (Rochaix et al., 1984). A number of C. reinhardtii transformants were isolated that maintained the introduced DNA in an unintegrated state. The unintegrated DNA was gradually lost when the cells were cultured under nonselective conditions, however, and the recovered plasmids, which presumably contained functional replication origins, did not result in higher transformation rates than the original transformation vector, suggesting that integration into the genome is not the step that limits nuclear transformation rates (Rochaix et al., 1984). Shortly thereafter, the E. coli nptII gene from Tn5 (encodes neomycin phosphotransferase) was reported to confer resistance to G418 and kanamycin and appeared to have promise as a dominant selectable marker (Hasnain et al., 1985). Expression of this gene was driven by the SV40 promoter in plasmids that contained the yeast 2 micron origin of replication. It and conferred G418 resistance at the rate of the plasmid DNA appeared to remain unintegrated. Unfortunately, this marker turned out not to be very useful, possibly because of the high rate of spontaneous resistance to kanamycin and G418 or silencing of the introduced DNA (see below). The breakthrough in C. reinhardtii nuclear transformation resulted from two factors: the use of C. reinhardtii genes as selectable markers and the development of efficient procedures for introducing the DNA into the nucleus. The isolation of genes encoding argininosuccinate lyase (Arg7; Debuchy et al., 1989) and nitrate reductase (Nit1; Fernández et al., 1989) provided markers with excellent selectable phenotypes. Particle bombardment allowed the recovery of small numbers of transformants ( cell) with fairly high numbers of integrated copies (Debuchy et al., 1989; Kindle et al., 1989). The rate

43 of cotransformation was very high (Kindle et al., 1989; Day et al., 1990), which facilitated the isolation of transformants expressing genes that had no selectable phenotype (Diener et al., 1990). The development of the glass bead method, in which cells are transformed simply by agitating them vigorously with DNA in the presence ofpolyethylene glycol and glass beads, made the technology accessible to anyone with a vortex mixer (Kindle, 1990). Since then, methods for electroporation (Brown et al., 1991; Tang et al., 1995) and agitation in the presence of silicon carbide whiskers (Dunahay, 1992) have been developed, as well as a number of molecular tools and specific applications, which are discussed in detail below.

III. Selectable Markers The most effective selectable markers are those for which there is a strong phenotypic selection, so that there is little growth of nontransformed cells and vigorous growth of transformant colonies. Further­ more, the rate of reversion (for complementation of auxotrophic or nonphotosynthetic mutants) or spontaneous resistance (for drug resistance markers) should be as low as possible. A relatively short coding region facilitates molecular manipulations to create new vectors, and analysis of insertion events following nuclear transformation is more straight­ forward if the gene does not contain repeated sequences. Depending on the application, it may be advantageous to have a marker where even a low level of gene expression affords a robust growth phenotype.

A. Complementation of C. reinhardtii Mutations The Arg7 gene encodes argininosuccinate lyase. It spans 7 kb and is interrupted by 12 introns, which contain highly repeated sequences (Debuchy et al., 1989; Purton and Rochaix, 1994a). It complements arg7 and arg2 mutations, which map to the two ends of the ARG7 locus, and transformants are selected on acetate plates that lack arginine. The insertion of a 400 bp fragment from bacteriophage into an intron of the Arg7 gene provides a molecular marker that facilitates DNA blot analysis of putative Arg7 transformants (Gumpel and Purton, 1994). An Arg7­ based cosmid has been constructed, and was used to independent produce a genomic library with

Karen L. Kindle

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clones, representing approximately 250 genome equivalents (Purton and Rochaix, 1994a). The Nit1 gene encodes nitrate reductase, which alleviates the ammonium requirement of nit1 mutants, to allow transformants to grow on media with nitrate as the sole nitrogen source (Fernández et al., 1989; Kindle et al., 1989; see Chapter 33, Fernández et al.; Nia1 is the same as Nit1). It contains 15 introns and spans a region of about 8.5 kb (D. Zhang, M. Lavoie, S. Christenson and P.A. Lefebvre, unpublished). For transformation with Nit1, a nit1 NIT2 strain must be used; Nit2 is a regulatory gene that is required for expression of Nit1. Many of the strains in the Chlamydomonas Genetics Center collection are derived from a ‘wild-type’ background that actually carries nit1 and nit2 mutations and hence are inappropriate for transformation with Nit1 (Harris, 1989). Mutants defective at the nit8 (nar2) locus also require ammonium for growth (Nelson and Lefebvre, 1995a), and transformants can be selected on nitrate plates following introduction of a 3.2-kb DNA fragment carrying the Nit8 (Nar2) gene (S.-C. Wang and P. A. Lefebvre, personal communication). The Nic7 gene complements a nicotinamiderequiring mutation (Ferris, 1995). Although it has not been used extensively, it should be a good transformation marker, since reversion of the nic7 mutation is rare and background growth of mutant cells can be reduced by growing them in the presence of 3-acetylpyridine. The Thi10 gene has also been used to complement a thiamine auxotroph, though untransformed cells apparently die slowly (Ferris, 1995). The CRlpcr-1 (Por1) gene encodes light-dependent NADPH:protochlorophyllide oxidoreductase, which carries out the final step in chlorophyll synthesis. It complements the yellow, light-sensitive phenotype of mutants such as pc1y7, which are defective in both light-independent and light-dependent protochloro­ phyllide reductase (Li and Timko, 1996). The pc1 and the gene is mutation reverts at a low rate relatively small, since a 3.6 kb fragment retains the ability to complement the pc1 mutation (our unpublished observations). A number of cloned genes complement the acetaterequiring phenotype of nonphotosynthetic mutants. For example, Oee1 encodes the 33 kD protein of the oxygen evolving complex and complements a transposon-induced mutation (Mayfield and Kindle, 1990). AtpC encodes the gamma subunit of chloroplast ATPase, and complements an insertion

mutation (Smart and Selman, 1991, 1992). RbcS1 and RbcS2 are linked genes that encode the small subunit of Rubisco and each complement a deletion mutation that was generated in the course of insertional mutagenesis (Khrebtukova and Spreitzer, is defective in chloroplast petA mRNA 1996). accumulation, and the gene that complements the defect has been cloned. (Gumpel et al., 1995). Finally, cytochrome the Ccs1 gene is required for synthesis and complements an insertion mutation (Inoue et al., 1997). Although all ofthese genes have good selectable phenotypes, they are not particularly useful as coselectable markers or for insertional mutagenesis in investigations of chloroplast biogenesis, since mutations that affect chloroplast biogenesis often impair photosynthesis themselves.

B. Drug Resistance Markers Drug resistance markers have a substantial advantage over markers that complement C. reinhardtii mutations since they can be used in any genetic background, thereby circumventing the need to construct appropriately marked strains as trans­ formation recipients. One such marker is the C. reinhardtii Cry1 gene, which confers resistance to the cytosolic translation inhibitors cryptopleurine and emetine, due to a mutation in the coding region of cytosolic ribosomal protein S14. Although the Cry1 gene confers cryptopleurine resistance at a high frequency, it is semi-dominant, so existing ribosomes must be depleted by a period of nitrogen starvation and regenerated with the mutant gene product before the resistance can be expressed (Nelson et al., 1994). A number of studies have utilized bacterial genes that confer resistance to aminoglycoside antibiotics, such as the nptII gene. When nptII was fused to the nopaline synthetase promoter and 3´ flanking regions from the Agrobacterium tumefaciens Ti plasmid, about half the transformants selected for growth on nitrate plates following cotransformation with Nit1 were resistant to low levels of kanamycin. At least one of these transformants appeared to synthesize a polypeptide with enzymatic activity (Hall et al., 1993). A very low frequency transformation event was observed with an octopine synthase/ nptII chimeric gene. In this transformant, the polypeptide was larger than expected, suggesting that it may have been a fusion protein resulting from a rare in-frame insertion into an expressed coding

Chapter 4 Nuclear Transformation region. Recently, it was reported that stable paromomycin-resistant C. reinhardtii transformants using were recovered at a low frequency the aminoglycoside 3´-phosphotransferase typeVIII gene from Streptomyces rimosus, which has a high GC content and weakly biased codon usage (Sizova et al., 1996). Higher frequency transformation (~ 1 % of the rate with endogenous C. reinhardtii genes) was recently reported when the Streptoalloteichus hindustanus ble gene, which encodes a bleomycin binding protein and confers resistance to phleomycin, was linked with 5´ and 3´ regulatory sequences from the RbcS2 gene (Stevens et al., 1996). Since antibiotic resistance seems to vary with different strains, media, and lots of antibiotics, it should be empirically determined for each application (J. Moseley and S. Merchant, unpublished; our unpublished observations). A similar construct containing the E. coli aadA coding region, which encodes aminoglycoside adenine transferase, confers resistance to spectinomycin and streptomycin (Cerutti et al., 1997b). High frequency transformation was reported with a construct in which the chloramphenicol acetyl transferase gene from Tn9 was cloned between the 35S promoter of cauliflower mosaic virus and the 3´ region of nopaline synthetase; it was introduced into C. reinhardtii cells by electroporation (Tang et al., 1995). Finally, when the hexose transporter gene from Chlorella was fused to the constitutive Volvox tubulin promoter and transformed into Volvox, it conferred increased sensitivity to the toxic glucose/ mannose analog 2-deoxyglucose. In Volvox the gene can be selected either negatively, as just mentioned, or positively, since the cells survived for prolonged periods in the dark in the presence of glucose when the gene was expressed (Hallman and Sumper, 1996). This could be a very useful negative selectable marker, if it can be expressed in C. reinhardtii. Thus, a wide variety of C. reinhardtii genes and bacterial drug resistance markers are available for nuclear transformation, and the number will certainly increase as wild-type versions ofthe genes disrupted by insertional mutagenesis are isolated (see below).

IV. Methods for Introducing DNA into the Nuclear Genome of C. reinhardtii Each of the methods that have been developed for nuclear transformation of C. reinhardtii has certain

45 advantages, depending on the application.

A. Particle Bombardment Reliable nuclear transformation was first reported using particle bombardment (Debuchy et al., 1989; Kindle et al., 1989; Diener et al., 1990; Mayfield and Kindle, 1990). In this method, DNA is precipitated onto the surface ofsmall particles (usually tungsten) and accelerated towards the target cells by a gun powder charge or high-pressure helium. The number of colonies per bombarded plate was fairly small with the gun powder version of the device (5–50); somewhat higher levels of transformation can be achieved with high pressure helium (~200 colonies per plate). A fairly large amount of DNA is probably delivered to cells that survive the trauma, and the number of gene copies that integrate into nuclear DNA is quite high. The particle gun works better with walled than wall-less strains, and seems to result in a slightly higher rate of homologous recombination than the glass bead method described below (Kindle, 1990; Sodeinde and Kindle, 1993).

B. Glass Beads, Silicon Whiskers High frequency transformation can be obtained by agitating cells on a vortex mixer in the presence of DNA, glass beads, and 5% polyethylene glycol (PEG; Kindle, 1990). The simplicity and efficiency of this procedure, as well as the fact that it requires no specialized equipment, has made it the method of choice for most applications. This method requires cell wall-deficient cells, either a cw mutant, or cells that have been rendered cell wall-deficient by treatment with gamete lytic enzyme (autolysin). Glass bead transformation with Nit1 can be carried out with walled cells, as long as the cells are incubated in ammonium-deficient medium for several hours prior to transformation (Nelson and Lefebvre, 1995b; Kindle, 1998). The conditions under which PEG stimulates transformation are still controversial. Stevens et al. (1996) have suggested that the nature of the cell wall mutation determines whether PEG improves transformation rates, though we have not found a strain or marker for which PEG is not effective (unpublished observations). Glass bead transformation results in a substantially lower number of integrated gene copies than particle bombardment, and the number of copies appears to be proportional to the amount of added DNA (Kindle, 1990). Single

Karen L. Kindle

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or low copy number integrations are highly advantageous for certain applications such as insertional mutagenesis, where a single insertion event greatly facilitates subsequent analysis and gene isolation. Silicon carbide whiskers can be employed in place of glass beads, and their use results in transformation with substantially lower cell rates of lethality during the vortexing period (Dunahay, 1992; Dunahay, 1993). Furthermore, it is not necessary to remove cell walls prior to whisker transformation. The main disadvantage ofthis method is the potential health hazard and waste disposal problem caused by the biohazardous nature of the silicon carbide microfibers.

C. Electroporation Low frequency stable nuclear trans­ formation has been reported by using single or double electrical discharges during electroporation (Brown et al., 1991; Keller, 1995) for wall-less or walled cells, respectively. Very high frequency nuclear was reported with a transformation bacterial gene encoding chloramphenicol acetyl transferase, with asomewhatdifferentelectroporation set-up involving a DC-shifted radio frequency wave (Tang et al., 1995). A fraction of transformants generated by this technique contained unintegrated copies of introduced plasmid DNA, though the unintegrated DNA declined after prolonged periods in culture. Among stable transformants, the number of integrated copies was low in most cases. Recently, an abstract also reported very high frequency transformation by electroporation (up to cell) ofcell wall-deficient strains with a 12 kb plasmid carrying Arg7 (Shimogawara et al., 1998). V. Reporters and Promoters The analysis of gene expression and the isolation of regulatory mutants is greatly facilitated by reporter genes encoding proteins that can easily be assayed, screened, or selected. For analysis ofprotein function or to make dominant negative mutants, it is important to express an altered coding region at a high level. If the expression of a given gene is anticipated to have a negative effect on the cell, it may be necessary to use a regulated promoter. The expression of antisense constructs or genes expected to have a dominant

negative effect has not yet been reported for Chlamydomonas, though a number of useful molecular tools that may have utility for these applications have emerged.

A. Arylsulfatase as a Reporter Probably the most useful reporter described to date is the periplasmic enzyme arylsulfatase (Ars), which functions in sulfur acquisition from aromatic compounds under conditions where other sulfur sources are unavailable (de Hostos et al., 1989; Chapter 32, Davies and Grossman). Expression of the Ars gene is completely repressed under normal sulfur-replete growth conditions, and sensitive, chromogenic substrates are available for plate and enzymatic assays (Davies et al., 1992; Ohresser et al., 1997). The Ars gene has been used as a reporter for gene expression from the promoters of TubB2 Davies et al., 1992), Cyc6 (cytochrome Quinn and Merchant, 1995), CabII-1 (Jacobshagen (mitochondrial carbonic et al., 1996), anhydrase; Villand et al., 1997), and Nit1 (nitrate reductase; Ohresser et al., 1997). Transformants containing the TubB2/Ars fusion gene expressed it at a much lower level than the endogenous TubB2 gene. Furthermore, no increase in Ars enzymatic activity was detected following deflagellation, although the abundance of the chimeric mRNA increased transiently (Davies et al., 1992), with a time course identical to endogenous TubB2 transcripts. The arylsulfatase protein is very stable, which may explain why Ars activity did not vary as much during a 24 h light-dark cycle as chimeric CabII-1/Ars mRNA (Jacobshagen et al., 1996). However, Ars enzyme activity increased in response to a shift from Cureplete to Cu-free medium in transformants containing a construct in which the Ars coding region had been fused to the Cyc6 promoter (Quinn and Merchant, 1995). Furthermore, a dramatic (> 100­ fold) increase in Ars activity was measured within four hours of a shift from ammonium-containing to nitrogen-free medium in transformants carryingNit1/ Ars chimeric constructs (Ohresser et al., 1997). A very sensitive method for detecting Ars activity has been reported recently in which is used as the substrate and a diazonium salt as the post-coupling reagent. This resulted in a 250-fold increase in sensitivity over 5-bromo-4-chloroindolylsulfate ( Ohresser et al., 1997). Because of the increase in sensitivity, it was possible to study

Chapter 4 Nuclear Transformation promoter function in pools of co-transformed colonies, rather than isolating and analyzing individual transformants, which should correct for variations in transcription activity due to different genomic insertion sites. and can be used to Both stain cells on agar plates to distinguish colonies that express Ars from those that do not. This is useful in identifying both transformants that express the reporter gene as well as mutants that fail to express it. Davies et al. (1994) have used this screen to isolate mutants defective in the expression ofthe endogenous Ars gene.

B. Other Reporters Another extracellular enzyme with the potential to be a good reporter is alkaline phosphatase, since sensitive enzymatic and plate assays are available. C. reinhardtii alkaline phosphatases have recently been characterized (Quisel et al., 1996). Expression of the gene encoding protochlorophyllide oxidoreductase is an easily screened visual marker that can be used both for selection (Li and Timko, 1996), as described above, or potentially as a reporter. The Rsp3 gene, which complements a motility defect in the paralyzed flagellar mutant pf14 (Diener et al., 1990), has recently been used as a reporter to identify mutants that express the Nit1 gene constitutively (Zhang and Lefebvre, 1997). A Nit1/ Rsp3 gene fusion was shown to confer motility when transformed into the pf14 strain, but only in nitrate medium, conditions under which the Nit1 gene is expressed. Twenty-one mutants that swam in the presence of ammonium were found to express both the reporter gene and the endogenous Nit1 gene constitutively, identifying at least two loci that regulate genes in the nitrate assimilation pathway.

C. Constitutive Promoters The most widely used constitutive promoter to date has been that from RbcS2 (Goldschmidt-Clermont and Rahire, 1986), which was used to express an epitope-tagged, non-acetylatable to 40– 70% of the total a five-fold increase over expression from the TubA1 promoter (Kozminski et al., 1993). The RbcS2 promoter has since been used in constructs to express the Cry1, ble, and aadA genes (Nelson et al., 1994; Stevens et al., 1996; Cerutti et al., 1997b). Other constitutively expressed

47 promoters that have been shown to be active in chimeric constructs include those from PetE (or Pcy1, encodes plastocyanin) and AtpC (encodes the gamma subunit of chloroplast ATPase) which have been fused to the coding region of Cyc6 (Quinn and Merchant, 1995), CabII-1, which has been fused to the nitrate reductase coding region (Blankenship and Kindle, 1992), and the and CabII-1 promoters, which were fused to arylsulfatase, as described above. RbcS2 is generally regarded as the strongest nuclear promoter tested to date. Although CabII-1 mRNA accumulates to a high level in vivo, transformants carrying a tagged version of this gene accumulated only a very low level of the marked transcript. Furthermore, chimeric CabII-1/Nit1 constructs that included up to 8.5 kb of upstream sequence were expressed only at a low level. However, wild-type levels of cytochrome accumulated when this gene was derived by the AtpC and PetE promoters (Quinn and Merchant, 1995). A comparison of the expression of the same reporter gene driven by each of these promoters would clearly be useful.

D. Regulated Promoters Many genes are regulated in response to environ­ mental conditions or during development and are therefore sources of potentially useful promoters for nuclear transformation. Some of these are expressed constitutively, but further induced in response to which is induced certain conditions, such as when cells are deflagellated. Others are expressed at a very low level until they are induced in response to specific stimuli. Table 1 shows a list of regulated promoters whose function has been tested by fusion to a reporter gene. The CabII-1 gene is expressed constitutively in mixotrophically grown cells, but is induced in response to light in synchronized cells grown under phototrophic conditions (Shepherd et al., 1983; Kindle, 1987), apparently by a circadian rhythm (Hwang and Herrin, 1994; Jacobshagen and Johnson, 1994). The Nit1 gene is induced by growth in nitrate medium and derepressed in nitrogen-deficient medium (Fernández et al., 1989; Quesada and Fernández, 1994; Chapter 33, Fernández et al.; Nia1 is the same as Nit1). Cyc6 expression is regulated at the level of transcription, which is induced when copper is limiting (Merchant et al., 1991; Quinn and Merchant, 1995). In addition to its response to gene expression varies deflagellation,

48

during the cell cycle (Silflow and Rosenbaum, 1981; Nicholl et al., 1988), while the mitochondrial carbonic anhydrase genes Ca1 and Ca2 are among a number of nucleus-encoded genes whose expression is (Villand et al., induced by low (air) levels of 1997). Conditional gene expression would be particularly important for certain experimental approaches. For example, with dominant negative mutants or antisense constructs, where the gene product might have a deleterious phenotype, it may be essential to keep the gene silent while the cells are growing. The gene could then be induced to determine its phenotypic effect. Alternatively, ifa gene is required for viability, one could introduce a conditionally expressed copy and then generate an insertion mutation in the endogenous (constitutively expressed) gene. It might then be possible to assess the phenotype of a null mutation by transferring the cells to conditions that prevent the expression ofthe introduced gene. Neither of these approaches has yet been taken to examine gene function in C. reinhardtii, partially due to problems with gene silencing and the difficulties in generating gene-targeted disruption mutants. However, the characterization of these regulated promoters is a first step toward developing these sophisticated genetic approaches. Several issues are important for the use of conditional promoters. The first is how much residual transcription takes place under repressed or noninduced conditions. This is critical if expression of the gene product is expected to be deleterious, since it might determine whether transformants capable of expressing the gene under inducing conditions can be recovered. The second issue is how highly expressed the genes are when they are induced/ derepressed, which may determine whether a phenotypic effect can be observed. Finally, since gene silencing seems to be a significant problem in C. reinhardtii (see below), it would be useful to know

Karen L. Kindle

how long a gene that is maintained under noninducing or repressing conditions maintains the ability to be activated upon transfer to appropriate conditions. When the Nit1 (Ohresser et al., 1997) and Cyc6 (Quinn and Merchant, 1995) promoters were fused to the arylsulfatase coding region and transformed into C. reinhardtii, the expression of arylsulfatase activity under non-inducing conditions varied from undetectable to very low. Ars activity increased by a factor of 100–1000 upon induction using the Nit1 promoter and up to 80-fold when the Cyc6 promoter was induced. Similarly, when the Ca1/Ca2 promoter was fused to Ars, both mRNA and enzymatic activity and increased were undetectable in 5% dramatically upon a shift to low (Villand et al., 1997). It would be interesting to compare expression at the mRNA level ofthese constructs under inducing and repressing conditions, to determine which promoters exhibit the tightest regulation and highest levels of induced gene expression. A priori Nit1, Cyc6 (and perhaps Ars) would seem to be good promoters for genes to be kept silent, since they are turned off in normal culture medium, which contains ammonium, copper, and sulfur. Alternatively, the Ca1 or Ca2 promoter would be useful for genes that should be maintained in an expressed state, since they are induced in air, but repressed in 5% (Villand et al., 1997).

VI. Characteristics of Transformation Events In order to survive selection, transformed cells must replicate the introduced DNA and express the selectable marker at a level high enough to allow cell growth. Whether expression of the introduced DNA remains stable appears to depend to some extent on whether it remains under selection. The difficulty in achieving stable expression of foreign and C.

Chapter 4 Nuclear Transformation reinhardtii genes at a high level seems to have several facets, which are discussed below.

A. Nature and Stability of Introduced DNA The best characterized markers (Nit1 and Arg7) and methods for introducing DNA into C. reinhardtii cells (glass bead transformation and particle bombardment) result primarily in transformants that contain integrated copies of the introduced DNA (Debuchy et al., 1989; Kindle et al., 1989; Kindle, 1990). The restriction pattern of introduced DNA is for the most part stable following extended periods in culture, whether or not the cells are maintained under selection (Kindle et al., 1989; Day et al., 1990; Cerutti et al., 1997a), though minor differences have occasionally been observed (Cerutti et al., 1997a). The restriction pattern of the introduced DNA is also stable through meiosis (Kindle et al., 1989; Diener et al., 1990). A recent report suggested that some transformants generated by electroporation contained unintegrated plasmid DNA (Tang et al., 1995). When undigested DNA from these transformants was separated by electrophoresis, a band that hybridized to the introduced plasmid migrated considerably ahead of chromosomal DNA. Furthermore, the introduced plasmid could be separated from the main nuclear DNA on CsCl gradients and recovered by electro­ poration into E. coli. It is unknown whether the observation of unintegrated DNA is a consequence of the electroporation method or the selectable marker. It is tempting to speculate that the high rate of may transformation with a foreign gene have resulted because a large fraction oftransformants harbor unintegrated gene copies, which might be less subject to silencing by chromosomal mechanisms (see below). If this electroporation method also results in the transient maintenance of introduced C. reinhardtii DNA in an unintegrated state, the isolation of genes by functional complementation could be facilitated, since it might then be possible to rescue into E. coli the gene responsible for complementing the defect.

B. Insertion Events Although the number of insertion events appears to depend on the amount of DNA delivered into the nucleus, the nature of the insertions appears to be similar, regardless of whether particle bombardment

49 or glass bead-mediated transformation is used to introduce DNA into the cell. Insertions nearly always take place in ectopic locations, since DNA blot analysis indicates that the endogenous gene is unaltered, and the introduced gene(s) segregate independently of the endogenous one in genetic crosses (Kindle et al., 1989; Diener et al., 1990; Mayfield and Kindle, 1990; Nelson and Lefebvre, 1995b). From these observations, it has been suggested that insertion occurs at random ectopic locations, though the high rate at which some insertional mutations are recovered suggests that integration may occur preferentially in some areas of the genome (Wilkerson et al., 1995). The insertion of linearized plasmid DNA appears to occur through its ends, usually with relatively small terminal deletions (Blankenship and Kindle, 1992; Cerutti et al., 1997b). Insertions can result in arrays with multiple copies of the introduced DNA or alternatively in a single integrated gene copy. Several unlinked single copy insertions have been documented in some transformants (Pazour et al., 1995). Multiple gene copies are usually inherited together in the progeny of genetic crosses, although occasionally one or more of multiple integrated DNA copies will segregate independently (Kindle et al., 1989; Pazour et al., 1995). A simple tandemly repeated array is ruled out by the complexity of hybridization patterns in DNA blots. Markers that are cotransformed on independent replicons are often genetically linked (Diener et al., 1990; Zhang and Lefebvre, 1997). It is likely that when the introduced plasmid DNA is present in the cell at a sufficiently high concentration, the molecules join together prior to integration. To determine whether homologous recombination between introduced molecules is efficient, two different truncated Nit1 or Arg7 genes were cotransformed into an appropriate mutant strain. Neither truncated gene could by itself complement the mutation in the recipient strain, but an extrachromosomal homologous recombination event would generate a fully functional gene. Using the two truncated genes, cotransformants were recovered at nearly as high frequency as with a single intact gene, suggesting that extrachromosomal homologous recombination is efficient (Sodeinde and Kindle, 1993; Gumpel et al., 1994). In high copy number transformants generated with the RbcS2/aadA/RbcS2 construct, genomic DNA fragments consistent with tail to tail, head to head, and head-to-tail repeats were observed (Cerutti et al., 1997b). Evidence for a small

50

number of tandemly repeated copies in low copy number transformants has been obtained both for targeted (homologous) integration events (Sodeinde and Kindle, 1993) and for integrations at ectopic sites (Cerutti et al., 1997b). Together, these results suggest considerable end-to-end intermolecular joining of the introduced plasmid DNA prior to integration.

C. Cotransformation and Expression of C. reinhardtii Genes Because both particle bombardment and glass bead transformation can result in the introduction of multiple copies of the transforming DNA, the rate of cotransformation of an unselected DNA is very high (Kindle et al., 1989; Day et al., 1990; Diener et al., 1990; Kindle, 1990). This is important for identifying transformants that contain genes that have an unknown or unselectable phenotype. In the case of C. reinhardtii genes, most cotransformants that contain the unselected DNA appear to express it, at least initially. However, the rate of expression of an gene in introduced non-functional transformants was increased significantly by putting both the selected and unselected genes on the same plasmid (Periz and Keller, 1997). We have noted that cotransformants that initially express plastocyanin genes with mutations that prevent chloroplast protein import and accumulation ofthe mature protein fail to express the gene at the mRNA level after various periods in culture (unpublished observations). Introduced genes may tend to be silenced during culture, so that only when gene expression confers a selective advantage, would gene expression persist. If gene expression is deleterious, cells in which gene expression has been silenced would be selected rapidly. Genes on the same piece ofDNA presumably integrate into the same locus, so that maintaining selection for one gene may prevent silencing of the unselected gene. There is a substantial difference in the levels of gene expression among different transformants with the same construct, e.g. (Blankenship and Kindle, 1992; Quinn et al., 1993; Davies et al., 1994), presumably due to differences in transcription caused by the particular genomic insertion site, so called position effects. In some cases, actively expressed reintroduced C. reinhardtii genes accumulate mRNA at a level comparable to the endogenous gene, e.g. Nit1 (Kindle et al., 1989) and PetE (or Pcy1,

Karen L. Kindle plastocyanin; Quinn et al., 1993) while for other genes, the level of mRNA or protein is much lower, e.g. CabII-1 (Blankenship and Kindle, 1992), Cyc6 (Quinn and Merchant, 1995), TubA Kozminski et al., 1993; Periz and Keller, 1997), and Davies et al., 1994). In the case of TubB2 chimeric genes containing the C. reinhardtii CabII­ 1 promoter linked to the Ars and Nit1 coding regions, the level of mRNA accumulation was somewhat higher than that of Ars and Nit1 under induced conditions, but significantly lower than CabII-1 (Blankenship and Kindle, 1992; Jacobshagen et al., 1996), even though 8.5 kb of upstream CabII-1 promoter sequences were integrated in some transformants. In most cases, the level ofexpression of introduced C. reinhardtii genes is not correlated with the number of introduced gene copies (Blankenship and Kindle, 1992; Quinn et al., 1993). An interesting exception was the Oee1 gene, which encodes the 33 kD polypeptide of the oxygen evolving complex. In this case, gene expression was proportional to the number of introduced gene copies, so that transformants harboring three to five introduced copies accumulated the polypeptide in excess of wild-type (Mayfield, 1991). Perhaps a locus control region, defined as an element that results in gene expression that is independent of integration site but proportional to the number of introduced gene copies (Dillon and Grosveld, 1993), lies close to Oee1 and was included in the cloned DNA that was used for transformation.

D. Expression of Foreign Genes/Silencing The difficulty inestablishing anucleartransformation system for C. reinhardtii was largely due to the difficulty, not yet fully understood, in expressing foreign genes. The failure to express non-C. reinhardtii genes may be attributed to a number of factors, including the inability to recognize regulatory sequences from heterologous sources, gene silencing of integrated foreign gene copies, or inefficient translation and/or instability of foreign transcripts, which could be a consequence of differences in codon usage compared to the very biased codon usage of C. reinhardtii nuclear genes. It seems likely that some of the problem with foreign gene expression is due to the coding region, since chimeric foreign genes with C. reinhardtii promoters and 5´ and 3´ untranslated regions are expressed at a low frequency (Stevens et al., 1996; Cerutti et al., 1997b; see below)

Chapter 4 Nuclear Transformation or at a frequency too low to be detected (Blankenship and Kindle, 1992). However, differences in codon usage seem unlikely to be the major reason for the difficulty in expressing foreign coding regions. The ble gene from Streptoalloteichus hindustanus has codon usage similar to that in C. reinhardtii, and although phleomycin-resistant transformants could be selected directly, the number of transformants was still about two orders of magnitude lower than those obtained using a homologous C. reinhardtii transformation marker (Stevens et al., 1996). A similarly low recovery of spectinomycin-resistant transformants was reported when the RbcS2 promoter was used to drive expression of aadA (Cerutti et al., 1997b). Furthermore, a careful study of aadA transformants indicated that the spectinomycin­ resistant phenotype of many of them was unstable when cells were grown under nonselective conditions, indicating that gene silencing can occur even after the initial selection (Cerutti et al., 1997b). Thus, there appear to be at least two levels at which foreign genes may fail to be expressed. Assuming that the same kinds of integration events occur regardless of the nature ofthe introduced DNA, it appears that 97– 99% of insertion events fail to lead to the expression of foreign genes. Alternatively, it is possible that expression of foreign genes is affected in all locations, and it is only those that integrate into extremely active sites in the genome that are expressed well enough to survive selection. The instability of expression following the initial integration and selection appears to be an independent problem, which affects about half of the selected transformants (Cerutti et al., 1997a). As mentioned above, even reintroduced C. reinhardtii genes may be subject to this kind of silencing. The extent of methylation of the CabII-1 promoter, assessed by digestion with methylation-sensitive restriction enzymes, was related to the expression status ofintroduced chimeric constructs: unexpressed ) CabII-1/uidA (uidA encodes constructs were hypermethylated relative to CabII1/Nit1 constructs that were expressed at a level high enough to allow growth on nitrate medium (Blankenship and Kindle, 1992). Cerutti and coworkers have analyzed gene silencing using the RbcS2/aadA gene. They have shown that silencing and reactivation are reversible and that they are not a consequence of loss or rearrangement of the integrated gene copies, as determined by digestion with restriction enzymes. Neither were the instances

51 of gene silencing examined correlated with changes in methylation status or chromatin structure, as assessed by sensitivity to DNase (Cerutti et al., 1997a). An intriguing possibility is to use the unstable expression of aadA to search for mutants that lose the ability to silence introduced gene copies. It might be possible to do this by selecting for strains that reactivate an unstably silenced gene copy to a stably expressed state. Such a mutant might be defective in gene silencing and therefore transformable by foreign genes at a high rate.

VII. Insertional Mutagenesis and Gene Tagging

A. Development and Application of the Approach The observation that transforming DNA integrates into the nuclear genome at apparently random locations by nonhomologous recombination sug­ gested that these insertions might be mutagenic if they disrupted a gene. Furthermore, the inserted DNA would act as a molecular tag that would allow the mutant gene to be isolated. (See Fig. 1 for an outline of this approach.) Tam and Lefebvre (1993) pioneered this powerful approach by generating a series of motility-defective mutants following transformation with theNit1 gene. They demonstrated phenotype that for more than 85% of them, the cosegregated with the motility defect in genetic crosses, suggesting that the insertion of the selectable marker was the mutagenic event. The C. reinhardtii DNA that flanked the inserted DNA was cloned for two mutants, using two different approaches. In the first case, the plasmid DNA was intact, so that it could be recovered in E. coli by transformation using genomic DNA that had been digested with an appropriate restriction enzyme and self-ligated. In the second instance, the vector sequences were not intact, but a partial genomic library was prepared and hybridized with a Nit1 probe to take advantage of the tightly linked Nit1 gene. In both cases, the wild-type alleles were screened from a genomic library, and the functional gene was demonstrated to complement the mutant phenotype. An analysis of three of the insertion mutations revealed that in all three cases, there were rearrangements at the tagged locus. In two cases, the rearrangements were deletions, which ranged in size from 3–4 kb to over 23kb.

52

Insertional mutagenesis has been utilized to generate mutants defective in many cellular processes, including photosynthesis (Adam et al., 1993; Gumpel et al., 1995; Khrebtukova and Spreitzer, 1996; Inoue et al., 1997; M. Goldschmidt-Clermont, unpublished), motility (Wilkerson et al., 1995; Smith and Lefebvre, 1996), phototaxis (Pazour et al., 1995), sulfur and nitrate assimilation (Davies et al., 1994; Prieto et al., 1996a), and sensitivity to cadmium or high salt (McHugh and Spanier, 1994; Tang et al., 1995; Prieto et al., 1996b). The efficiency with which mutants were recovered varied dramatically in different studies, depending in part on the number of genes involved in a given process, i.e. the target size for mutagenesis, and on the conditions used for selection. For example, the recovery of nonphotosynthetic mutants has been reported to vary between 0.22% of transformants, when transformants were selected in dim light and then screened for failure to grow without acetate (Adam et al., 1993) to 1.5%, when transformants were selected in the dark and then screened for an acetate-requiring phenotype (Khrebtukova and Spreitzer, 1996). Many of the

Karen L. Kindle

mutants recovered in the latter study could be maintained only in the dark, suggesting that selection in the dark is important for recovering certain kinds of nonphotosynthetic mutants. The recovery of motility-impaired mutants was exceptionally high in one study (2.5%; Tam and Lefebvre, 1993) and the number of mutations in the single copy gene that encodes IC78, the intermediate chain of outer arm transformants) dynein, was so high (0.25% of the that it must represent a hotspot for integration and/or deletion (Wilkerson et al., 1995). Thus, it is very likely that insertional mutagenesis is not as random as once thought. A priori, there is no way to tell whether a gene is more or less likely than average to be disrupted. Therefore, it is impressive that mutations sought in two single copy genes required for photosynthesis, PsaF and AtpC, were recovered using this method. In both studies, the authors were originally trying to target mutations to these genes by homologous recombination. The mutations that were recovered, a deletion in PsaF (Farah et al., 1995) and what appears to be an insertion in AtpC (Smart and Selman, 1991), were probably initiated

Chapter 4 Nuclear Transformation by a random insertion event, though the deletion in PsaF could have resulted from abortive homologous recombination.

B. Technical Considerations It is advantageous to carry out insertional mutagenesis in such a way as to maximize the number of mutants in which a single insertion event has occurred, and in which both the marker and the vector sequences of the introduced DNA are intact. This facilitates genetic analysis, since the mutant phenotype and selectable marker should cosegregate, and increases the chance that the genomic DNA that flanks the insertion can be cloned by a plasmid rescue strategy. To accomplish this, the plasmid DNA should be linearized before transformation. This increases the likelihood that recombination will occur through the ends, rather than at a random break in the vector sequences (J. Woessner and M. Campbell, personal commun­ ication); the selectable marker must be intact in order to recover the transformant. Furthermore, the minimum amount of DNA necessary to generate a reasonable number of transformants should be used, to decrease the likelihood of concatemer formation prior to insertion or multiple integration events (Kindle, 1990). The Nit1 and Arg7 genes have both been used for insertional mutagenesis, and so far the rate of tagging for Nit1 appears to be quite high, generally about 80% (Tam and Lefebvre, 1993; Pazour et al., 1995). In some of the mutants characterized in detail by Pazour et al. (1995), the mutant phenotype cosegregated with an integrated piece of DNA, even though the functional Nit1 gene integrated elsewhere. In fact, it seems likely that even untagged mutations are due to deletions generated as a byproduct of insertion events, since the likelihood of a random mutation among the relatively small number of transformants examined in most studies (2000– 20,000) is very low. As already noted, deletions of the target locus are often associated with insertions of plasmid DNA (Tam and Lefebvre, 1993; Wilkerson et al., 1995).

C. Isolation of the Gene Responsible for the Mutant Phenotype Before embarking on a strategy to isolate the C. reinhardtii DNA that flanks the plasmid insertion, it

53 is important to establish that the selectable marker, or at least a part of the introduced plasmid DNA, cosegregates with the mutant phenotype in genetic crosses. A restriction analysis of the inserted DNA can then establish whether the vector is intact so that a plasmid rescue approach is likely to succeed or whether a hybridization strategy must be employed. Once the DNA flanking the insertion has been isolated, it can be used as a hybridization probe to demonstrate a restriction fragment length poly­ morphism (RFLP) between the mutant strain and the transformation recipient and also to isolate a wildtype genomic clone from an appropriate library. If there has been no deletion of genomic DNA at the insertion site, the cloned wild-type DNA should be able to complement the mutant phenotype, providing that the entire gene is present on the cloned fragment. If there has been a deletion, then the gene responsible for the mutant phenotype could be some distance away from the fragment that flanks the insertion site. It might therefore be necessary to perform a short walk to find the DNA fragment capable of complementing the mutant phenotype. If the mutant locus is tagged by an intact plasmid copy, the entire mutagenesis and gene isolation can be carried out within a few months, though in practice it often takes a while to hit upon the ideal screening strategy.

D. Perspectives The real power of insertional mutagenesis by nuclear transformation lies in the ease with which huge numbers of potentially tagged mutants can be created. It is easy to generate 10,000 transformants and not unreasonable to contemplate making 100,000. Much more labor may be involved in screening this large pool of potential mutants, so visual screens, such as those for high chlorophyll fluorescence are highly advantageous (Farah et al., 1995; Bennoun and Béal, 1997; Chapter 23, Bennoun and Béal). Because of the large number of mutants that can be generated, it should be possible to tag every gene in a given pathway that is not required for viability, assuming that insertional mutagenesis is reasonably random. Even if some genes cannot be tagged because they lie in a part of the genome where insertions do not readily occur or near genes that are required for viability, the number of mutations in interesting genes is likely to be large, as has already been demonstrated by information generated in the few

54 years since the technique has been available. Despite the potential of insertional mutagenesis, not all interesting genes can be isolated using this approach, which for the most part generates null mutations. Fortunately, alternative strategies for isolating genes by their ability to complement recessive mutations have been developed, and are described further below.

VIII. Gene Isolation by Complementation of a Mutant Phenotype Shotgun cloning requires a high efficiency trans­ formation system as well as a representative genomic library with large inserts. Complementation of the arg7 mutation with a total genomic cosmid library demonstrated the feasibility of selecting C. reinhardtii genes from a complex mixture by function (Purton and Rochaix, 1994b). Isolation of the transforming DNA from such a rescued mutant would be easier if it replicated autonomously in the host and could be recovered by transformation in E. coli. Since DNA introduced into the nuclear genome of C. reinhardtii usually integrates, an alternative strategy has been employed for isolating genes by function: the use of an indexed cosmid library. Zhang et al. (1994) produced a genomic cosmid library containing 11,280 clones with an average insert size of 38 kb. The individual cosmids were picked into the wells of 120 microtiter dishes and cosmid DNA was prepared from pools of clones and used in transformation experiments to complement the arg7 mutation. By identifying the individual microtiter dish that contained the DNA that gave rise transformants and then pinpointing the row to and column, a cosmid containing a functional Arg7 gene was identified. This library has recently been used to isolate the gene that complements the high -requiring mutant ca-1 (Funke et al., 1997). The gene that was recovered encodes an intracellular carbonic anhydrase and is identical to the previously identified Cah3 gene (Karlsson et al., 1995). The same library has also been used to isolate genes that rescue a nonphotosynthetic mutant that lacks psbB mRNA (F. Vaistij, M. Goldschmidt-Clermont, J.-D. Rochaix, unpublished) and a psaA trans-splicing mutant (C. Rivier, M. Goldschmidt-Clermont, and J.-D. Rochaix, unpublished). A genomic clone capable of complementing an arg7 nac2-26 cw15 mutant (Kuchka et al., 1989) and another one complementing

Karen L. Kindle a psaA trans-splicing mutant were isolated from a genomic cosmid library prepared in an Arg7 vector (Purton and Rochaix, 1994a; J. Nickelsen, K. Perron, M. Goldschmidt-Clermont and J.-D. Rochaix, unpublished) Although constructing the cosmid library and preparing the DNA pools necessary for this approach are rather labor-intensive, the transformations are straightforward, providing that the mutation is recessive and has a low reversion rate, and that transformants have a good selectable phenotype. Identifying the cosmid that complements the mutation is a matter of only two or three rounds of transformations. It is in some respects simpler than insertional mutagenesis strategies, since relatively little characterization of the mutant is required. Furthermore, this approach should allow the isolation of genes that complement mutations generated by chemical or UV mutagenesis, as well as insertional mutants that are not tagged or that have a complex insertion pattern. If the mutation of interest is dominant, e.g. a dominant suppressor mutation, the same strategy can be used, though it would be necessary in this case to prepare the genomic library from the suppressor strain and transform the DNA into the appropriate mutant strain.

IX. Homologous Recombination and Gene Targeting The ability to target endogenous genes for homolo­ gous recombination raises the possibility of disrupting their function or replacing them with altered versions. Therefore, considerable effort has been expended to develop technology for gene targeting in C. rein­ hardtii. As mentioned earlier, it is clear that C. reinhardtii contains the enzymatic machinery necessary for efficient homologous recombination, since transformation is nearly as frequent using two plasmids with non-overlapping mutations in Nit1 or Arg7 as with intact versions of the gene (Sodeinde and Kindle, 1993; Gumpel et al., 1994). Moreover, homologous recombination has been shown to occur between extrachromosomal DNA and the cognate endogenous gene, by using a Nit1 gene truncated at the 5´ end to repair the genomic copy. Molecular evidence for homologous recombination was presented and was consistent with single or multiple insertion events as well as gene replacement events. The estimated ratio of targeted integration and

Chapter 4 Nuclear Transformation replacement events to random insertion events was significantly higher for the particle gun (1:40) than for glass bead transformation (1:1000), though the reason for this was not established (Sodeinde and Kindle, 1993). For glass bead transformation, 70% of the events were consistent with single or multiple insertions, while about 30% were replacement events (Kindle and Sodeinde, 1994). Rare gene-targeted insertions have also been documented at the Arg7 locus (Gumpel et al., 1994). Several groups have tried to generate mutations by gene-targeting strategies, but have met with very limited success. Attempts to obtain a mutant with a disrupted AtpC gene by performing glass bead transformation in the presence of excess herring sperm DNA resulted in the isolation of a mutant that was apparently the consequence of insertion of the herring sperm DNA within the AtpC gene (Smart and Selman, 1991; our unpublished observations). Similarly, Farah et al. (1995) tried to target a mutation to PsaF by cotransforming an arg7 strain with the Arg7 gene and a disruption plasmid that contained two short deletions at either end of the PsaF gene. The disruption plasmid was linearized between these deletions so as to produce a gap. Among 22,000 transformants, 110 colonies with anomalous fluorescence induction kinetics suggestive of a possible PsaF defect were identified. They were then screened by hybridization to identify those that carried a PsaF deletion corresponding to the gap in the original transforming plasmid. The PsaF deletion mutant that was isolated did not have a structure consistent with a targeting event and probably arose as a consequence of a deletion following random insertion of one of the transforming plasmids or possibly from an abortive homologous recombination event. The gap would probably have been repaired during accurate homologous recombination in C. reinhardtii, as it is in yeast and mammals (Szostak et al., 1983; Valancius and Smithies, 1991). The only convincing evidence for a mutagenic gene targeting event was reported by Nelson and Lefebvre (1995a), who used a targeting plasmid in which the Nit8 gene was interrupted by the selectable Cry1 gene. Two substrates were used: 1) a fragment containing only the disrupted C. reinhardtii Nit8 gene; and 2) a plasmid linearized within C. rein­ hardtii DNA downstream of the disrupted Nit8 gene. The linearized plasmid would be expected to integrate by a single crossover, giving rise to a gene duplication that would not be mutagenic unless recombination

55

subsequently took place between the duplicated copies to eliminate the wild-type copy. The isolated fragment would be expected to replace the endo­ genous gene by a double recombination or gene conversion event, which would be mutagenic. (See Fig. 2 for an illustration of mutagenesis by insertion and replacement events.) Potential nit8 strains were enriched for by selecting for chlorate resistance and screened for a phenotype. No nit8 strains were recovered using linearized plasmid DNA, while eight nit8 mutants were recovered using the isolated fragment. Three of them showed evidence of a homologous recombination event, but only on one

56 side of the Nit8 gene. In each case the other end was generated by a more complex process, probably secondary to a nonhomologous recombination event. There were no simple homologous replacements. Double recombination events may be extremely rare in C. reinhardtii. Earlier work (Sodeinde and Kindle, 1993; Kindle and Sodeinde, 1994), suggested that single; recombination events are more frequent than homologous replacements. Furthermore, even the homologous replacement events that were observed may have arisen in two steps: an insertion followed by homologous recombination between the integrated copies. In the work with Nit1, either insertion or phenotype, replacement would have resulted in a while for disruption of Nit8, only a complete Nit8 replacement would have led to a chlorate-resistant phenotype. The frequency of such double recom­ bination events may be so low that even random insertions into the single copy gene or insertioninduced deletions are more frequent. It is interesting that in mammalian systems, insertion and replacement vectors target with similar efficiency (Thomas and Capecchi, 1987). In conclusion, it is clear that gene-targeting events in C. reinhardtii are very rare, occurring with a frequency comparable to that for random insertion in a specific gene. Perhaps the efficiency could be improved by performing transformation in syn­ chronized cells at a specific time in the cell cycle, when the chromatin might be less condensed and more likely to interact with exogenous DNA. Gene targeting might also be enhanced by transforming gametes and then immediately mating them, to increase the chance that they would contain a relatively high concentration of introduced DNA during meiosis, a time when chromosomes are known to undergo homologous recombination. It might also be helpful to induce DNA-repairpathways by treating cells with DNA damaging agents. The best template for gene targeting appears to be a linearized or gapped plasmid, with mutations engineered upstream and downstream of the linearization point, as shown in Figure 2. However, the introduced mutations should not lie too close to the linearization site or gap, since the targeting DNA may be digested from the ends prior to double-strand gap repair, which could correct the introduced mutations. Nelson and Lefebvre’s results (1995a) suggest that it may be advantageous to use plasmid DNA prepared from E. coli strains that are deficient in methylation. A strong selection or screen for targeting events is essential, given their

Karen L. Kindle low incidence. A scheme that would select for very rare homologous recombination events in genes for which there is no easy screen would clearly be very useful (Mansour et al., 1988).

X.The Use of Nuclear Transformation to Study Promoter Function Nuclear transformation has the potential to facilitate the analysis of a wide variety of cellular processes, some ofwhich are discussed elsewhere in this volume. Since promoter structure and function are important issues for technical aspects ofnuclear transformation, I will conclude this review with a more detailed examination of promoter structure and function, as assessed by transforming various constructs into the C. reinhardtii nucleus. As mentioned above and summarized in Table 1, a number of promoters have been linked to reporter genes and shown to confer transcriptional regulation. Regulatory sequences appear to be located fairly close to the transcription start sites, within 200–300 bp, in most cases (Davies et al., 1994; Quinn and Merchant, 1995; Ohresser et al., 1997; Periz and Keller, 1997; Villand et al., 1997). The promoters of four genes have been examined ), TubA1 in some detail: TubB2 (encodes ), Cyc6, and Nit1. The TubB2 (encodes promoter contains seven copies of a repeat, termed the tub box, which is also found upstream of other flagellar genes. Davies and Grossman (1994), made fusions of the TubB2 promoter and part of the 5´ untranslated region to the arylsulfatase coding region and examined accumulation of Ars mRNA in response to deflagellation and during the cell cycle in individual transformants that expressed Ars protein. They found that sequences between –95 and +65 relative to the mRNA 5´ end were sufficient for accumulation of Ars mRNA in response to deflagellation, which occurred with a time course indistinguishable from transcripts derived from the endogenous TubB2 gene (see Table 2). Transformants with sequences between –64 and +65 expressed the gene at a very low level, and there was no response to deflagellation. The –95 to +65 region contains 4 copies of the tub box, while the –64 to +65 region has two copies. By testing constructs in which various tub boxes were removed or mutated, it appeared that multiple copies ofthe tub box were necessary for regulated expression, and that the pair in the central region was most critical.

Chapter 4 Nuclear Transformation

Transformants expressing chimeric TubB2/Ars genes accumulated Ars mRNA during a specific part of the cell cycle, but considerably earlier than the peak of TubB2 mRNA. Constructs that contained sequences from –64 to +65 accumulated Ars mRNA, but showed a dampened rhythmicity during the cell cycle, while the region from –36 to +65 allowed low level constitutive expression. Thus, the region required for basal expression is very small, while the sequences required for cell cycle regulation and response to deflagellation are partially overlapping but distinct, lying within 100 bp of the transcription start site. Since only steady-state mRNA levels were examined, the possibility of differential mRNA stability, due to sequences from +1 to +65 cannot be eliminated. Since the differences in expression among individual transformants from a single construct were greater than the variation between constructs, it was not possible to define regions that affect TubB2 expression quantitatively. Periz and Keller made a series of 5´ deletions in a TubA1 gene that had been marked by a 233 bp deletion in the coding region (Periz and Keller, 1997).

57

The plasmids also contained the Arg7 gene, and 60– 70% of transformants expressed the test gene. By testing both pools of transformants and individual transformants for induction ofthe test and endogenous TubA1 genes in response to acid-induced deflagel­ lation, two regions were found to be important for the deflagellation response. An element between –176 and –122 was important for determining the magnitude of the response, but not the time course, while deleting a region between –85 and –56 abolished the deflagellation response. The region between –85 and –16 was sufficient to confer a deflagellation response on a Cyc6/Ars chimeric construct. Addition of tub box elements to this construct had no apparent effect, but it was noted that the –85 to –16 region contains elements common to other rubulin promoters that could play an important role in the deflagellation response, including an ATB (tub-associated) box (TTCGGGG), and a GC-like box (CGGGCG.) Quinn and Merchant (1995) identified two elements between –127 and –56 relative to the transcription start site that regulate Cyc6 transcription in response to copper limitation (see Table 2). These elements

58 appear to be transcriptional activators, since when they were fused to the basal TubB2/Ars construct, they stimulated expression in response to copper limitation, but did not reduce expression in the presence of copper. Sequences upstream of–127 and within the Cyc6 coding region played no role in the copper response, while constructs that contained the region between –54 and –7 did not express the reporter. Because ofthe wide variability in expression of the gene in individual transformants, it was not possible to define additional sequence elements that played a role in quantitative, as opposed to copperregulated, levels of Cyc6 expression. Because of the sensitivity of the chromogenic substrate for assaying Ars activity, Ohresser et al. (1997) were able to measure enzymatic activity in pools of transformed colonies, which circumvents the need to analyze multiple individual transformants to correct for variations in gene expression due to position effects. For this approach to be meaningful, the co-transformation frequency must be similar for different constructs. By comparing pools of transformants generated with different promoter deletion mutations, they were able to identify elements that affected expression of the reporter gene quantitatively. Analysis of transformants expressing fusions of the Nit1 promoter to the Ars coding region suggested that positive enhancer-type elements are localized between –751 and –353, and between –282 and –198, while a negative element resides between –342 and –282 (see Table 2). The role of introns and 5´ and 3´ untranslated regions of nuclear transcripts in regulating gene expression has not been investigated systematically in C. reinhardtii, to my knowledge. However, a nitA (nitrate reductase) mutation in Volvox carteri was shown to affect the 5´ splice site of intron 2, leading to non-functional splice variants. Furthermore, nuclear transformation of a nitA cDNA into Volvox was increased 10-fold by including intron 1 or introns 9 and 10, suggesting that introns may play an important regulatory role in gene expression in this closely related alga (Gruber et al., 1996). A radial spoke protein (Rsp3) minigene, which included the cDNA sequence and ~665 and 380 bp from the 5´ and 3´ flanking regions of genomic Rsp3 DNA, successfully complemented a motility defect due to a mutation at PF14 (Diener et al., 1993). Although the level of gene expression was not directly examined, the gene functions well enough that it does not limit the synthesis or assembly of flagellar proteins.

Karen L. Kindle XI. Conclusion The development of efficient methods and molecular tools for nuclear transformation has revolutionized the analysis of cellular processes in C. reinhardtii. Nuclear transformation provides powerful approaches for assessing gene function, generating large numbers of mutants, and isolating the genes that are affected in these mutants. However, technical challenges remain. Despite recent advances, the expression of foreign genes is still problematic. Gene silencing may be part of the problem, which may also affect the stability of expression of unselected genes, both from C. reinhardtii and other organisms. Furthermore, there is, as of yet, no good way to mutate or downregulate the expression of endogenous copies of cloned genes. An effective gene targeting strategy or development of antisense technology would be very helpful. The progress in the last decade has been dramatic, and the enthusiasm for this organism as a model for a variety of developmental and metabolic pathways suggests that this progress will continue.

Acknowledgments I am grateful to the National Science Foundation for supporting my research program, most recently through grants from the Cell Biology Program (MCB­ 9406540) and the Biochemical Genetics Program (with David Stern; MCB-9406550). I am also grateful to former and present members of the Stern and Kindle labs for helpful discussions and to Donna Esposito for critiquing the manuscript.

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60 Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 87: 1228–1232 Kindle KL (1998) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Meth Enzymol (in press) Kindle KL and Sodeinde OA (1994) Nuclear and chloroplast transformation in Chlamydomonas reinhardtii: strategies for genetic manipulation and gene expression. J Appl Phycol 6: 231–238 Kindle KL, Schnell RA, Fernández E and Lefebvre PA (1989) Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol 109: 2589–2601 Kozminski KG, Diener DR and Rosenbaum JL (1993) High level expression of non-acetylatable alpha tubulin in Chlamydomonas reinhardtii. Cell Motil Cytoskeleton 25: 158–170 Kuchka MR, Goldschmidt-Clermont M, van Dillewijn J and Rochaix J-D (1989) Mutation at the Chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PS II. Cell 58: 869–876 Li J and Timko MP (1996) The pc-1 phenotype of Chlamydomonas reinhardtii results from a deletion mutation in the nuclear gene for NADPH:protochlorophyllide oxidoreductase. Plant Mol Biol 30: 15–37 Mansour SL, Thomas KR and Capecchi MR (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336: 348–352 Mayfield SP (1991) Over-expression of the oxygen-evolving enhancer 1 protein and its consequences on Photosystem II accumulation. Planta 185: 105–110 Mayfield SP and Kindle KL (1990) Stable nuclear transformation of Chlamydomonas reinhardtii by using a C. reinhardtii gene as the selectable marker. Proc Natl Acad Sci USA 87: 2087– 2091 McHugh JP and Spanier JG (1994) Isolation of cadmium-sensitive mutants in Chlamydomonas reinhardtii by transformationinsertional mutagenesis. FEMS Microbiol Lett 124: 239–244 Merchant S, Hill K and Howe G (1991) Dynamic interplay between two copper-titrating components in the transcriptional EMBO J 10: 1383–1389 regulation of cytochrome Nelson JA and Lefebvre PA (1995a) Targeted disruption of the N I T 8 gene in Chlamydomonas reinhardtii. Mol Cell Biol 15: 5762–5769 Nelson JAE and Lefebvre PA (1995b). Transformation of Chlamydomonas reinhardtii. In: Dentler W and Witman G (eds) Methods in Cell Biology, Vol 47: Cilia and Flagella, pp 513–517. Academic Press, New York Nelson JAE, Savereide PB and Lefebvre PA (1994) The CRY1 gene in Chlamydomonas reinhardtii: Structure and use as a dominant selectable marker for nuclear transformation. Mol Cell Biol 14: 4011–4019 Nicholl DST, Schloss JA and John PCL (1988) Tubulin gene expression in the Chlamydomonas reinhardtii cell cycle: Elimination of environmentally induced artifacts and the measurement of tubulin mRNA levels. J Cell Sci 89: 397–403 Ohresser M, Matagne RF and Loppes RL (1997) Expression of the arylsulfatase reporter gene under the control of the Nit1 promoter in Chlamydomonas reinhardtii. Curr Genet 31: 264– 271 Pazour GJ, Sineshchekov O and Witman GB (1995) Mutational

Karen L. Kindle analysis ofthe phototransduction pathway of Chlamydomonas reinhardtii. J Cell Biol 131: 427–440 Periz G and Keller LR (1997) DNA elements regulating tubulin gene induction during regeneration of eukaryotic flagella. Mol Cell Biol 17: 3858–3866 Prieto R, Dubus A, Galván A and Fernández E (1996a) Isolation and characterization of two new negative regulatory mutants for nitrate assimilation in Chlamydomonas reinhardtii obtained by insertional mutagenesis. Mol Gen Genet 251: 461–471 Prieto R, Pardo JM, Niu X, Bressan RA and Hasegawa PM (1996b) Salt-sensitive mutants of Chlamydomonas reinhardtii isolated after insertional tagging. Plant Physiol 112: 99–104 Purton S and Rochaix J-D (1994a) Characterization of the ARG7 gene of Chlamydomonas reinhardtii and its application to nuclear transformation. Eur J Phycol 30: 141–148 Purton S and Rochaix J-D (1994b) Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library. Plant Mol Biol 24: 533–537 Quesada A and Fernández E (1994) Expression of nitrate assimilation related genes in Chlamydomonas reinhardtii. Plant Mol Biol 24: 185–194 Quinn JM and Merchant S (1995) Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators. Plant Cell 7: 623–638 Quinn J, Li HH, Singer J, Morimoto B, Mets L, Kindle K and Merchant S (1993) The plastocyanin-deficient phenotype of Chlamydomonas ac-208 results from a frame-shift mutation in the nuclear gene encoding preapoplastocyanin. J Biol Chem 268: 7832–7841 Quisel JD, Wykoff DD and Grossman AR (1996) Biochemical characterization ofthe extracellular phosphatases produced by phosphorus-deprived Chlamydomonas reinhardtii. Plant Physiol 1 1 1 : 839–848 Rochaix J-D and vanDillewijn J (1982) Transformation of the green alga Chlamydomonas reinhardtii with yeast DNA. Nature 296: 70–73 Rochaix J-D, van Dillewijn J and Rahire M (1984) Construction and characterization of autonomously replicating plasmids in the green unicellular alga Chlamydomonas reinhardtii. Cell 36: 925–931 Shepherd HS, Ledoigt G and Howell SH (1983) Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in Chlamydomonas reinhardtii. Cell 32: 99–107 Shimogawara K, Fujiwara S, Grossman AR and Usuda H (1998) High efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics, in press Silflow CD and Rosenbaum JL (1981) Multiple and genes in Chlamydomonas and regulation of tubulin mRNA levels after deflagellation. Cell 24: 81–88 Sizova IA, Lapina TV, Frolova O, Alexandrova NN, Akopiants KE and Danilenko VN (1996) Stable nuclear transformation of Chlamydomonas reinhardtii with a Streptomyces rimosus gene as the selective marker. Gene 181: 13–18 Smart EJ and Selman BR (1991) Isolation and characterization of a Chlamydomonas reinhardtii mutant lacking the of Mol Cell Biol 1 1 : 5053– chloroplast coupling factor 1 5058 Smart EJ and Selman BR (1992) Complementation of a Chlamydomonas reinhardtii mutant defective in the nuclear

Chapter 4 Nuclear Transformation gene encoding the chloroplast coupling factor 1 subunit (atpC). J Bioenerg Biomembr 25: 275–284 Smith EF and Lefebvre PA (1996) PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella. J Cell Biol 132: 359–370 Sodeinde OA and Kindle KL (1993) Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 90: 9199–9203 Stevens DR, Rochaix J-D and Purton S (1996) The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Mol Gen Genet 251: 23–30 Szostak JW, Orr-Weaver TL and Rothstein RJ (1983) The doublestrand-break repair model for recombination. Cell 33: 25–35 Tam L-W and Lefebvre PA (1993) The use of DNA insertional mutagenesis to clone genes in Chlamydomonas. Genetics 135: 375–384 Tang DKH, Qiao S-Y and Wu M (1995) Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and hetero­ logous DNA. Biochem Molec Biol Int 36: 1025–1035 Thomas KR and Capecchi MR (1987) Site-directed mutagenesis

61 by gene targeting in mouse embryo-derived stem cells. Cell 51: 503–512 Valancius V and Smithies O (1991) Double-strand gap repair in a mammalian gene targeting reaction. Mol Cell Biol 11: 4389– 4397 Villand P, Eriksson M and Samuelsson G (1997) Carbon dioxide and light regulation of promoters controlling the expression of mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii. Biochem J 327: 51–57 Wilkerson CG, King SM, Koutoulis A, Pazour GJ and Witman GB (1995) The 78,000 Mr intermediate chain of Chlamy­ domonas outer arm dynein is a WD-repeat protein required for arm assembly. J Cell Biol 129: 169–178 Zhang D and Lefebvre PA (1997) FAR1, a negative regulatory locus required for the repression of the nitrate reductase gene in Chlamydomonas reinhardtii. Genetics 146: 121–133 Zhang H, Herman P and Weeks DP (1994) Gene isolation through genomic complementation using an indexed library of Chlamydomonas reinhardtii DNA. Plant Mol Biol 24: 663– 672

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Chapter 5 Modes and Tempos of Mitochondrial and Chloroplast Genome Evolution in Chlamydomonas: A Comparative Analysis Aurora M. Nedelcu and Robert W. Lee Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada

Summary I. Introduction II. Phylogenetic Position of Chlamydomonas Ill. Monophyletic versus Polyphyletic Origin of Mitochondria and Plastids: The Chlamydomonas Case IV. Evolution of Mitochondrial and Chloroplast Genome Size in Chlamydomonas A. Factors Contributing to Variation in Genome Size 1. Changes in Intergenic Spacer Size 2. Changes in Intron Number 3. Changes in Gene Content 4. Changes in the Amount of Repeated DNA B. Mechanisms Possibly Involved in the Evolution of Genome Size 1. Length Mutations 2. Intron Mobility 3. Gene Transfer V. Evolution of Mitochondrial and Chloroplast Genome Organization in Chlamydomonas A. Mitochondrial and Chloroplast Genome Structure B. Mitochondrial and Chloroplast Gene Order 1. Factors Contributing to Gene Rearrangement 2. Mechanisms Possibly Involved in Gene Rearrangement VI. Evolution of Mitochondrial and Chloroplast Gene Structure and Organization in Chlamydomonas A. Intron-containing Coding Regions B. Fragmented Coding Regions VII. Evolution of Mitochondrial and Chloroplast DNA Sequences in Chlamydomonas VIII. Conclusions Acknowledgments References

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Chlamydomonas mitochondrial and chloroplast genomes, in contrast to the land plant counterparts, exhibit concerted modes and tempos of evolution. The 1.5-fold variation currently observed in the size of both organelle genomes is mostly accounted for by changes in the spacer DNA and intron number, with less contribution from changes in gene content and amount of repeated DNA. Gene order is highly variable in both mitochondrial and chloroplast genomes of Chlamydomonas, the level of gene rearrangement being correlated with the abundance of short dispersed repeated sequences throughout the genome. Intron-containing-, J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 63–91. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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fragmented-, and fragmented and scrambled coding regions are common features of mitochondrial and chloroplast gene structure and organization within the group. The level of ribosomal RNA gene sequence divergence in both mitochondrial and chloroplast genomes is higher in the Chlamydomonas lineage than in land plants and is most likely due to higher rates of nucleotide substitution in Chlamydomonas organellar DNAs. The mechanisms as well as the selective pressures that shaped the organellar genomes in the Chlamydomonas lineage remain to be explained. I. Introduction The increasing accumulation of information on organellar genome sequence, structure and organi­ zation in various lineages makes it possible to address questions such as: (i) how conserved are the organellar genomes in terms of DNA sequence, structure and organization among different evolutionary lineages; (ii) what are the mechanisms underlying the evolutionary processes in organellar genomes; (iii) are the mechanisms acting on mitochondrial and chloroplast genomes similar or different; (iv) do the mitochondrial and chloroplast genomes have the same tempo and mode of evolution in a given lineage; (v) what are the evolutionary forces shaping the organellar genomes. Land plants and green algae are the only two groups that possess both mitochondria and chloro­ phyll a/b-containing chloroplasts. As the complete DNA sequence of several land plant chloroplast (see Shimada and Sugiura, 1991 for references) and mitochondrial (Oda et al., 1992) genomes has become available and more and more genes have been mapped and sequenced it has become obvious that the two organellar genomes exhibit different modes and tempos of evolution in this group: chloroplast genomes are more conserved in size and gene order but more variable in DNA sequence than the mitochondrial counterparts (Palmer, 1990). No extensive analyses have been done, however, to assess the rates and patterns of evolutionary change of green algal organellar genomes, mainly due to incompleteness or disparity of the available data. Given that Chlamydomonas is the only green algal lineage for which information on the structure, organization and DNA sequence of both mitoAbbreviations: cpDNA – chloroplast DNA; CW – clockwise flagellar configuration; DIR – direct inverted repeat; DO – directly opposed flagellar configuration; IR – inverted repeat; ITS – internal transcribed spacer; LSU – large subunit; mtDNA – mitochondrial DNA; ORF – open reading frame; rDNA – ribosomal RNA coding sequence; SSU – small subunit; TIR – terminal inverted repeat

chondrial and chloroplast genomes is available, it provides us with the opportunity to address such issues. This chapter is not intended to review the vast amount of information on Chlamydomonas organellar genomes; rather, it will present only the information considered to have evolutionary significance from a comparative point of view. Extensive general reviews dealing with the structure, organization and evolution of both mitochondrial and chloroplast genomes have been published in the last decade (Palmer et al., 1985; Palmer, 1987, 1990, 1991; Gray, 1992, 1993, 1995;Gillham, 1994;Rochaix, 1995; Wolstenholme and Fauron, 1995; Gray and Spencer, 1996). Any attempt to assess features of organelle genome evolution in a given group requires a good understanding of the phylogeny of that group. On the other hand, a better understanding of phylogeny grows from knowledge about organellar genome evolution. The second section of this chapter, therefore, will present a phylogenetic framework of green algae focusing, however, only on the information necessary for understanding the phylogenetic position of Chlamydomonas within the group. The third section will address the issue of the mono- versus polyphyletic origin of mitochondria and plastids with reference to the Chlamydomonas case. In the remaining sections of this chapter we will attempt to (i) define evolutionary trends in the two organellar genomes of Chlamydomonas; (ii) compare the mitochondrial genome mode and tempo of evolution to that of the chloroplast counterpart within the Chlamydomonas lineage and among green algae; and (iii) contrast the rates and patterns of evolutionary change in the organellar genomes of Chlamydomonas and land plants. II. Phylogenetic Position of Chlamydomonas The green algal genus Chlamydomonas Ehrenberg consists of over 450 species (Ettl, 1976) with considerable morphological, physiological and reproductive interspecific variability (Schlösser,

Chapter 5 Mitochondrial and Chloroplast Genome Evolution 1984). Systematic studies of the genus defined nine distinct morphological groups that differ primarily in chloroplast morphology (Ettl, 1976), as well as 15 distinct sporangial autolysin groups (Schlösser, 1984). The flagellate genus Chlamydomonas belongs to the order Chlamydomonadales of the class Chloro­ phyceae sensu Mattox and Stewart (1984). Mattox and Stewart’s classification system divides the green algae (Chlorophyta) into five classes: Chlorophyceae, Pleurastrophyceae, Ulvophyceae, Charophyceae and Micromonadophyceae. Cladistic analyses using organismal data are consistent with the evolutionary hypotheses underlying Mattox and Stewart’s classification system, and indicate that (i) the Chloro­ phyceae is a sister group to the Pleurastrophyceae; (ii) the Ulvophyceae is a sister group to the Chloro/Pleurastrophyceae clade; and (iii) the charophycean group, the Ulvo-/Chloro-/Pleurastrophyceae clade, and the micromonadophycean taxa all emerge from an unresolved node (Kantz et al., 1990). The phylogeny of green algae is being progressively deciphered and the new information gathered through molecular approaches will probably trigger the reconsideration of traditional green algal systematics (Chapman and Buchheim, 1991; Friedl, 1995). Phylogenetic analyses based on nuclear ribosomal RNA gene sequence (rDNA) data confirm the presence of five main evolutionary lineages among green algae, but they increasingly reveal more inconsistencies between the phylogenetic position and the polyphyly of many generic- and ordinal-level lineages on the one hand, and the traditional taxonomy on the other (Buchheim and Chapman, 1992; Friedl, 1995). Similar analyses indicate that the class Chlorophyceae itself consists of two distinct evolutionary lineages (Steinkötter et al., 1994; Friedl, 1995) that are consistent with the two flagellar apparatus configurations described among flagellate chlorophycean taxa, the directly opposed (DO) and clockwise (CW) types. The same phylogenetic analyses, however, suggest that while some members of the non-flagellate (autosporic) taxa (such as Scenedesmus obliquus) traditionally included in the chlorophycean order Chlorococcales, affiliate with DO chlorophycean taxa, other members (e.g., Prototheca wickerhamii) form amonophyletic group with advanced lineages of the class Pleurastrophyceae (sensu Mattox and Stewart, 1984); this latter group has been recently defined by Friedl (1995) as a new class, the Trebouxiophyceae (Fig. 1a). In addition, the primitive green flagellates grouped by Mattox

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and Stewart (1984) and Moestrup and Throndsen (1988) into the class Micromonadophyceae and Prasinophyceae, respectively, do not form a monophyletic assemblage of taxa (Steinkötter et al., 1994). Furthermore, phylogenetic analyses using nuclear and chloroplast rDNA sequences clearly show that the flagellate genus Chlamydomonas is not a natural assemblage of taxa: multiple lineages exist within the group, some of them containing both Chlamydomonas and non-Chlamydomonas taxa from distinct families or orders (Buchheim et al., 1990, 1996). Despite the continuous reconsideration of the phylogenetic relationships among green algae, there is no question that the Chlorophyta (green algae) and Embryophyta (land plants) form a monophyletic group (see McCourt, 1995 for a review). Nevertheless, phylogenetic analyses based on mitochondrial data, both rDNA nucleotide and cytochrome oxidase subunit 1 amino acid (COXI) sequences, reveal an unexpected dichotomy among green algae with respect to their relationships with land plants: while at least in COXI phylogenetic analyses, some green algae most closely affiliate, as expected, with the land plant group, the chlamydomonadalean lineage branches inconsistently with ciliates, fungi or animal counterparts in both rDNA and COXI trees (Wolff et al., 1993; Antamarian et al., 1996; Denovan-Wright et al., 1996) (Fig. 1b).

III. Monophyletic versus Polyphyletic Origin of Mitochondria and Plastids: The Chlamydomonas Case It is well accepted now that at least two of the eukaryotic cell’s organelles, namely the mitochondria and plastids, have a eubacterial and cyanobacterial, respectively) endosymbiotic origin, although their mono-or polyphyletic ancestry is still debated (see Gray, 1992 for a review). Single endosymbiotic events accounting for the origin of mitochondria and plastids, respectively, would imply that some common ancestral characters should be present in all the extant lineages, and that distinct derived traits should be developed within and shared among related lineages. In addition, monophyletic origins for the mitochondria and plastids, respectively, would also require that phytogenies based on organellar traits be consistent with the ones based on nuclear or nucleus-encoded features; in other words

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68 all the compartments within a eukaryotic cell should resemble their corresponding counterparts in the same compared lineage. Although the phylogenetic relationships within the chlamydomonadalean group as well as between the group and other green algal lineages are not fully deciphered, Chlamydomonas taxa appear to be legitimate members of green algae. However, in an early archaebacterial-eubacterial-chloroplastmitochondrial-nuclear phylogenetic tree inferred from small subunit ribosomal RNA (SSU rRNA) gene sequence data, the Chlamydomonas reinhardtii nuclear and mitochondrial sequences suggested different phylogenetic affiliations when compared to the closest relatives of green algae, namely, the land plants (Gray et al., 1989). In the nuclear subtree, C. reinhardtii formed a clade with the plant sequences (as it did also in the chloroplast subtree) and branched off at about the same point as animals and fungi. In contrast, in the mitochondrial subtree, C. reinhardtii branched with the ciliate/fungal/animal sequences, far away from higher plants, which clustered very clade. near the root, close to the The affiliation of the nuclear SSU rRNA gene sequences of higher plants and C. reinhardtii was seen as consistent with traditional phylogenies that consider green algae as being the closest relatives of land plants (Chapman and Ragan, 1980; Chapman and Buchheim, 1991), whereas the green algal/land plant dichotomy in the mitochondrial tree was interpreted as an anomaly. This anomaly in branching topology was, however, attributed to the plant rather than C. reinhardtii mitochondrial sequences and was considered not to be a treeing artifact due to the relatively rapid rate of sequence divergence of nonplant mitochondrial rRNA sequences (Gray et al., 1989). To explain the different branching position of plants within the nuclear and mitochondrial lineages, respectively, and to account for the strong eubacterial features of their mitochondrial rRNAs, Gray et al. (1989) suggested two possibilities: either (i) the mitochondrial rRNA genes of plants have diverged relatively little from the rRNA genes of the ancient eubacterial ancestor of all mitochondria (mono­ phyletic origin) or (ii) the higher plant mitochondrial rRNA genes or the mitochondria itself have been acquired more recently than those ofother eukaryotic lineages (biphyletic origin). In addition, due to the very different way in which genes are organized and expressed in the mitochondrial genomes of C. rein­ hardtii and land plants, the authors concluded that

Aurora M. Nedelcu and Robert W. Lee there is no indication that the two shared a common mitochondrial ancestor as recently as they shared a common nuclear (or chloroplast) ancestor. Because all the mitochondria investigated seemed to affiliate with only one subgroup of the biphyletic or polyphyletic concept as used by Gray et al. (1989) did not, however, imply more than one primary original endosymbiosis but rather a more recent secondary endosymbiotic event for the land plant mitochondria or its rRNA genes. In contrast to these results, Van de Peer et al. (1990) presented a phylogenetic tree based on SSU rRNA gene (rrnS) sequences of eukaryotic, archaebacterial, eubacterial, chloroplast, and mitochondrial origin and argued that mitochondria appeared polyphyletic: one cluster contained all the animal mitochondria; a second cluster, was formed by the C. reinhardtii, fungal and ciliate mitochondria; and the third cluster was comprised of the land plant mitochondria and was embedded in the eubacterial as the cluster with the Proteobacteria closest relative. The input of other green algal mitochondrial rDNA sequences, namely, of Prototheca wickerhamii (Wolff and Kück, 1990; Wolff et al., 1993) and Chlamy­ domonas eugametos (Denovan-Wright et al., 1996) did not resolve Chlamydomonas and P. wickerhamii sequences as a green algal clade sharing a most recent common ancestor with the land plants to the exclusion of other groups (Fig. 1b). Phylogenetic trees based on COXI amino acid sequence suggested, however, that the plant and green algal mitochondrial lineage including P. wickerhamii (Wolff et al., 1993) and the prasinophycean (sensu Moestrup and Throndsen 1988) Platymonas (Tetraselmis) subcordi­ formis (Kessler and Zetsche, 1995) do form a monophyletic group. The expected congruency of nuclear, plastid and mitochondrial phylogenetic trees appears thus verified in the case of trebouxiophycean (sensu Friedl, 1995) and prasinophycean (sensu Moestrup and Throndsen, 1988) green algae and land plants, whereas the chlamydomonadalean taxa branch with land plants in nuclear and chloroplast trees and with unrelated taxa (e.g., fungi or ciliates) in mitochondrial trees. To explain such findings, a polyphyletic origin for the green algal mitochondria was suggested (Wolff and Kück, 1993). Gray and Spencer (1996) also considered that there is little or no evidence that land plants and Chlamydomonas shared a common mitochondrial ancestor as recently as they shared a common chloroplast or nuclear ancestor; however, they proposed that the differences

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between the Chlamydomonas and Prototheca/land plant mitochondrial genome types are ‘best explained by a relatively rapid and extreme evolution’ of the former genome from the ancestral pattern represented by the more conservative genomes in the latter group. Similarly, current evidence seems to favor the view of a primary monophyletic cyanobacterial origin of plastids followed by an early subsequent diversification of the accessory pigments (Gray, 1993). Nevertheless, it appears that the rhodophyte, cryptophyte and chromophyte plastids are more closely related to each other than to their chlorophyte and land plant counterparts (Douglas, 1994). Phylogenetic trees constructed from the small and large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS and rbcL) amino acid sequences indicated that the plastids of non-green plants are most closely related to proteobacteria whereas the green algal and land plant counterparts are most closely related to cyanobacteria (Morden et al., 1992; Delwiche et al, 1995); potential explan­ ations for this apparent dichotomy are discussed by Gray and Spencer (1996). Phylogenetic analyses using either SSU rDNA sequences or translation elongation factors and other protein amino acid sequences are contradictory and do not provide confident support for either a monophyletic or polyphyletic origin of plastids (reviewed by Douglas, 1994 and Gray and Spencer, 1996). Although the chlorophyte/embryophyte connection is relatively well supported in all chloroplast phylogenetic analyses, it is noteworthy that in a recent phylogenetic analysis, Chlamydomonas SSU rDNA nucleotide sequences did not branch as expected with other green algae, but rather suggested a very early divergence of this lineage relative to all other chloroplast counterparts examined (Gray and Spencer, 1996) (Fig. 1c).

IV. Evolution of Mitochondrial and Chloroplast Genome Size in Chlamydomonas The size of mitochondrial and chloroplast genomes appears quite different, both in the same lineage, as well as among lineages. Land plant mitochondrial genomes are large and extremely variable in size, ranging from 186 kb to 2500 kb (reviewed by Palmer, 1990). In contrast, land plant chloroplast genomes are smaller and seem to be rather conservative in size, varying from 120 kb to 160 kb, in only few

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cases reaching 220 kb (reviewed by Palmer, 1991). On the other hand, animal mitochondrial genomes are very small and extremely conserved in size, varying generally from 15.7 kb to 21 kb (see Wolstenholme and Fauron, 1995 for a review), with the exception of scallop mitochondrial genomes that vary from 16.2 kb to 41 kb (see Gjetvaj et al., 1992 for references). Surprisingly, the chlamydomonadalean mito­ chondrial genomes are 10–100-fold smaller than their land plant homologs, thus approximating the size of their metazoan counterparts, whereas the chlamydomonadalean chloroplast genomes are slightly larger than most of their angiosperm homologs. Our analysis of the known mitochondrial and chloroplast genome sizes within the chlamy­ domonadalean group suggests a 1.5-fold variation in both organelles, from a 15.8-kb mitochondrial DNA (mtDNA) in C. reinhardtii (Michaelis et al., 1990) to a 22.9-kb counterpart in C. eugametos (DenovanWright et al., in press), and from a 187-kb chloroplast DNA (cpDNA) in Chlamydomonas pitschmannii (Boudreau and Turmel., 1995) to a 292-kb homolog in Chlamydomonas moewusii (Boudreau et al., 1994). It is noteworthy that the same level ofchloroplast and mitochondrial genome size variation is also observed in each of the two very divergent evolutionary lineages within the Chlamydomonas group, i.e., the ‘C. rein­ hardtii’ and the ‘C. eugametos’ lineages. Exceptional variation in genome size has, however, been described among the sexually incompatible members of the colonial chlamydomonadalean taxon, Pandorina morum, whose mitochondrial and chloroplast genome sizes vary from 20 kb to 38 kb, and from 150 kb to 450 kb, respectively (Moore and Coleman, 1989; Moore, 1990). It appears, therefore, that mito­ chondrial genome size is more conservative among chlamydomonadalean taxa than among land plants (1.5-fold in the former, relative to 12.5-fold in the latter), whereas chloroplast genomes vary slightly more (1.5-fold) than do their land plant counterparts (1.3-fold). It is noteworthy that in contrast to land plants, in Chlamydomonas both organellar genomes exhibit the same level of size variation. Furthermore, it appears that the two organelle genomes followed parallel evolutionary pathways not only within the group but also within a given species: the mito­ chondrial and chloroplast genomes are both either small (e.g., in C. reinhardtii and C. pitschmannii) or large (e.g., in C. eugametos and C. moewusii) relative to the currently known size range in the Chlamy­ domonas group.

70 Among green algae, however, there is a five-fold variation in mitochondrial genome size (excluding the 220-kb mitochondrial genome of Bryopsis), from 15.8 kb in C. reinhardtii (Michaelis et al., 1990) to 80 kb in Chlorella pyrenoidosa (Bayen and Rode, 1973). Similarly, there is at least a five-fold variation in chloroplast genome size among the green algal lineages investigated so far, from 89 kb in Codium fragile to 400 kb in a few members of three out of the five green algal classes, namely, the Charophyceae, Ulvophyceae, and Chlorophyceae (see Palmer, 1991 for references). Although the degree of variation in organelle genome size is overall higher among green algae compared to Chlamydomonas, it is interesting that both genome types seem to exhibit the same level of variation in size, a situation similar to that observed in Chlamydomonas but in contrast to that noted among land plants. Because the mitochondrial genome in Platymonas subcordiformis (Kessler and Zetsche, 1995), a green flagellate that retains ancestral-like features, is larger (i.e., 42.8 kb) than in chlamydomonadalean taxa, it is most likely that the reduced genome size in the latter represents a derived condition among green algae. Moreover, one can hypothesize an evolutionary trend towards a smaller mitochondrial genome within the chlorqphycean group, from a 45-kb mitochondrial genome as in, for example, Scenedesmus obliquus (Kück, 1989), to a 15.8 kb homolog in C. reinhardtii. Although limited, the current data do not support a similar trend among other green algal lineages: at 42.8 kb, the mitochondrial genome of the primitivelike green flagellate Platymonas subcordiformis is smaller than the 55.3-kb homolog in Prototheca wickerhamii (Wolff et al., 1994) and the 80-kb counterpart in Chlorella pyrenoidosa. There is no indication of a tendency towards a smaller chloroplast genome among chlorophycean taxa as suggested for the mitochondrial counterparts; however, chloroplast genome sizes much smaller than the average have been reported among ulvophycean (89 kb in Codium fragile, Manhart et al., 1989) and charophycean (130 kb in Spirogyra maxima, Manhart et al., 1990) taxa.

A. Factors Contributing to Variation in Genome Size Generally, changes in genome size are the result of changes in sequence complexity and/or changes in the amount of repeated DNA (Palmer, 1990). Changes in genome complexity occur through the deletion

Aurora M. Nedelcu and Robert W. Lee and insertion of unique sequences (intergenic regions, introns and open reading frames). The great variation in mtDNA size among land plants is mostly accounted for by changes in the complexity of spacer DNA. In contrast, cpDNAs in the same group vary relatively little in size, with less contribution from changes in intergenic region size, intron number or gene content, but more (i.e., nearly two-thirds of cpDNA size variation) from the expansion/contraction of the inverted repeat (see Palmer 1991, for a review).

1. Changes in Intergenic Spacer Size The intergenic spacers are very reduced in Chlamy­ domonas mitochondrial genomes (i.e., 16–17% of the genome). In C. eugametos, with the exception of two large intergenic regions of 902 bp and 1057 bp, respectively, the intergenic spacers range from 0 to 466 bp, with most of them being smaller than 72 bp. In the more compact mitochondrial genome of C. reinhardtii, with the exception of the terminal noncoding regions of about 530 bp each, most of the intergenic regions are smaller than 200 bp and missing whenever two rRNA gene pieces are adjacent. On the other hand, the intergenic regions in the mitochondrial genome of P. wickerhamii represent 29% of the genome, and in the majority of cases are 100-150 bp long with only two considerably longer regions (1118 bp and 1993 bp) (Wolff et al., 1994). Only about 12% of the 7-kb difference in size between the two completely sequenced Chlamydomonas mitochondrial genomes can be accounted for by differences in intergenic region size, whereas the 30­ kb difference in mitochondrial genome size between the mitochondrial genomes of Chlamydomonas and Prototheca is a consequence of variation in both the intergenic region size as well as gene content (discussed later). In contrast, the substantial difference in chloroplast genome size between closely related Chlamy­ domonas taxa (i.e., C. moewusii and C.pitschmannii as well as C. reinhardtii and C. gelatinosa) is mainly a consequence of multiple deletions/additions in the intergenic spacers (Boudreau and Turmel, 1995, 1996). Similarly, most of the observed differences in cpDNA restriction patterns between the interfertile C. reinhardtii and C. smithii are correlated with insertions/deletions of short dispersed repeated sequences of 50 bp to 200 bp, which are ubiquitous in the intergenic regions of C. reinhardtii cpDNA (Rochaix, 1978; Gelvin and Howell, 1979; Palmer et

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al., 1985). On the other hand, 12% of the 50-kb difference in size between the interfertile taxa C. moewusii and C. eugametos is accounted for by a 6-kb insertion in one of the single copy-regions of the C. moewusii chloroplast genome (Lemieux et al., 1985); the rest is the consequence of a 21 -kb insertion in its large inverted repeat (Turmel et al., 1987). Another type of insertion is represented by the two copies of a 2.4 kb DNA sequence, the Wendy element, that has many of the features of transposable elements (discussed later) and has only been described in the chloroplast genome of C. reinhardtii (Fan et al., 1995). The absence of a counterpart in any of the other chlamydomonadalean or land plant chloroplast genomes examined to date suggests that Wendy is a relatively recent acquisition in the C. reinhardtii lineage (Fan et al., 1995). The difference in chloroplast genome size between Chlamydomonas and land plants is correlated with the presence of enlarged intergenic spacers in the former relative to the latter (Boudreau et al., 1994).

2. Changes in Intron Number There is quite a variation among chlamydo­ monadalean taxa in terms of the number of introns present in their organellar genomes. The difference in size between the mitochondrial genomes, otherwise co-linear (Boynton et al., 1987), of the two interfertile taxa, C. reinhardtii and C. smithii, is solely the result of a unique 1-kb intron inserted in the cob sequence of C. smithii (Matagne et al., 1988; Colleaux et al., 1990). In addition, it appears that the difference in mitochondrial genome size between the members of the other pair of interfertile Chlamydomonas taxa, that is C. eugametos and C. moewusii, might also be correlated with the presence of optional introns (Denovan-Wright and Lee, 1993). Moreover, 88% of the difference in size between the C. reinhardtii and C. eugametos mitochondrial genomes can be accounted for by the presence of nine intervening sequences in the coding regions of the latter (Fig. 2). There seems to be a variation also in the number of introns harbored by homologous genes among chlamydomonadalean taxa: two introns are present in the cob sequence of a chlamydomonadalean taxa more closely related to C. eugametos than to C. reinhardtii (Buchheim et al., 1996), namely Chlorogonium elongatum (Kroymann and Zetsche, 1997), whereas only one intron is found in C. eugametos (Denovan-Wright et al., 1998) and C.

71

smithii (Colleaux et al., 1990) cob, but none in the homologous C. reinhardtii gene (Michaelis et al., 1990). More data have to become available before one can decide whether the Chlamydomonas ancestor had or did not have introns in its mitochondrial genome, but variation in intron content is clearly a significant factor responsible for the observed variation in mitochondrial genome size among Chlamydomonas taxa. Similarly, there is quite a variation in intron content among Chlamydomonas chloroplast genomes (Turmel et al., 1991). It is noteworthy that the chloroplast large subunit (LSU) rRNA genes from 17 Chlamydomonas taxa investi­ gated contain a total of 39 group I introns representing 12 insertion sites (Turmel et al., 1993). While C. pitschmannii and C. reinhardtii have no and only one intron, respectively, in their chloroplast LSU rRNA gene, C. moewusii and C. eugametos have five and six, respectively. On the other hand, four introns are present in the C. reinhardtii psbA gene, which is intronless in C. eugametos (Erickson et al., 1984; Lemieux et al., 1985), and one of the four introns present in C. reinhardtii is missing in the interfertile C. smithii (Palmer et al., 1985); additional optional introns are also present in rrnS, psaB and psbC (see Turmel et al., 1993 for references). Another case of optional insertions contributing to variation in size of the open reading frames in Chlamydomonas chloroplast genes is represented by the presence of (i) one or two large insertion sequences that are not spliced out at the mRNA level in the genes coding for the catalytic subunit of the ATPdependent Clp protease (clpP) of C. reinhardtii and C. eugametos, respectively (Huang et al., 1994), and (ii) in-frame long sequences of unknown identity juxtaposed within the gene coding for the RNA polymerase subunit C (rpoC2) of C. reinhardtii cpDNA (Fong and Surzycki, 1992).

3. Changes in Gene Content One of the most distinctive features of the mitochondrial genome in Chlamydomonas is its very reduced gene content relative to other green algal and land plant counterparts. The two Chlamydomonas mitochondrial genomes completely sequenced to date, i.e., those of C. reinhardtii (Michaelis et al., 1990, and references therein) and C. eugametos (Denovan-Wright et al., 1998) have the same set of standard genes: seven respiratory protein-, three transfer RNA (tRNA)- as well as fragmented and

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Aurora M. Nedelcu and Robert W. Lee scrambled SSU and LSU rRNA-coding regions (Fig. 2). However, a reverse transcriptase-like gene (rtl) (Boer and Gray, 1988b) and an additional tRNA coding region (Denovan-Wright et al., 1998) have been identified in C. reinhardtii and C. eugametos, respectively. Nevertheless, different codon usage and deduced amino acid composition of the reverse transcriptase-like coding region led Boer and Gray (1988b) to suggest that rtl in C. reinhardtii had an independent more recent origin relative to the standard mitochondrial genes. Although in Oenothera berteriana mtDNA an independent open reading frame showing reverse transcriptase-like similarity has also been described (Schuster and Brennicke, 1987), it is noteworthy that the rtl in C. reinhardtii mtDNA is flanked by intergenic regions that contain sequence motifs present at the splice sites of group II introns (Nedelcu and Lee, unpublished). The additional coding region present in C. eugametos mtDNA flanks one of the two copies of a large direct repeat (Fig. 2) and may be the result of a duplication/inversion-related event given that the other end of the large direct repeat is also flanked gene. Duplicated tRNA genes or tRNA by a pseudogenes have been previously reported at inversion ends in wheat and rice chloroplast genomes (Howe et al., 1988; Shimada and Sugiura, 1989). The presence of virtually the same set of coding regions in the mitochondrial genomes of two Chlamydomonas taxa that belong to lineages considered to have diverged very early in the evolution of the Chlamydomonas group allows us to speculate that (i) the feature ofa very reduced gene content was already present in the most recent chlamydo­ monadalean common ancestor and (ii) gene content is not a significant contributor to the mitochondrial genome size variation within the Chlamydomonas group. No Chlamydomonas chloroplast genome is fully described, but half of the C. moewusii and C. eugametos chloroplast genomes have been sequenced and the analysis of these genomes revealed half of the land plant cpDNA gene content (Boudreau

Chapter 5

Mitochondrial and Chloroplast Genome Evolution

et al., 1994). Moreover, the two very divergent Chlamydomonas taxa, C. reinhardtii and C. moewusii, appear to share a similar gene complement (Fig. 3) (Boudreau et al., 1994). Among the 75 genes mapped on the two Chlamydomonas chloroplast genomes, five coding regions have not been reported in any land plant cpDNA, and four of these have also not been identified in other green algal counterparts (Boudreau et al., 1994). On the other hand, the genes encoding the chlororespiratory NADH dehy­ drogenase subunits present in land plant cpDNA may be lacking in the chloroplast genome of Chlamydomonas. The information accumulated so far led Boudreau et al. (1994) to suggest that the presence of additional genes accounts only for a very small fraction of the increased size of the chlamy­ domonadalean chloroplast genome relative to the land plant homologs. It is noteworthy that some chloroplast genes in C. reinhardtii display unusual structures and the corresponding transcripts are undetectable, suggesting that they might not be functional (Fong and Surzycki, 1992). Although there seems to be some variation in gene content among green algal chloroplast genomes (e.g., several additional genes involved in nucleotide metabolism reported in the cpDNA of the ulvo­ phycean taxon Acetabularia mediterranea and a more reduced gene content in that of Codium fragile, see Palmer, 1991 for discussion), the most striking differences in gene content have been observed among mitochondrial genomes. Unexpectedly, the mito­ chondrial genomes of green algal taxa outside the chlamydomonadalean group do not seem to share the very reduced gene content of their Chlamydo­ monas counterparts. The Platymonas subcordiformis mtDNA encodes at least 12 respiratory proteins, seven tRNAs, two ribosomal proteins as well as continuous LSU and SSU rRNAs (Kessler and Zetsche, 1995). Moreover, the mitochondrial genome of Prototheca wickerhamii codes for 16 respiratory proteins (including three subunits of the ATPase complex which is entirely non-mitochondriallyencoded in Chlamydomonas), 26 tRNAs, 13 ribosomal proteins as well as 5S, LSU and SSU rRNAs (Wolff et al., 1994). Given that the mitochondrial genome of the ancestral-like green algal lineage Platymonas as well as of the advanced trebouxiophycean (sensu Friedl 1995) lineage, Prototheca, has a gene content more similar to the land plant counterparts, one can hypothesize that the most recent common ancestor

73

of green algae and land plants contained quite a large number of genes in its mtDNA and that a reduction in gene content occurred in the chlorophycean lineage leading to Chlamydomonas. However, given that Chlamydomonas and Prototheca may not be as closely related as previously thought (Friedl, 1995) (i.e., they are in fact members of the chlorophycean and trebouxiophycean lineages [sensu Friedl, 1995], respectively, whose divergence is probably very old) the differences in gene content between Chlamy­ domonas and Prototheca are less surprising. The dichotomy in mitochondrial gene content within the green algal group is also reflected in different degrees of resemblance to their counterparts in land plants, the group considered their closest relatives at the nucleo-cytosolic level (Chapman and Ragan, 1980; Chapman and Buchheim, 1991). While the mitochondrial genomes of C. reinhardtii and C. eugametos (and most likely of all the chlorophycean taxa) resemble more closely their ciliate/fungal/ animal counterparts in terms of gene content, those of Platymonas subcordiformis and Prototheca wickerhamii (and most likely of all trebouxiophycean lineages) have a gene content similar to their land plant counterparts.

4. Changes in the Amount of Repeated DNA Large repeated sequences have been found in mitochondrial genomes of Chlamydomonas. The terminal non-coding regions of the C. reinhardtii linear mtDNA each contain a copy of an inverted repeat (TIR) of about 530 bp (Fig. 2) (Vahrenholz et al., 1993). On the other hand, the two largest intergenic regions in the C. eugametos circular-mapping mitochondrial genome each contain a copy of a 260­ bp direct repeat (DR) (Fig. 2) (Denovan-Wright et al., 1998). TIRs were described also in the linear mitochondrial genome of the colonial chlamy­ domonadalean taxon Pandorina morum (Moore and Coleman, 1989). Given that among sexually incompatible members of P. morum the TIRs range in size from 1.8 to 3.3 kb, it seems, at least in this taxon, that the variation in the amount of repeated DNA could be responsible in part for the observed intraspecific variation in mitochondrial genome size (from 20 kb to 38 kb). However, complete mitochondrial genome sequence data for more chlamydomonadalean taxa are needed in order to make any suggestion as to how significant, if at all, is the amount of repeated DNA in genome size evolution

74

Aurora M. Nedelcu and Robert W. Lee

Chapter 5

Mitochondrial and Chloroplast Genome Evolution

within the chlamydomonadalean group. Among the large repeated chloroplast DNA sequences in Chlamydomonas, the most common is a two-copy inverted repeat (IR) found always as two identical but oppositely oriented copies that divide the chloroplast genome into two rather equal singlecopy regions (Fig. 4a) (Rochaix, 1978; Lemieux et al., 1985; Turmel et al, 1987; Boynton et al., 1992). As in land plants, the Chlamydomonas IR varies in size by spreading or shrinkage (Turmel et al., 1991). The 50-kb difference in chloroplast genome size between the very closely related C. moewusii and C. eugametos is mostly accounted for by an enlarged inverted repeat in C. moewusii, which was shown to be the consequence of a 21 -kb insertion (Turmel et al., 1987). The expansion/contraction of the inverted repeat also accounts for the gain/loss of a 7-kb sequence containing the atpB gene between the two closely related C. reinhardtii and C. gelatinosa (Boudreau and Turmel, 1996). On the other hand, although there is a 53-kb difference in size between the chloroplast genomes of the distantly related C. eugametos and C. reinhardtii, their inverted repeats are about the same size in both taxa. Nevertheless, similar size does not necessarily imply identical sequence complexity, given that the inverted repeat in C. reinhardtii lacks the rbcL gene, which is located instead in a single-copy region (Malnoë et al., 1979; Dron et al., 1982). In contrast, although the inverted repeats of the interfertile C. reinhardtii and C. smithii are identical in gene content and similar in overall gene organization, they differ at the fine structure level by an extensive series of small deletions/ additions: a minimum of 11 length mutations (20– 1600 bp) are distributed throughout the IR of C. smithii, which makes it almost 1 kb larger that the 22-kb repeat of C. reinhardtii (Palmer et al., 1985). Comparisons between the inverted repeats of the members of each of the interfertile Chlamydomonas pairs, C. reinhardtii/C. smithii and C. eugametos/ C. moewusii, have revealed more deletion/addition differences than nucleotide substitutions (Palmer et al., 1985; Lemieux et al., 1985), which is opposite to the situation observed in higher plants (Zurawski et al., 1984). Among green algae there is even more variation than seen in Chlamydomonas in terms of the amount of repeated DNA in the organellar genomes. While there are no large repeats in the circular-mapping mitochondrial genome of Prototheca wickerhamii, a two-copy inverted repeat of ca. 1.5 kb was reported in the Platymonas subcordiformis homolog (Kessler

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and Zetsche, 1995). Similarly, among green algae, the chloroplast IR ranges in size between 20 and 41 kb and seems to be lacking in a few charophycean and ulvophycean lineages whose cpDNAs are also known to be smaller than the chloroplast genome size average (see Palmer, 1991, for references).

B. Mechanisms Possibly Involved in the Evolution of Genome Size 1. Length Mutations Changes in organellar genome sequence complexity occur primarily by length mutations, i.e., the addition of new sequences or the deletion of existing ones. Many of the small-length mutations in organelle genomes were found to be flanked by or close to short direct repeats, suggesting their occurrence during DNA replication or repair according to the ‘slippage-mispairing’ model (Takaiwa and Sugiura, 1982; Zurawski et al., 1984). The great majority of length mutations are small, only 1–10 bp in size, and occur predominantly in noncoding DNA (intergenic spacers and introns). Length mutations of 10–1,200 bp in size occur less frequently than smaller ones and are more likely to occur by recombination than by replication; unequal crossing-over between mis­ aligned tandem repeats could produce both deletions and additions, and intramolecular recombination between short direct repeats could produce deletions (see Palmer, 1991 for a review). Repetitious DNA is very abundant in the mitochondrial genome of Chlamydomonas. Short dispersed repeats have been reported in five of the intergenic spacers ofthe C. reinhardtii mitochondrial genome (Boer and Gray, 1991). A more abundant and complex set of repetitive sequences (in both direct and inverted orientation) has been found in the mitochondrial genome of C. eugametos; more than 80 repetitive elements, ranging in size from 6 bp to 17 bp, are dispersed throughout the intergenic regions as well as within several introns (Denovan-Wright et al., 1998; Nedelcu and Lee, 1998). Identical copies or closely related sequences of the same repeat are present tandemly repeated in various combinations. The C. reinhardtii chloroplast genome also possesses a family of about 40 short (100–300 bp) repeated sequences scattered throughout most of the genome (Rochaix, 1978; Gelvin andHowell, 1979), at least 13 of them being localized within the IR (Palmer et al., 1985). The short dispersed repeats account for ca. 22% of the total C. reinhardtii

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chloroplast genome and each is composed of shorter repeated motifs that occur in direct or inverted orientation and in various combinations (Gillham, 1994). Given that a number of the observed smalllength mutations mapped close to regions showing differences in the number of these small repeats, Palmer et al. (1985) suggested that enhanced recombination within and between repeat elements may be related to the increased incidence of length

Aurora M. Nedelcu and Robert W. Lee

mutations in Chlamydomonas relative to angiosperm cpDNA. Studies of experimentally induced mutants showed that the endpoints of most of the structural mutations, both deletions and inversions, mapped in the general vicinity of the 100–3 00-bp repeat elements scattered throughout the inverted repeat (Palmer et al., 1985). The high frequency of symmetrical alterations suggested the existence of a copycorrection mechanism for maintaining identity

Chapter 5 Mitochondrial and Chloroplast Genome Evolution between the two copies of the inverted repeat. The mechanism for insertion of new sequences within the intergenic regions of Chlamydomonas cpDNA is not known. Because no dispersed repeats have been detected by Southern blot hybridization in the cpDNAs of C. pitschmannii and C. eugametos, Boudreau and Turmel (1995) did not favor the proliferation of existing sequences throughout the genome through unequal recombination as a mechanism responsible for the 56-kb difference in size between the intergenic regions in the cpDNAs of these two taxa. It is noteworthy that in land plant cpDNA, repeats were thought to have been created and spread by duplicative transposition (Tsai and Strauss, 1989). No classical transposable element has been, however, isolated from any land plant chloroplast. In contrast, the repeated sequences and the ORFs (whose deduced amino acid sequence show some similarity with transposases and integrases of other mobile elements) associated with the Wendy element in the C. reinhardtii chloroplast genome argue for the existence, present or past, of a transposable element in this lineage. Short repetitive sequences are also present in the organellar genomes of other green algae. The mitochondrial genome of P. wickerhamii is rich in very complex repetitive motifs consisting of AT-rich tandem repeats in both intergenic spacers and introns; although it has been suggested that they might be implicated in transcription and/or processing, their evolutionary origin is not known (Wolff et al., 1994). In the Chlorella ellipsoidea chloroplast genome, insertions of repeated sequences as well as ORFs with terminal repeated sequences account for larger intergenic spacers in its IR relative to the C. reinhardtii counterpart (Yamada, 1991). Because the smallest chloroplast IRs known to date contain at least the rRNA (rrn) operon, Yamada (1991) suggested that the IR might have been originally created from the duplication (in an inverted orientation) of the rrn operon, followed by its expansion to incorporate additional coding regions. The mechanism proposed for the expansion of the IR involves a double reciprocal recombination during the replication step and requires the presence of repetitive sequences acting as recombination hot spots within and around the IR (Yamada, 1991). Such a model is consistent with the location of the repetitive sequences in the inverted repeat of the Chlorella ellipsoidea chloroplast genome.

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2. Intron Mobility Although gain or loss of introns could be considered a special case of length mutation (Palmer, 1991), the mechanisms underlying these processes are very different. It was proposed that the loss of introns is the result of two processes: (i) the reversetranscription of an RNA whose intron sequences have been removed by splicing and (ii) homologous recombination between the intronless cDNA and the native gene (Dujon, 1989). In mitochondria, the putative reverse transcriptases (RTs) encoded either by the group II intronic ORFs or certain non-intronic ORFs with reverse transcriptase similarity, as found in C. reinhardtii (Boer and Gray 1988b) and a few angiosperms (see Moenne et al., 1996 for references), could produce an intronless copy from a spliced RNA. Although the above mentioned processes seem more likely to account for the loss of group II introns, Wolff et al. (1993) considered that the reversetranscriptases encoded in group II introns could have also been responsible for the loss of group I introns, culminating in their complete elimination from higher land plant mtDNA. It is noteworthy that although the overexpressed gene product of C. reinhardtii rtl does not appear to have a reverse transcriptase activity (Faßbender et al., 1994), such an activity has been recently detected in the mitochondria of potato (Moenne et al., 1996). On the other hand, in the Chlamydomonas chloroplast, evidence for reverse transcriptase activity is still lacking, but recom­ bination processes seem to be well developed (Dürrenberger et al., 1996). To explain the transfer of group I introns to novel locations (intron transposition), two mechanisms have been proposed: one occurs at the DNA level and is promoted by an intron-encoded endonuclease (Dujon, 1989), whereas the other starts at the RNA level and involves (i) the introduction of an intron RNA sequence into a foreign RNA by a reversal of a selfsplicing reaction, (ii) the reverse-transcription of the recombined RNA and (iii) the integration of the cDNA sequence into the genomic DNA by homolo­ gous recombination (Woodson and Cech, 1989). Turmel et al. (1993) presented evidence that reverse self-splicing might have played the major role in the creation of novel intron insertion sites in the LSU rRNA genes (rrnL) as well as elsewhere in the chloroplast genome of Chlamydomonas. However, these authors also suggested that certain group I

78 introns might have been introduced via lateral transfer facilitated by the site-specific endonuclease encoded in these introns. Such a mechanism is considered to be responsible for the mobility (intron homing) of the chloroplast rrnL intron of C. eugametos (Lemieux and Lee, 1987; Gauthier et al., 1991; Bussières et al., 1996) and C. reinhardtii (Dürrenberger and Rochaix, 1991) as well as of the mitochondrial cob intron of C. smithii (Boynton et al., 1987; Matagne et al., 1988; Colleaux et al., 1990; Ma et al., 1992) to cognate positions within the corresponding intronless genes. Another case indicative of intron mobility is the apparent evolutionary transfer of a group I intron between the mitochondrial rrnL of Acanthamoeba castellanii and the chloroplast counterpart of Chlamydomonas, either intracellularly in a remote photosynthetic common ancestor of the two lineages, or intercellularly, as a result ofa recent lateral transfer event (Lonergan and Gray, 1994; Turmel et al., 1995b).

3. Gene Transfer Among the processes of genetic flux (such as gene transfer, reverse gene transfer, gene substitution, gene sharing, gene recruitment, and gene loss—see Palmer, 1991 for a review) that account for the differences in gene content among lineages, gene transfer seems to be the main contributor to changes in genome complexity in organellar genomes. However, Palmer (1991) considers that the incor­ poration of a foreign gene into an organelle genome (reverse gene transfer) is not very rare and includes events such as the invasion of chloroplast genes into the higher plant mtDNA as well as the recent acquisition of a number of ORFs within land plant and green algal chloroplast introns (see Palmer, 1991 for references). To explain the gene transfer from one cellular compartment to another, Obar and Green (1985) proposed a stepwise model involving: (i) duplication of an organellar gene followed by the transfer of one copy to the nucleus; (ii) activation of the nuclear copy still keeping active the organellar counterpart; and (iii) inactivation and subsequent loss of the organellar gene copy. Gene transfer from the organelle to the nuclear genome seems to have been more important in the evolution of the mitochondrial than the chloroplast genome in Chlamydomonas. The presence of a higher number of genes in the mitochondrial genome of Platymonas subcordi-

Aurora M. Nedelcu and Robert W. Lee formis, considered a descendant of the primitive green flagellates from which all the advanced green algal lineages have evolved, suggests that the feature of a very reduced gene content in Chlamydomonas is a derived trait among green algae. The mechanisms and causes responsible for such a massive reduction in the gene content of mtDNA in Chlamydomonas are not known, although a few suggestions have been made. Recombination between short direct repealed sequences was proposed to have been responsible for the ‘excision’ of mitochondrial coding regions during the evolution of the Chlamydomonas-like genomes (Nedelcu, 1997; Nedelcu and Lee, 1998). Short direct repeated sequences and short particularly recombinogenic GC-rich clusters within AT-rich spacers regions have been shown to be involved in site-specific intra­ molecular recombination events resulting in the excision of small subgenomic circles in plant and fungal mitochondrial genomes, respectively (Fig. 4b) (Zinn et al., 1988; Hartmann et al., 1994 and references therein; Benslimane et al., 1996; JamietVierny et al., 1997 and references therein). It is possible that the accumulation of GC-rich short direct repeated sequences with recombinogenic properties in the lineage leading to Chlamydomonas could have promoted recombination events responsible for the deletion of protein-coding genes as well as tRNA- or rRNA-coding regions (Fig. 4d). Nevertheless, the processes accounting for the transfer of genetic information into the nucleus remain to be deciphered. Theoretically, there are two ways one can envision such a transfer: at the DNA or RNA level. The transfer of a DNA molecule from one compartment to another could be comparable to the transfer of an episome from one eubacterial cell to another, following the excision, conjugation, and integration steps described in a transformation cycle. Alternatively, the genetic information transcribed into an RNA molecule could be reverse-transcribed into a DNA molecule either before or after leaving the organelle, and subsequently integrated into the nuclear DNA. It is noteworthy that gene transfer from the mitochondria to the nucleus seems to be an on-going process among flowering plants and at least in this group, the transfer seems to happen at the RNA level, because the nuclear copies resemble more the edited rather than unedited versions of the mitochondrial genes (Brennicke et al., 1993; Schuster and Brennicke, 1993; Gray, 1995).

Chapter 5 Mitochondrial and Chloroplast Genome Evolution V. Evolution of Mitochondrial and Chloroplast Genome Organization in Chlamydomonas

A. Mitochondrial and Chloroplast Genome Structure An unexpected dichotomy in mitochondrial genome conformation has been observed among Chlamy­ domonas taxa: linear mtDNA molecules have been isolated from C. reinhardtii (Boer et al., 1985), C. smithii (Boynton et al., 1987) and the colonial chlamydomonadalean alga Pandorina morum (Moore and Coleman, 1989) but circular-mapping mtDNAs have been reported for C. eugametos (DenovanWright and Lee, 1992), C. moewusii (Lee et al., 1991) and C. pitschmannii (Boudreau and Turmel, 1995). However, as discussed by Bendich (1993), electrophoretic migration patterns of the circularmapping mitochondrial genomes of C. moewusii and C. eugametos (Boer et al., 1985; Lee et al., 1991) leave open the possibility of their existing in vivo as linear, larger-than-unit-size genomes. Circularmapping mitochondrial genomes have also been reported in other chlorophycean taxa such as Chlorella and Scenedesmus obliquus, as well as trebouxiophycean and prasinophycean taxa like Prototheca wickerhamii and Platymonas subcordi­ formis, respectively (Kück, 1989; Moore and Coleman, 1989; Waddle et al., 1990; Kessler and Zetsche, 1995). It is note worthy that although circularmapping mitochondrial genomes have also been reported in land plants, they may exist in vivo predominantly as larger-than-unit-genome-size linear structures (Bendich, 1993, 1996). If the circular-mapping mtDNAs are circular molecules in vivo, their linearization in some chlamydomonadalean lineages could have been the consequence of a recombination event between short repeated sequences on a small linear episome and their homologs on the circular chromosome, as described during the linearization of the maize mitochondrial genome (Schardl et al., 1984). In this connection, it is intriguing that one of the long terminal inverted repeats in the linear C. reinhardtii mtDNA is flanked by small inverted repeats thus arguing for a potential previous episomal existence of this TIR (Nedelcu and Lee, 1998). In contrast, chloroplast genomes appear to be circular in conformation in all Chlamydomonas

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investigated to date; however, they exist as a 50:50 mixture of two genetically identical but physically distinct molecules that differ only in the relative orientation of their single-copy regions as a result of high-frequency intramolecular recombination events between the two copies of the IR (Fig. 4a) (Aldrich et al., 1985; Palmer et al., 1985). In addition, intermolecular recombination events involving the short repeated sequences in the C. reinhardtii cpDNA have been suggested to yield dimer and multimer cpDNA molecules (Boynton et al., 1992; Boudreau and Turmel, 1996).

B. Mitochondrial and Chloroplast Gene Order Although the Chlamydomonas mitochondrial genome size and gene content is more animal-like than plant­ like, the gene order among taxa appears to be as variable as observed among the vascular plant mitochondrial genomes. None of the coding regions is flanked by homologous counterparts in both the C. reinhardtii and C. eugametos mitochondrial genomes, i.e., there is no gene cluster common to the two genomes and the protein-coding genes are highly interspersed with tRNA genes as well as rRNA gene pieces in both Chlamydomonas genomes (Boer and Gray, 1988a; Denovan-Wright et al., 1998) (Fig. 2). Given that in C. eugametos the protein-coding genes are more interspersed with rRNA-coding regions than they are in C. reinhardtii, it seems likely that the mitochondrial genome of C. eugametos has undergone additional gene rearrangements relative to its C. reinhardtii counterpart. It is interesting that, in contrast to land plants, the chloroplast gene order is quite variable among Chlamydomonas taxa. Although the chloroplast genomes of the interfertile members of each pair of taxa, C. eugametos/C. moewusii and C. reinhardtii/ C. smithii are co-linear (Turmel et al., 1987; Boynton et al., 1992), the cpDNAs are so extensively rearranged between the two pairs (i.e., C. eugametos/ C. moewusii and C. reinhardtii/C. smithii) that rearrangements cannot be described in terms of simple individual events (Fig. 3). It has been suggested that the great evolutionary distance separating these algae might be responsible for such a high level of rearrangements although the possibility that cpDNA rearranges at a fast rate in Chlamydomonas cannot be disregarded (Lemieux and Lemieux, 1985). Interestingly, a comparative analysis of chloroplast

80 gene order in the two closely related Chlamydomonas taxa, C. moewusii and C. pitschmannii, has revealed a level of rearrangement close to that observed among all land plants: one or two inversions and possibly one or three events of expansion/contraction of the inverted repeat (Boudreau and Turmel, 1995). Unexpectedly, however, the level of rearrangements appears much more extensive between C. reinhardtii and C. gelatinosa (i.e., at least nine inversions and one expansion/contraction event of the IR), although chloroplast LSU rDNA sequence-based phylogenies suggest they are as closely related as are C. moewusii and C. pitschmannii (Boudreau and Turmel, 1996). Most changes in gene order in the two pairs of closely related Chlamydomonas are located in the single-copy region bordering the rrnS gene (Boudreau and Turmel, 1995, 1996). In the course of these rearrangements, a few cpDNA sequences have moved from one single copy region to the other, a phenomenon not observed in higher plant cpDNA. Chlamydomonas cpDNAs lack the extensive operon structure of their land plant counterparts; the best example is represented by the six atp genes, which are organized into two operons in all other chloroplasts but are scattered singly around the genome in a species-specific manner in Chlamy­ domonas (Palmer, 1991). It has been suggested (Turmel et al., 1988) that the numerous rearrange­ ments during Chlamydomonas cpDNA evolution resulted in the disruption of the polycistronic transcription units inherited from the prokaryotic ancestor; in contrast, different evolutionary pressures during land plant evolution determined an increased number of polycistronic transcription units and a more compact genome organization. Most of the ancestral operons still present in land plant cpDNAs have been lost in the Chlamydomonas lineage; of 76 genes mapped on five Chlamydomonas cpDNAs, 40 represent 15 conserved clusters, four of which are similar to the primitive operons present in land plant chloroplast genomes, and one has exactly the same gene content as the land plant equivalent (Boudreau et al., 1994; Boudreau and Turmel, 1996). The level of mitochondrial gene rearrangement among green algae is difficult to assess due to the lack of complete mitochondrial genome sequences for green algal lineages other than Chlamydomonas and Prototheca, as well as the very reduced gene content of the mitochondrial genome of the former relative to the latter. Nevertheless, among the 12 genes common to both green algal lineages, there is only one gene cluster that is common to Prototheca

Aurora M. Nedelcu and Robert W. Lee wickerhamii and C. reinhardtii, i.e., the nad5-nad4 cluster, and none between P. wickerhamii and C. eugametos. It is noteworthy that among the genes that have been mapped on the mitochondrial genome of Platymonas subcordiformis, the only gene cluster that is shared with Prototheca is the same nad5-nad4 cluster. Probably the most unexpected variation in gene order among the green algal mitochondrial genomes has to do with one of the most conserved gene clusters in the land plant counterparts, i.e, the one comprising the rRNA genes: in Platymonas, as in most other mitochondrial systems, rrnL and rrnS are located on the same DNA strand, while in Prototheca and land plant mitochondrial genomes the rrnS and rrnL are encoded on opposite DNA strands. It is also interesting that there might be at least five polycistronic units in the mtDNA of Platymonas, but as few as two in Prototheca and C. reinhardtii mtDNA, and only one in C. eugametos. Similarly, although limited, studies on cpDNA in green algae outside the chlamydomonadalean group have disclosed a highly variable gene order with only few conserved gene clusters, suggesting that the evolutionary pattern of green algal cpDNA is less conservative than that of their land plant counterparts (Palmer, 1991). The few green algal chloroplast genomes mapped to date do not share similar gene orders with either one another or with Chlamy­ domonas counterparts. Some gene clusters, however, are present in more than one lineage: for instance, the petA-petD gene cluster present in Chlamydomonas is also found in Scenedesmus obliquus (Kück, 1989; Kück et al., 1990) and it was suggested that a more extended gene cluster, including might have been present in the most recent common ancestor of Chlamydomonas and Scenedesmus (Boudreau et al., 1994). Moreover, two clusters, psaA-psaB and psbC-psbD, are shared by Spirogyra and Codium (Manhart et al., 1990). DNA rearrange­ ments involving ancestral polycistronic units also occurred d u r i n g the evolution of green algal chloroplasl genomes; rrnS and rrnL that are cotranscribed in all bacteria, as well as Chlamydomonas and land plant chloroplasts, are separately transcribed in Spirogyra (Manhart et al., 1990), Codium (Manhart et al., 1989) and Chlorella ellipsoidea (Yamada and Shimaji, 1987).

1. Factors Contributing to Gene Rearrangement Palmer (1990) suggested several factors that are likely to promote more inversional recombination in

Chapter 5 Mitochondrial and Chloroplast Genome Evolution land plant mtDNA relative to cpDNA: (i) more short dispersed repeats that could serve as points for homologous recombination; (ii) larger intergenic regions that could tolerate inversions; and (iii) more monocistronic rather than multicistronic mito­ chondrial operons. It is interesting that short repeated sequences have been found in the intergenic spacers of both C. reinhardtii and C. eugametos mtDNAs. Furthermore, the presence of fewer and more reduced gene clusters in the C. eugametos mitochondrial genome relative to the C. reinhardtii homolog, as well the disruption of the conserved nad5-nad4 gene cluster in the former, are correlated with a more abundant and complex set of short GC-rich repetitive sequences in the intergenic regions of the former relative to the latter (Nedelcu and Lee, 1998). Short repeated sequences are also present in the mito­ chondrial genome of P. wickerhamii, but they are highly AT-rich (Wolff et al., 1994). It is noteworthy in this connection that small dispersed repeated sequences of ca. 50–1000 bp have been reported in the land plant mitochondrial genome and shown to be involved in intramolecular recombination events promoting gene rearrangements (Hartmann et al., 1994 and references therein; Benslimane et al., 1996). Not surprisingly, short dispersed repeats are also particularly abundant in the highly rearranged cpDNAs of Chlamydomonas. The dispersed repeats in the C. reinhardtii and C. gelatinosa cpDNA are composed of short repeated units occurring in different combinations at different loci in the genome (Boynton et al., 1992; Boudreau and Turmel, 1996). The insertion of repeated sequences in intergenic spacers has been suggested to have been a significant factor in promoting the disruption of the ancestral polycistronic operons in the lineage leading to Chlamydomonas (Boudreau et al., 1994). The very different number of repeated sequences between the cpDNAs of the members of two apparently equally distant pairs of closely related Chlamydomonas taxa, C. reinhardtii/C. gelatinosa and C. moewusii/ C. pitschmannii, correlates directly with the very different level of gene rearrangement between the cpDNAs of the two lineages (Boudreau and Turmel, 1996). It is noteworthy that the tobacco and Marchantia chloroplast genomes, which differ in gene order by a single 30 kb inversion despite about 400 million years of evolutionary separation, contain no dispersed repeats larger than 50 bp (Palmer, 1990). However, small dispersed repeats of 50–1000 bp are unusually abundant in most of the land plant genomes that are highly rearranged. Short terminal degenerate

81

inverted repeats and four-nucleotide directly repeated sequences as well as additional degenerate copies of these sequences in direct or inverted orientation have been also found within the two copies of the Wendy element suggested to be involved in rearrangement events in the chloroplast genome of C. reinhardtii (discussed later) (Fan et al., 1995).

2. Mechanisms Possibly Involved in Gene Rearrangement In higher plant mitochondria, recombination has been invoked to explain the genomic rearrangements accounting for their complex mitochondrial genome structures. It has been proposed that the frequency of homologous recombination is related to the presence of recombinogenic repeated sequences (Palmer and Shields, 1984). A model invoking large direct and inverted repeat-mediated inter- and intramolecular recombination events involving various isomeric forms of the mitochondrial genome has been proposed to illustrate the multipartite structure and the evolution of the maize mitochondrial genome (Fauron et al., 1995). Because there is no evidence for a multipartite structure of the mitochondrial chromosome in Chlamydomonas, recombination events similar to those proposed for the land plant mitochondrial genome do not seem very likely to have happened during the evolution of the Chlamydomonas counterparts. Rather, we suggest that intramolecular recombination between one and two sets of inverted repeats, which results in the inversion and interchange of the regions flanked by the repeats, respectively, (Fig. 4b and c), might have played an important role in the evolution of mitochondrial genomes in Chlamydomonas. Moreover, we think that periods of instability associated with mtDNA replication occurring at multiple sites, first competing with, and later replacing the conventional origin of mtDNA replication could have also been contributed to the extensive gene rearrangement observed in Chlamy­ domonas, in a manner similar to that proposed for the vertebrate mitochondrial genome (Macey et al., 1997). The GC-rich palindromic sequences present in the intergenic spacers of C. reinhardtii and C. eugametos could act as surrogate origins of replication of the mitochondrial DNA (Nedelcu and Lee, 1998). In land plant chloroplast genomes, on the other hand, homologous or illegitimate recombination between short repeats (as small as 11–16 bp) and/or

82 tRNA genes (trn), both functional and pseudogenes, is considered the major cause of gene rearrangements (see Boudreau and Turmel, 1995, 1996 for references). The relative abundance of appropriately oriented and located (between, rather than within transcription units) short dispersed repeats seems to be a major factor in determining the prevalence of cpDNA inversions in land plants (Palmer, 1991). Similarly, recombination events between short dispersed repeats have also been proposed to account for the various rearrangements described in the Chlamydomonas chloroplast genome (discussed by Boudreau and Turmel, 1996). Moreover, the finding that trn-specific oligonucleotide probes hybridized near the endpoints of an inversion in C. pitschmannii cpDNA led Boudreau and Turmel (1995) to raise the possibility of intra- or intermolecular recombination events between duplicated tRNA genes being responsible for the observed inversion. In addition, in C. reinhardtii, the Wendy element is considered to have played a major role in the shuffling of chloroplast gene clusters in this lineage relative to other Chlamydomonas lineages; the fact that both copies of Wendy were found to be flanked by gene clusters that are contiguous in C. moewusii but are separated and inverted relative to each other in C. reinhardtii argues for such an involvement. The mechanisms involved in such rearrangements might have involved Wendy-dependent illegitimate homologous or site-specific recombination events, or both (Fan et al., 1995).

VI. Evolution of Mitochondrial and Chloroplast Gene Structure and Organization in Chlamydomonas

A. Intron-containing Coding Regions Both mitochondrial and chloroplast coding regions in chlamydomonadalean taxa are interrupted by introns. No introns of the group II type have been identified in the mitochondrial genes of the group, but a trans-spliced group II intron has been found in the chloroplast psaA gene of several Chlamydomonas taxa (see Turmel et al., 1995a for references). Introns of the group I family have been found in mitochondrial rRNA- and protein- but not tRNAcoding regions of Chlamydomonas. It is interesting, however, that the mitochondrial genome of C. rein­ hardtii is devoid of any intronic sequences. A group I

Aurora M. Nedelcu and Robert W. Lee intron has been also reported in the only other available mtDNA sequence of a chlorophycean taxon outside the chlamydomonadalean group, i.e., a partial sequence of mitochondrial rrnL from Scenedesmus obliquus (Kück et al., 1990). Because the insertion site of the rrnL group I intron in S. obliquus is four nucleotides downstream of the insertion site of one of the C. eugametos mitochondrial rrnL group I introns, it is difficult to make any suggestions as whether the most recent common ancestor of these two taxa, which belong to the two main evolutionary lineages within the chlorophycean group, namely the DO and CW lineages, had or did not have group I introns in its LSU rRNA gene. Nevertheless, two group I introns have been found in the mitochondrial rrnL of the trebouxiophycean (sensu Friedl, 1995) taxon Prototheca wickerhamii: one intron is present at the same position as the mitochondrial rrnL group I intron in S. obliquus (Wolff et al., 1993), where as the other intron shares an identical insertion site with one of the three mitochondrial rrnL group I introns in C. eugametos. This finding could argue for the presence of at least two introns in the mitochondrial LSU rRNA gene in the most recent common ancestor of the trebouxiophycean and chlorophycean lineages, followed by the loss of one of the introns as well as the acquisition of new introns at new insertion sites in each of the two lineages. It seems likely, therefore, that the most recent common ancestor of green algae had introns in its mitochondrial coding regions, and subsequently introns were independently lost or acquired in distinct evolutionary lineages. Interestingly, two of the three group I introns present in cox1 of Prototheca wickerhamii are located at positions identical to the sites of insertion of liverwort mitochondrial cox1 introns, and it has been suggested that they were already present in the common chlorophyte/embryophyte ancestor (Wolff et al., 1993). Moreover, both Scenedesmus obliquus and liverwort mtDNAs contain introns ofthe group II family, which represent the only type of intron found in the mitochondrial genes of angiosperms. It is interesting to note that the reverse transcriptase-like coding region present in the C. reinhardtii mtDNA seems to be in fact the intronic ORF of a degenerate group II intron (Nedelcu and Lee, 1998). These observations may suggest that the most recent common ancestor ofthe green algal/land plant group contained both group I and II introns in its mitochondrial coding regions and that a massive loss of all of the former, and most of the latter, has

Chapter 5

Mitochondrial and Chloroplast Genome Evolution

occurred in the tracheophyte (vascular plant) as well as C. reinhardtii lineage. In contrast, Chlamydomonas chloroplast genes contain both group I and II introns. The distribution of 39 group I introns representing 12 insertion sites is highly variable among 17 Chlamydomonas taxa and does not suggest the same phylogenetic relationships among Chlamydomonas lineages as do the chloroplast rDNA sequences (Turmel at al., 1993). Because the rrnL of cyanobacteria and of the Chlorella and land plant chloroplast lineages lack introns, it was suggested that all of the intron insertion positions in Chlamydomonas rrnL are of recent origin and that some of them might have arisen after the divergence ofthe two main Chlamydomonas lineages (Tunnel et al., 1993). Moreover, because the chloroplasts of both land plants and their closest relatives, the charophycean green algae, display overall a very small number of group I introns (Palmer, 1991), it has been suggested that the proliferation of group I introns occurred in the Chlamydomonas lineage after the charophycean divergence (Turmel et al., 1993). However, it was recently reported that the Chlorella vulgaris chloroplast rrnL is interrupted by a group I intron inserted at the same position as the single group I intron in the C. reinhardtii rrnL; intronic sequence comparisons as well as amino acid sequence similarities of their intronic ORFs suggested that the two closely related self-splicing rrnL group I introns in Chlorella vulgaris and C. reinhardtii descended from the same group I intron present in the most recent common ancestor of these two lineages (Kapoor et al., 1997). The only introns with features of the group II family identified to date in Chlamydomonas chloroplast genes are those found in psaA of several Chlamydomonas taxa (see Turmel et al., 1995a). While Chlamydomonas chloroplast genes have multiple introns in most of the genes possessing introns, land plant chloroplast genes contain single introns. Also, although land plant chloroplast tRNA genes harbor long single introns, no split tRNA genes have been found in algal chloroplasts. It is interesting that some of the land plant mitochondrial and chloroplast protein-coding genes consist of scattered exons flanked by 5´- or 3´segments ofgroup II introns; the exons are separately transcribed and spliced in trans (see Turmel et al., 1995a for references). In Chlamydomonas, such an organization has only been described among chloroplast genes. Chlamydomonas psaA coding

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regions are made up of three exons scattered around the genome and spliced in trans (Kück et al., 1987; Choquet et al., 1988; Turmel et al., 1991; Goldschmidt-Clermont et al., 1991; Turmel et al., 1995a). Although the location of the three exons in the genome is different between two very divergent taxa, C. moewusii and C. reinhardtii, the information contained is similar, indicating that the most recent common ancestor of these two lineages possessed a psaA coding region interrupted in a similar manner (Boudreau et al., 1994). In contrast, psaA is not trans-spliced in land plants or other genera of algae (with the exception of Euglena gracilis), but rps12 (which is uninterrupted in Chlamydomonas, Euglena and Cyanophora) is trans-spliced in all examined land plants (Sugiura, 1989). An additional feature contributing to variation in chloroplast gene structure in Chlamydomonas is the presence of translated large insertion sequences in the clpP gene of C. reinhardtii and C. eugametos (Huang et al., 1994) as well as chimeric RNA polymerase coding regions juxtaposed in-frame with long sequences of unknown origin in C. rein­ hardtii (Fong and Surzycki, 1992).

B. Fragmented Coding Regions In Chlamydomonas, both mitochondrial and chloroplast rRNA genes are fragmented into coding modules whose mature transcripts are not spliced together into covalently continuous rRNA molecules. However, the rRNA gene organization and expression as well as evolutionary origins are very different between the two organellar genomes. Mitochondrial rRNA genes in Chlamydomonas are not only highly fragmented but also scrambled (i.e., the gene pieces are interspersed with other coding regions and do not follow the 5'–3' transcriptional order of their counterparts in conventional continuous genes) (Fig. 2). The SSU and LSU rRNA-coding regions are fragmented into four and eight gene pieces, respectively, in C. reinhardtii (Boer and Gray, 1988a) and into three and six gene pieces, respectively, in C. eugametos (Denovan-Wright and Lee, 1994). The rRNA-coding modules are extensively interspersed with each other as well as with protein-coding and tRNA genes, and the rRNA fragments are most likely excised from longer multicistronic transcripts following precise endonucleolytic scissions (Boer and Gray, 1988a). Although the rRNA pieces are not spliced together,

84 they have the ability to interact through intermolecular base pairing, restore the conserved core of the rRNA secondary structure (Boer and Gray, 1988a; DenovanWright and Lee, 1994), and assemble into mito­ chondrial ribosomes (Denovan-Wright and Lee, 1995). Discontinuous mitochondrial rRNAs have been reported not only in other Chlamydomonas taxa (Denovan-Wright et al., 1996), but also in other chlorophycean taxa with a CW or DO flagellar configuration as well as in chlorococcalean taxa phylogenetiqally related to them (Nedelcu et al., 1996). A trend in the evolution of this trait, that is, a tendency towards an increase in the degree of discontinuity from continuous mitochondrial rRNAs to the highly fragmented mitochondrial rRNAs in C. eugametos and C. reinhardtii was suggested (Nedelcu et al., 1996; Nedelcu, 1997). Although mitochondrial rRNA genes are highly fragmented and scrambled in both C. reinhardtii and C. eugametos, the distribution of the coding in formation among their coding modules, as well the order of these modules within the genome, is different between the two species (Denovan-Wright and Lee, 1994). Calculations of the minimal number of transpositions required to convert hypothetical ancestral rRNA gene organizations to the arrange­ ments present in the two Chlamydomonas taxa, as well as a limited survey of the size of mitochondrial LSU rRNAs in other Chlamydomonas species, led Denovan-Wright et al. (1996) to propose that the last common ancestor of Chlamydomonas algae pos­ sessed fragmented mitochondrial rRNA genes whose coding modules were nearly co-linear with their counterparts in conventional continuous rRNA genes. The model presented by the authors predicted that in taxa basal to the Chlamydomonas group, mito­ chondrial rRNA genes would be fragmented but not scrambled. The presence of scrambled but not highly fragmented mitochondrial LSU rRNA coding regions in Scenedesmus obliquus, however, suggested that scrambling may have developed at an early stage in the evolution of discontinuous and scrambled rRNA genes within the chlorophycean green algal group, probably in parallel with the fragmentation events (Nedelcu, 1997). The mechanisms responsible for either the fragmentation or the scrambling ofthe mitochondrial rRNA coding regions in Chlamydomonas are not known yet, although several suggestions have been made. The GC-rich repeat clusters identified in

Aurora M. Nedelcu and Robert W. Lee C. reinhardtii mitochondrial DNA were suspected to have contributed to the extensive rRNA gene arrangements through a mechanism analogous to bacterial transposition (Boer and Gray, 1991). The absence of a reverse transcriptase-like open reading frame in C. eugametos mitochondrial DNA led Denovan-Wright and Lee (1994) to favor the view that the mitochondrial rRNA coding regions in Chlamydomonas became scrambled by recom­ bination between non-homologous regions of mtDNA molecules such as the dispersed repeated elements found in C. reinhardtii (Boer and Gray, 1991) and C. eugametos (Nedelcu and Lee, 1998) rather than by reverse transcription (Boer and Gray, 1988a). The authors further assumed that the unusual gene structure in Chlamydomonas mitochondria arose from conventional, continuous rRNA genes by two separate, consecutive processes: the introduction of processing signals and the scrambling of coding regions defined by these signals. Nedelcu (1997), however, proposed a recombination model that could disrupt and scramble a coding region in a single step. The proposal is an extension of the model presented by Fauron et al. (1995) for the evolution of the maize mitochondrial genome and involves an intramolecular homologous recombination event between two sets of two-copy inverted repeats. Generally, such an event would result in an interchange of the sequences situated between the two sets of inverted repeats (Fig. 4c). The way in which recombination events such as the one suggested above, as well as those proposed by Fauron et al. (1995), could have been involved in the evolution of chlorophycean mito­ chondrial rRNA genes is illustrated in Fig. 4d. Comparisons among the locations of the short repeated elements within the mitochondrial genome of C. reinhardtii, C. eugametos and the available DNA sequences of other chlorophycean green algae revealed similarities regarding the positions of these repeats relative to the rRNA-coding units within the respective genomes (Nedelcu, 1997; Nedelcu and Lee, 1998). It was suggested, therefore, that the fragmented and scrambled mitochondrial rRNA coding regions in the chlorophycean green algal group may have been generated through multiple recombination events triggered by the accumulation of short repeated sequences within the variable regions of the rRNA genes and the intergenic spacers of these mitochondrial genomes. To illustrate how recombination events similar to those proposed above could be entirely responsible for the extensive

Chapter 5 Mitochondrial and Chloroplast Genome Evolution mitochondrial rRNA gene rearrangements, a hypothetical pathway to gradually convert conven­ tional continuous mitochondrial rRNA genes to the rRNA gene arrangement described in C. eugametos was envisioned (Nedelcu, 1997). It is interesting that chloroplast LSU but not SSU rRNA-coding regions are also fragmented in Chlamydomonas. Three internal transcribed spacers (ITSs) located at the same position in the chloroplast rrnL of all 17 Chlamydomonas taxa investigated by Turmel et al. (1993) interrupt this gene into four gene pieces whose transcripts are not covalently linked after the removal of the ITSs from the primary transcript. Unlike the introns, but like the break points in the mitochondrial rRNA-coding regions, the ITSs are located within highly variable regions of primary and/or secondary structure. The ITSs in the Chlamydomonas chloroplast rrnL are usually less than 300 bp long and differ substantially in size and base composition. Although they are always excised post-transcriptionally from a precursor RNA to yield four mature rRNA species, no common sequence motif to account fora similar processing recognition signal has been identified, suggesting that either different recognition signals or specific threedimensional topology of the ribosome might be involved in the processing of ITSs (Tunnel et al., 1993). It is noteworthy that the size and base composition differences among corresponding ITSs in different Chlamydomonas taxa are not consistent with the phylogenetic relationships suggested by the LSU rRNA-coding sequences (Turmel et al., 1993). This feature of fragmented chloroplast LSU rRNAcoding regions is not confined to Chlamydomonas taxa. In Chlorella ellipsoidea, an insert that does not have the characteristics of an intron has been reported at the same position as ITS3 of Chlamydomonas (Yamada and Shimaji, 1987). Moreover, Nedelcu et al. (1996) showed that the chloroplast LSU rRNAs in green algal lineages from three green algal classes (sensu Mattox and Stewart, 1984), the Chlorophyceae, Pleurastrophyceae, and Micromonadophyceae, have fragmented chloroplast LSU rRNAs, in most cases the fragmentation pattern being similar to that described in Chlamydomonas. The distinct patterns observed in some lineages are most likely due to the absence or inability to process one of the ITSs. On the other hand, although three ITSs, one of which accounts for the 4.5S rRNA species, have been identified in the maize chloroplast LSU rRNA gene (Kössel et al., 1985), they are situated at different

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positions than in Chlamydomonas. It is interesting that the chloroplast rrnL of Chlamydomonas as well as the mitochondrial rrnL of higher plants are both missing the variable region in which the ITS that accounts for the 4.5S rRNA species is situated (see Turmel, 1993 for references). It was proposed that there is a direct evolutionary connection between variable regions and ITSs, in the sense that variable regions might have in fact evolved from the ITSs separating the rRNA coding modules in the progenote (Gray and Schnare, 1995). Although most of the ITSs in contemporary rRNA genes represent most likely derived rather than primitive traits, the acquisition of the processing sites responsible for the excision of the contemporary ITSs could be considered a ‘reversion to a primitive state’ (Gray and Schnare, 1995). Another unusual gene organization has been reported for the chloroplast RNA polymerase genes (rpo) of C. reinhardtii. The rpoB coding region is divided into two ORFs separated by a 616 bp spacer. Although evidence for the transcription of these ORFs is missing, if expressed, they most likely encode separate polypeptides (Fong and Surzycki, 1992).

VII. Evolution of Mitochondrial and Chloroplast DNA Sequences in Chlamydomonas In land plants, estimated rates of synonymous (silent) nucleotide substitution per site in mitochondrial and chloroplast protein-coding genes are lower than in nuclear genes (reviewed by Palmer, 1991; Bousquet et al., 1992; Laroche et al., 1997). This trend contrasts with the situation in mammals where synonymous substitution rates in mitochondrial genes are higher than in nuclear genes (discussed by Palmer, 1991). No extensive studies on point mutation levels in Chlamydomonas mitochondrial, chloroplast and nuclear genes have yet been published. However, K. J. Prendergast and R. W. Lee (unpublished) have noted that in Chlamydomonas, the number of synonymous substitutions per site, in contrast to land plants, is higher in mitochondrial and chloroplast protein-coding genes than in the one nuclear proteincoding gene (rbcS) for which such a value can be calculated at present. Moreover, in Chlamydomonas, the number of synonymous substitutions per site is higher in mitochondrial than in chloroplast genes, which is opposite to the situation observed in land

86 plants. Further support for such a trend in Chlamydomonas is provided by the mitochondrial and chloroplast LSU rRNA sequences (Turmel et al., 1993; Denovan-Wright et al., 1996). It is interesting that the number of substitutions in Chlamydomonas mitochondrial SSU and LSU rRNA sequences is several-fold higher than the accumulated substitutions in land plant mitochondrial counterparts (Denovan-Wright et al., 1996). Mitochondrial rRNA sequences of P. wickerhamii also seem to have a high rate of nucleotide substitution and, together with the Chlamydomonas, ciliate, fungal and yeast counter­ parts, constitute a rapidly evolving group (associated with long branches in phylogenetic analyses), in marked contrast to the slowly evolving land plant mitochondrial rRNA sequences. Although the number of transitional substitutions is probably saturated in the rapidly evolving mitochondrial rRNA sequences, Denovan-Wright et al. (1996) showed that the apparent affiliation of the Chlamydomonas sequences with ciliate/fungal/yeast counterparts, and therefore their separation from the land plant sequences, is not due to a ‘long-branch length attract’ artifact (i.e., the grouping of rapidly evolving sequences together, in spite of their true phylogenetic relatedness). Surprisingly, Chlamydomonas chloroplast rrnL sequences also display extensive sequence diver­ gence; the various Chlamydomonas lineages studied by Turmel et al. (1993) revealed at least twice the range of sequence variation seen in land plants. Moreover, within some Chlamydomonas lineages (including those leading to C. reinhardtii or C. eugametos) the level is greater than that found between the bryophyte Marchantia and the monocot Oryza (Turmel et al., 1993). Although the chloroplast genomes of the closely related C. pitschmannii and C. eugametos/C. moewusii are extremely similar in gene order they appear to be very divergent in DNA sequence, as deduced from differences in their cpDNA restriction patterns (Boudreau and Turmel, 1995). The ratio of point mutations to length mutations in Chlamydomonas cpDNA is, however, substantially lower than in angiosperm chloroplast DNA, due to the increased level of length mutations in the former (Palmer et al., 1985). The lack of knowledge as to the exact time of divergence of different Chlamydomonas lineages makes it difficult to assess absolute rates of nucleotide substitutions in Chlamydomonas organellar genomes and to compare their tempo of DNA sequence evolution with that of other counterparts. Although fossil evidence suggests that the chlamydo-

Aurora M. Nedelcu and Robert W. Lee monadalean group is at least 350 million years old, the first chlorophycean green algal fossils are around 900 million years old (Tappan, 1980). It seems reasonable to assume, therefore, that the C. reinhardtii/C. eugametos divergence could be as old as 900 million years or as recent as 400 million years. On the other hand, the bryophyte/tracheophyte divergence is believed to have occurred about 400 million years ago (Schopf, 1970; Shear, 1991). If nucleotide substitution levels in organelle DNAs of Chlamydomonas were roughly equal to or up to twice the level observed in their land plant counterparts, comparable rates of nucleotide substitutions in Chlamydomonas and land plants could be hypothesized. In contrast, levels of nucleotide substitution exceeding twice the level noted among land plant counterparts would suggest a higher rate of nucleotide substitution within Chlamydomonas. The observed levels of sequence divergence in organellar rRNA genes in Chlamy­ domonas, i.e., several-fold and at least two-fold higher in mitochondrial and chloroplast rRNA genes, respectively, indicate higher and slightly higher rates of nucleotide substitution in Chlamydomonas mitochondrial and chloroplast rRNA genes, respec­ tively, relative to their land plant counterparts. However, more knowledge about the levels of nucleotide substitution in protein-coding genes from all three genetic compartments from various lineages within the chlamydomonadalean group have to be available before the tempo of DNA sequence evolution in Chlamydomonas organellar genomes can be assessed with confidence. It is not fully understood why the DNA sequences in different genomes of a given lineage or among different groups have different evolutionary rates. Palmer (1990) suggested that error-free replication mechanisms, better postreplication repair systems or copy-correction mechanisms might explain the overall low substitution rates in plant organelle genomes. It is noteworthy that in contrast to land plants, the rDNA nucleotide substitution levels in both Chlamydomonas organellar genomes appealrelatively high. It is possible that this opposite trend could be determined by the same factors proposed for land plant organellar genomes but acting in an opposite direction: error-prone replication mechan­ isms, inefficient postreplication repair systems or copy-correction mechanisms. To explain the observation that mitochondrial and chloroplast genomes in land plants both have low rates of DNA sequence evolution, it was suggested that they might

Chapter 5

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be under common nuclear control (Palmer, 1990). Such a control can also be hypothesized for Chlamydomonas given that both organellar DNA sequences seem to have evolved at high rates.

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study group in attempts to elucidate the evolutionary processes that shape organellar genomes.

Acknowledgments VIII. Conclusions Land plant organellar genomes revealed ‘contrasting modes and tempos of genome evolution’ (Palmer, 1990). Our comparative analysis of evolutionary trends in the mitochondrial and chloroplast genomes of Chlamydomonas, however, allows us to suggest concerted modes and tempos of evolution of these two organellar genomes in this green algal lineage. While land plant mitochondrial genomes are more variable in size and organization but more stable in DNA sequence than their chloroplast counterparts, both organellar genomes in Chlamydomonas seem to have evolved at comparable rates with respect to genome size, organization as well as DNA sequence, as summarized in Table 1. The mechanisms and the selective pressures responsible for both the overall high rate of evolution in Chlamydomonas organellar genomes relative to the land plant counterparts, as well as their apparent concerted evolution are not fully understood (Nedelcu, 1998). The increasing amount of information on the biology, biochemistry, molecular biology, and genetics of these green flagellates (reviewed in this book), as well as the development of new technologies in manipulating their cellular genomes, make Chlamydomonas a good

We thank M. W. Gray for helpful discussion and critical reading of an earlier version of this paper and D. Bhattacharya, E. Boudreau, E. M. DenovanWright, and M. W. Gray for permission to use modified versions of figures previously published. A. M. N. was supported from a Natural Sciences and Engineering Research Council (N.S.E.R.C.) of Canada grant to R. W. L., a Dalhousie University Graduate Scholarship, and an Izaac Walton Killam Memorial Scholarship. Research in the laboratory of R. W. L. is supported by N.S.E.R.C. of Canada.

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Van de Peer Y, Neefs J-M and De Watcher R (1990) Small ribosomal subunit RNA sequences, evolutionary relationships among different life forms, and mitochondrial origins. J Mol Evol 30: 463–476 Waddle JA, Schuster AM, Lee KW and Meints RH (1990) The mitochondrial genome of an exosymbiotic Chlorella-like green alga. Plant Mol Biol 14: 187–195 Wolff G and Kück U (1990) The structural analysis of the mitochondrial S S U r R N A implies a close phylogenetic relationship between mitochondria from plants and from the heterotrophic alga Prototheca wickerhamii. Curr Genet 17: 347–351 Wolff G and Kück U (1993) Organization and coding capacity of mitochondrial genomes of algae. In: Brennicke A and Kück U (eds) Plant Mitochondria with Emphasis on RNA Editing and Cytoplasmic Male Sterility, pp 1 0 1 – 1 1 3 . VCH Verlagsgesellschaft,Weinheim Wolff G, Burger G, Lang BF and Kück U (1993) Mitochondrial genes in the colourless alga Prototheca wickerhamii resemble plant genes in their exons but fungal genes in their introns. Nucleic Acids Res 21: 719–726 Wolff G, Plante I, Lang BF, Kück U and Burger G (1994) Complete sequence of the mitochondrial DNA of the chlorophyte alga Prototheca wickerhamii. Gene content and genome organization. J Mol Biol 237: 75–86 Wolstenholme DR and Fauron CM-R (1995) Mitochondrial genome organization. In: Levings III CS and Vasil IK (eds) The Molecular Biology of Plant Mitochondria, pp 1–59. Kluwer Academic Publishers, Dordrecht Woodson SA and Cech TR (1989) Reverse self-splicing of the Tetrahymena group I intron: Implication for the directionality of splicing and for intron transposition. Cell 57:335–345 Yamada T (1991) Repetitive sequence-mediated rearrangements in Chlorella ellipsoidea chloroplast DNA: Completion of nucleotide sequence of the large inverted repeat. Curr Genet 19: 139–147 Yamada T and Shimaji M (1987) An intron in the 23S rRNA gene of the Chlorella chloroplasts: Complete nucleotide sequence of the 23S rRNA gene. Curr Genet 11:347–352 Zinn AR, Pohlman JK, Perlman PS and Butow RA (1988) In vivo double-strand breaks occur at recombinogenic GC-rich sequences in the yeast mitochondrial genome. Proc Natl Acad Sci USA 85: 2686–2690 Zurawski G, Clegg MT and Brown AHD (1984) The nature of nucleotide sequence divergence between barley and maize chloroplast DNA. Genetics 106: 735–749

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Chapter 6 Uniparental Inheritance of Chloroplast Genomes E. Virginia Armbrust University of Washington, Marine Molecular Biotechnology Laboratory, School of Oceanography, Box 357940, Seattle, WA 98195, U.S.A.

Summary I. Introduction II. Historical Overview of the Uniparental Inheritance of Chloroplast DNA A. Genetic Evidence for the Selective Elimination of Minus Chloroplast DNA in Early Zygotes B. Physical Evidence for the Selective Elimination of Minus Chloroplast DNA in Early Zygotes III. Mating-Type Control of Life Cycle Events A. Gamete Differentiation and Fusion B. Zygote Development C. Chloroplast DNA Inheritance IV. Protection of Plus Chloroplast DNA A. Evidence for a Specific Gene Required for Protection 1. Diploid Crosses 2. The mtl1 Mutation of C. monoica B. Is Plus Chloroplast DNA Protected by Methylation? V. Zygote Specific Elimination of Minus Chloroplast DNA Specific Factor A. Dependence on a UV Sensitive, but not Gametes Enhances Biparental Inheritance 1. Brief UV Irradiation of 2. The Ezy1 Gene 3. The uvsE1 Mutation 4. Involvement of RecA? Parent B. Dependence on the Chloroplast DNA Content of the 1. Treatment with an Inhibitor of Chloroplast DNA Replication, 5-fluorodeoxyuridine 2. The mat3 Mutation C. The Ezy2 Gene D. The sup1 Mutation VI. Regulation of Chloroplast DNA Inheritance A. Regulation of Protection B. Regulation of Destruction VI. Evolution of the Uniparental Inheritance of Organelle Genomes Acknowledgments References.

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Summary An intriguing feature of most eukaryotes is that chloroplast and mitochondrial genomes are inherited almost exclusively from one parent. This can be explained for those organisms that produce gametes of different sizes as chloroplasts and mitochondria are mostly excluded from the sperm or pollen. However, uniparental inheritance also typifies those organisms that produce gametes of identical sizes. In Chlamydomonas, the J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 93–113. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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uniparental inheritance of chloroplast genomes is achieved by a series of mating type-controlled events that culminate in the early zygote with the selective degradation of chloroplast DNA (but not the chloroplast) parent. Thus, only the chloroplast DNA from the mating-type plus contributed by the mating-type minus parent persists in the zygote to be transmitted to meiotic progeny. How Chlamydomonas selectively degrades a subset of organelle genomes has long fascinated researchers and is the subject of this chapter. The molecular mechanisms underlying this phenomenon remain elusive, but are hypothesized to entail two distinct events that occur during different stages of the life cycle: a ‘protection’ of plus chloroplast DNA, perhaps during gametogenesis, and a ‘destruction’ of unprotected minus chloroplast DNA during early zygote development. The gene(s) required for protection have yet to be isolated but all evidence to date indicates that they will be cells only. The degradation of unprotected minus chloroplast DNA appears to be accomplished, present in in part, by a zygote-specific nuclease targeted to the chloroplast during early zygote development. Interestingly, multiple genes have now been identified that all appear to play a role in accomplishing this degradation. As will be shown, the specific elimination of minus chloroplast DNA during early zygote development is a complex and carefully orchestrated phenomenon likely requiring the activity of several proteins. Potential mechanisms for regulating this process are highlighted.

I. Introduction Eukaryotes employ two fundamentally different mechanisms to transfer genetic information from parent to offspring. Nuclear genes are transmitted according to Mendel’s laws. The meiotic spindle ensures that nuclear alleles segregate from one another and genes located on nonhomologous chromosomes sort independently. The existence ofthese laws means that the inheritance patterns of nuclear genes are predictable. Organelle genomes, on the other hand, appear to defy Mendel’s laws and display their own mode of inheritance. In the vast majority of multicellular organisms, the egg and sperm or pollen are dramatically different in size such that organelles and their genomes are typically transmitted to offspring via the egg. Moreover, in mammals at least, any male mitochondria that do enter the zygote appear to be quickly destroyed (Yaneda et al., 1995). Thus most plants and animals inherit organelle genomes from the female parent only, in a process traditionally referred to as maternal inheritance. Conifers, the main exception to this rule, inherit organelle genomes from the male parent and thus display paternal inheritance (Whatley, 1982; Neale et al., 1989). In most unicellular organisms, organelle genomes are also inherited from only one parent even though the two gametes are generally the same Abbreviations: CsCl – cesium chloride; DAPI – 4´,6-diamidinophenylindole; ezy — early zygote; FdUr – 5-flurodeoxyuridine; – mating type minus; – mating type plus; mtl – mating type limited; RFLP – restriction fragment length polymorphism; sup – suppressor of uniparental inheritance

size and contribute equal numbers of organelles to the zygote. In these instances, uniparental inheritance is accomplished by selectively destroying one set of organelles (Braten, 1971, 1973) or organelle genomes (Harris, 1989; Kuroiwa, 1991; Meland et al., 1991) in the developing zygote. Although uniparental inheritance dominates in most eukaryotes, organelle DNA can also be inherited from both parents in a process known as biparental inheritance (Sears, 1980; reviewed in Birky, 1995). Biparental inheritance can occur when the mechan­ isms that typically exclude or destroy organelle genomes are either inefficient (Gyllensten et al., 1991), or else nonexistent (Sears, 1980). Because neither mitochondria nor chloroplasts possess a spindle, when organelle genomes of two types coexist within the zygote, they partition randomly during meiosis such that some progeny inherit the organelle DNA from one parent, some progeny inherit from the other parent, and some progeny inherit from both parents. The biparentally transmitted genomes further segregate during subsequent mitotic divisions such that eventually, the mitotic offspring are pure for the organelle genomes from one parent or the other. This mitotic segregation also results from a stochastic partitioning of genomes to progeny (Birky, 1983). And finally, perhaps the most intriguing version of organelle inheritance, known as doubly uniparental inheritance, is displayed by mussels: female mussels transmit mitochondrial DNA to both male and female offspring while male mussels selectively pass on to their male offspring the mitochondrial DNA they have inherited from their father (Hoeh et al., 1996).

Chapter 6

Chloroplast DNA Inheritance

Non-Mendelian transmission patterns and the mitotic segregation of alleles are now accepted as defining features of organelle inheritance. Beginning at the turn of the century, however, when seemingly aberrant inheritance patterns were first independently described by Correns and Bauer (reviewed in Birky, 1995) and continuing until the mid 1950s and 1960s, the possible existence of genetic information that did not obey Mendel’s laws was somewhat heretical (reviewed in Sager, 1965). DNA within the nucleus could be seen, but there was no notion that organelles could also contain their own genomes. Research with Chlamydomonas reinhardtii was instrumental in transforming organelle inheritance studies from a descriptive science to an experimental science and today C. reinhardtii remains a model organism for the study ofmechanisms underlying chloroplast DNA transmission (Rochaix, 1995). In Chlamydomonas spp., the basis of uniparental inheritance is a selective degradation in the early zygote of the chloroplast DNA (but not the chloroplast) from the mating-type parent, presumably due to the action of minus a zygote-specific nuclease. The chloroplast DNA from the mating-type plus parent is somehow protected from degradation and persists in the zygote to be transmitted to the meiotic progeny. Thus, meiotic progeny inherit chloroplast DNA uniparentally from parent only. the The molecular basis for the selective destruction of minus chloroplast DNA has been the subject of continual study since the phenomenon was first discovered in 1954 (Sager, 1954), but details of the underlying mechanisms remain elusive. What remains even more mysterious, however, is how the uniparental inheritance of organelle genomes evolved in the first place. A number of excellent reviews of chloroplast DNA inheritance have been published in recent years by Harris (1989), Gillham et al. (1991), Kuroiwa (1991), Gillham (1994), and Sears and VanWinkleSwift (1994). This chapter will focus on possible molecular mechanisms underlying the uniparental inheritance of chloroplast genomes. Unless otherwise stated, the work described involves C. reinhardtii since this has traditionally been the study organism of choice. However, it should be noted that all species of Chlamydomonas appear to destroy selectively the chloroplast DNA from one parent in the early zygote (McBride and McBride, 1975; Coleman and Maguire, 1983; VanWinkle-Swift and Aubert, 1983).

95 II. Historical Overview of the Uniparental Inheritance of Chloroplast DNA

A. Genetic Evidence for the Selective Elimination of Minus Chloroplast DNA in Early Zygotes The field of chloroplast genetics began in 1954 when Ruth Sager described the inheritance patterns of two UV-induced mutations, sr1 and sr2 (reviewed in Boynton et al., 1992). The sr1 mutation confers resistance to low levels ofthe antibiotic streptomycin and the sr2 mutation confers resistance to high levels of streptomycin (Sager, 1954; Table 1). Regardless or the parent, ofwhether sr1 is carried by the when a resistant strain is crossed to a sensitive strain, two of the meiotic progeny are resistant to low levels of streptomycin and two of the progeny are sensitive, just as predicted for a nuclear mutation inherited in a parent Mendelian fashion. In contrast, when a carrying the sr2 mutation is crossed to a sensitive parent, all the meiotic progeny are resistant to high levels of streptomycin; only rarely is a sensitive progeny obtained. When the reciprocal cross is conducted, and a resistant parent is crossed to a sensitive parent, the meiotic progeny are all streptomycin sensitive (Fig. 1). Sager (1954) performed a series of backcrosses with each type of sr2, sensitive, F1 progeny (– sr2, sensitive) to prove that these seemingly aberrant inheritance patterns were stable and that the resistance phenotype conferred by the sr2 mutation was transmitted to meiotic progeny only when carried by parent. It is now known that the sr2 mutation the is localized within the rps12 gene of the chloroplast genome (Liu et al., 1989) and that these inheritance patterns reflect the uniparental inheritance of parent. Those rare chloroplast DNA from the zygotes that transmit minus chloroplast DNA to meiotic progeny are referred to as exceptional since they represent the exception to the rule. Sager (1954) briefly raised the possibility that she might have discovered a Chlamydomonas version of maternal inheritance, but she ended this landmark paper by lamenting that ‘the nature of the physical carrier of this inheritance as well as the mechanism of transmission remains obscure.’ Over the next 10–15 years, additional uniparentally inherited mutations were isolated, all of which appeared genetically linked (Sager and Ramanis, 1963; Gillham, 1965; Sager and Ramanis, 1965; Gillham and Fifer, 1968), suggesting that the ‘physical

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carrier’ must be DNA (for discussion, see Gillham, 1969). During this early period, further evidence for the existence of chloroplast DNA came from work by Ris and Plaut (1962). Using a combination of electron and light microscopy, they described regions in the chloroplast stroma that stained with Feulgen and acridine orange, both indicative of the presence of DNA. Moreover, these DNA-staining regions, or nucleoids, contained 25Å microfibrils that were

E. Virginia Armbrust

eliminated by incubation with DNase. Just one year later, Chun et al. (1963) and Sager and Ishida (1963) described the isolation ofchloroplast DNA by cesium chloride(CsCl) densitygradient centrifugation.Thus, it seemed increasingly likely that chloroplasts con­ tained DNA and that it was this chloroplast DNA that was uniparentally transmitted to meiotic progeny.

Chapter 6

Chloroplast DNA Inheritance

B. Physical Evidence for the Selective Elimination of Minus Chloroplast DNA in Early Zygotes By the early 1970s it had been clearly documented that the two chloroplasts (one from each parent) fuse in their entirety in the early zygote (Cavalier-Smith, 1970) and yet mutations presumably located on the minus chloroplast genome were not transmitted to meiotic progeny. What happened to this minus chloroplast DNA in the zygote? To address this question, Sager and Lane (1972) labeled the DNA and gametes with either from and used CsCl density gradient centrifugation to monitor the fate of nuclear and chloroplast DNA in 6 h and 24 h zygotes. Six hours into zygote develop­ ment, the nuclear DNA contributed by both parents could be recovered. In contrast, the chloroplast DNA parent had disappeared from this same from the timepoint and the chloroplast DNA from the

97 parent had undergone a shift in density (Sager and Lane, 1972). Here, at last, was a physical explanation for the genetic results: the chloroplast DNA from the parent could not be transmitted to meiotic progeny because this DNA was somehow eliminated during early zygote development. But why was the gamete left chloroplast DNA contributed by the intact? Sager and Lane (1972) postulated that the observed density shift of the plus chloroplast DNA specific methylation event that was the result of a somehow protected this DNA from elimination in the zygote, a possibility discussed in more detail in a later section. Conclusive proof that minus chloroplast DNA was eliminated from zygotes came in the early 80s from two new sources—molecular biology and fluorescent microscopy. The first molecular evidence was provided when Grant et al. (1980) discovered that a C. reinhardtii mutant strain, ac-u-g-23 (Shepherd et al., 1979), carried two small deletions in its chloroplast DNA. The presence or absence ofthe deletions could be easily monitored by an analysis of chloroplast DNA restriction fragment length polymorphisms (RFLPs). Grant et al. (1980) found that both the deletions in the chloroplast DNA and the independent nonphotosynthetic phenotype were uniparentally inherited. A few years later, Dron et al. (1983) determined that the mutation, rcl-u-1-10-6C, which is in the gene encoding the large subunit of ribulose bisphosphate carboxylase (Spreitzer and Mets, 1980), was localized to chloroplast DNA. Mets and Geist (1983) showed that this gene was uniparentally inherited. The next year, Lemieux et al. (1984) found that RFLPs between the chloroplast genomes of the two interfertile species, C. eugametos and C. moe­ wusii, cosegregated with uniparentally inherited antibiotic resistance markers. Thus, both photo­ synthetic genes and antibiotic resistance mutations were physically localized to the chloroplast genome and it was this DNA that was uniparentally inherited. Beautifully convincing fluorescent microscopy data for the selective elimination of minus chloroplast DNA in the zygote was published during this same period by Kuroiwa et al. (1982). Chloroplast DNA in Chlamydomonas is localized into 8–10 nucleoids, easily distinguished from the much larger cell nucleus when stained with the DNA fluorochrome, 4, 6diamindio-2-phenylindole or DAPI (Fig. 2). Within about 40 min of zygote formation, each chloroplast parent begins to nucleoid contributed by the ‘dissolve’ from its periphery until the DAPI

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E. Virginia Armbrust III. Mating-Type Control of Life Cycle Events

fluorescence disappears completely while the parent remain chloroplast nucleoids from the intact. The two nuclei fuse within the next hour, followed 1–2 h later by the fusion of the two chloroplasts. By 10 h after zygote formation, the nucleoids have coalesced and migrated to form a single large nucleoid near the pyrenoid (Kuroiwa et al., 1982). The chloroplast DNA content ofthe zygote was shown later to remain roughly constant throughout zygote maturation (Coleman, 1984). A burst of chloroplast DNA synthesis occurs prior to meiosis so that each newly formed progeny will possess a full complement of chloroplast DNA (Coleman, 1984). Uchida et al. (1992b) have recently extended this visual analysis of minus chloroplast DNA degradation. By using electron microscopy to determine the distribution of immunogold labeled DNA-specific antibodies, they confirmed that the parent is completely chloroplast DNA from the eliminated and not simply dispersed during early zygote development. This then is the stepping-off point for this chapter. Yes, the chloroplast genome from the parent is specifically eliminated during early zygote develop­ ment, but how is this accomplished? What is known about underlying molecular mechanisms and how are these processes controlled? Many potential players in chldroplast inheritance have been identified, and as will be seen, an understanding of the roles played by these proteins is just emerging.

The simplest model for the uniparental inheritance of chloroplast DNA is that the process entails two distinct events that are likely restricted to different stages of the life cycle: a ‘protection’ of plus chloroplast DNA perhaps during gametogenesis and a degradation (‘destruction’) of unprotected minus chloroplast DNA during early zygote development (reviewed by Gillham et al., 1991; Gillham, 1994; Sears and VanWinkle-Swift, 1994). The genes required for the protection of plus chloroplast DNA and the destruction of minus chloroplast DNA are thought to be controlled by the mating-type locus, described in detail below. In fact, three complex lifecycle events — gamete differentiation, zygote maturation and organelle inheritance — are all governed by the mating-type locus, perhaps reflecting early steps in the evolution of sexual reproduction (reviewed by Goodenough et al., 1995). To understand the regulation of processes occurring during the uniparental inheritance of chloroplast DNA, the life cycle and its control by the mating-type locus will first be examined. Under nutrient-replete conditions, haploid vegetative cells reproduce mitotically and are unable to mate. In response to nitrogen starvation (Sager and Granick, 1954) and a blue light signal (reviewed by Beck and Haring, 1996), vegetative cells or differentiate into mating-competent gametes. When gametes of the opposite mating type are mixed, they rapidly fuse to form diploid zygotes. After an obligate period of maturation, zygotes undergo meiosis and germination to form four haploid progeny that can once again reproduce mitotically. Thus, those genes required for gamete differentiation are expressed in response to nitrogen starvation and those required for zygote maturation are expressed in response to gamete fusion (Goodenough and Ferris, 1987). Life-cycle events are governed by genes of two general types: ‘sex­ linked’ genes are physically located at only one mating-type locus and ‘sex-limited’ genes are unlinked to the mating-type locus but are nonetheless expressed in cells of only one mating type (reviewed in Galloway and Goodenough, 1985; Goodenough and Ferris, 1987). As will be seen, both sex-linked and sex-limited genes are likely involved in the uniparental inheritance of chloroplast DNA. The mating-type locus in C. reinhardtii is located

Chapter 6 Chloroplast DNA Inheritance on the left arm of linkage group VI (Ebersold et al., 1962) and in genetic crosses displays Mendelian and segregation of the two apparent alleles, (Smith and Regnery, 1950). In fact, however, the mating-type locus is a complex region of suppressed recombination that spans nearly 850 kb of DNA, almost 1% of the entire genome (Gillham, 1969; Ferris and Goodenough, 1994). Twelve genes, five required for life-cycle events and seven ‘house­ keeping’ genes, have to date been localized to the mating-type locus (Ferris and Goodenough, 1994); the physical locations of three housekeeping genes, Nic7, Thi10, and Ac29, have recently been described (Ferris, 1995). Our understanding ofthe mating-type control of life-cycle events has advanced rapidly in the past few years due primarily to the work of Ferris and Goodenough. In 1994, they published the results of a one megabase chromosome walk through the entire region of suppressed recombination (Ferris and Goodenough, 1994). They found that this region and cells except for a is homologous in central domain of about 190 kb, designated the R domain, in which chromosomal rearrangements have occurred and segments unique to one or the other mating type are located (Fig. 3). Genes required for mating type-controlled life-cycle events are hypothesized to reside within these unique segments. Presumably, the extensive rearrangements that have occurred within the R domain explain the lack of recombination around mating-type, an arrangement that may ensure that genes required for sexual differentiation are inherited as a unit (Ferris and Goodenough, 1994).

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A. Gamete Differentiation and Fusion The recent cloning oftwo mating type-specific genes, Fus1 and Mid, provides insight into the regulation of sexual differentiation and gamete fusion. Surprisingly, the differentiation circuitry appears to be even simpler than imagined originally (Goodenough and Ferris, 1987). Apparently, only the two sex-linked genes, Fus 1 and Mid, are necessary to determine whether a or a gamete, although a cell will mate as a third gene defined by the iso1 mutation and unlinked to mating type (Campbell et al., 1995), may be required for proper Mid function (Goodenough et al., 1995). The Fus 1 gene is found exclusively within locus (Fig. 3) and is the R domain at the hypothesized to encode a structural protein localized to the plus mating structure and necessary for gamete fusion (Ferris et al., 1996). If a cell possesses a defective Fus 1 gene, as occurs in imp1 strains, the gamete will recognize and agglutinate with imp1 a partner, but will fail to undergo cell fusion (Goodenough et al., 1976; Goodenough et al., 1982; Ferris et al., 1996). The Mid gene is found exclusively locus (Fig. 3) and is in the R domain at the hypothesized to encode a transcription factor that both turns on minus sexual differentiation and turns off plus sexual differentiation (Galloway and Goodenough, 1985; Ferris and Goodenough, 1997). cell possesses a defective Mid gene, as in the If a differentiation will not be turned imp 11 mutant, on and the cell will instead differentiate into a plus gamete. This ‘pseudo-plus’ gamete can do everything that a true gamete can do except it cannot fuse partner (Goodenough et al., 1982; Ferris with a

100 and Goodenough, 1997). It is now clear that the reason the imp11 cell can not fuse is that it lacks the necessary Fus1 gene found at the locus (Ferris et al., 1996). Remarkably, if the wild-type Fus1 gene is transformed into an imp 11 strain, this locus will differentiate into a func­ cell with a gamete to tional plus gamete that can fuse with a form viable ‘minus/minus’ zygotes (Fus1 imp11 able to undergo meiosis (Ferris et al., 1996). cell is transformed with a wildReciprocally, if a type Mid gene, this cell will differentiate into a fully functional minus gamete that can mate with a gamete to create ‘plus/plus’ zygotes (Mid that are also able to undergo meiosis (Ferris and Goodenough, 1997). As will be discussed in more detail later, this ability to change the mating type of any given cell and create sexual zygotes homozygous for mating type has revitalized our ability to examine the function of genes located exclusively within one or the other mating-type.

B. Zygote Development Less is know about the control of zygote-specific gene expression in Chlamydomonas. Fifteen genes that are expressed specifically during zygote differentiation have now been identified. Presumably, a cascade of expression is initiated by zygote formation since nine of the genes are transcribed almost immediately upon gamete fusion, three are transcribed about 1–3 h later (Ferris and Goodenough, 1987; Woessner and Goodenough, 1989; Matters and Goodenough, 1992; Uchida et al., 1993), and three are transcribed during late zygote maturation (Wegener and Beck, 1991). The almost immediate transcription of the early zygote genes occurs long before the two nuclei fuse and is independent ofnew protein synthesis (Ferris and Goodenough, 1987). Originally, it was hypothesized that zygote-specific gene expression was dependent upon an interaction in the zygote of mating type-specific regulatory gamete-specific, and M, proteins known as P, for for gamete-specific (Goodenough and Ferris, 1987). This hypothesis must be now modified to explain the zygote-specific expression that occurs in minus/minus zygotes. A P protein encoded exclu­ sively at the locus cannot exist in the minus/ locus. minus zygotes since they do not posses a parent in this cross Since the imp11 Fus1 differentiates as a plus gamete, however, the data are consistent with the existence ofa sex-limited gene P

E. Virginia Armbrust which is turned on during plus gamete differentiation (Goodenough et al., 1995).

C. Chloroplast DNA Inheritance The simplest models for the mating-type control of uniparental inheritance hypothesize that nuclear genes locus are required for located exclusively at the both the protection of plus chloroplast DNA and the degradation of minus chloroplast DNA (Gillham et al., 1991; Gillham, 1994). Until quite recently, all the data supported this simple hypothesis. As will be described in detail in the following sections, it now appears that genes located outside of the matingand the loci type locus as well as at both the are involved in controlling the uniparental inheritance of chloroplast DNA. Two points are clear, however, from the early studies. First, all the genes required for the selective elimination of minus chloroplast DNA are nucleusencoded; inhibitors of either the transcription of nuclear genes or the translation of nuclear gene products prevent destruction while comparable chloroplast-specific inhibitors have no affect (Kuroiwa et al., 1983a,b). Second, the elimination of minus chloroplast DNA occurs only in true zygotes, not in diploids generated by other means. For example, a low percentage (~5%) of sexually generated diploid cells fail to differentiate into zygotes and rather than undergo meiosis, they instead divide mitotically as stable vegetative diploids (Ebersold, 1967). Most (50–90%) of these vegetative diploids initially possess chloroplast DNA from both parents (Gillham, 1963). As the diploids divide mitotically, the two parental chloroplast genomes segregate to produce progeny that are eventually homoplasmic for one chloroplast genome or the other (Gillham, 1963; reviewed in Sears and VanWinkle-Swift, 1994). Vegetative diploids can also be generated artificially by incubating cells in the presence of polyethylene glycol (PEG) (Matagne et al., 1979). In these vegetative diploids, about one third of the fusion products are initially biparental, one third possess the chloroplast genome from one parent and one third possess the chloroplast genome from the other parent regardless cells, two of whether the fusion is between two and cell (Matagne and cells or a Hermesse, 1980; Matagne, 1981). Matagne (1980) suggests that the reduced frequency of biparental inheritance observed in the PEG fused diploids may occur because the first mitotic division is delayed in

Chapter 6

Chloroplast DNA Inheritance

these cells. Van Winkle- Swift (1978) had shown earlier that a delay in the timing of this first division somehow decreases the subsequent number of diploids that are initially biparental. Thus, a segregation of chloroplast alleles is observed in both types of vegetative diploids, but the directed elimination of the minus chloroplast genome occurs only in sexual zygotes.

IV. Protection of Plus Chloroplast DNA The degradation of minus chloroplast DNA in early zygotes is due presumably, at least in part, to the action of a nuclease (Kuroiwa et al., 1983a). The two most obvious ways that minus chloroplast DNA alone could be eliminated from the zygote are to specifically ‘mark’ the minus chloroplast DNA for elimination or else to specifically protect the plus chloroplast DNA from elimination. As will be shown, the most straightforward interpretation of the evidence is that the plus chloroplast DNA is specifically protected prior to zygote formation. But what exactly does it mean to be protected? It is still not known whether protection entails a covalent modification of plus chloroplast DNA that somehow protects the DNA from degradation (e.g., Sager and Ramanis, 1973; Sager and Kitchin, 1975) or whether protection instead entails the presence ofprotector proteins that somehow prevent the nuclease from gaining access to plus chloroplast DNA (e.g., Gillham et al., 1974; Kuroiwa, 1991). Regardless ofthe exact mechanisms underlying protection, the process must be reversible. Under conditions of strict uniparental inheritance, all the chloroplast DNA transmitted to meiotic progeny was once protected from elimination. At some point during the life cycle this protection must be erased so that the chloroplast DNA in the progeny will once again be susceptible to degradation during the next round of zygote formation.

A. Evidence for a

Specific Gene Required

for Protection

1. Diploid Crosses The earliest evidence that a specific gene is involved in protection came from crosses with a diploid gamete heterozygous for mating type. When heterozygous vegetative diploid is nitrogena gamete starved, the resulting gamete mates as a and thus is phenotypically minus (Ebersold, 1967).

101 In other words, is dominant to for sexual differentiation, a phenomenon known as minus dominance. As described in an earlier section, minus dominance is conferred by the Mid gene (Ferris and Goodenough, 1997). Over thirty years ago, Gillham (1963) showed that when a heterozygous gamete, the diploid is crossed to a haploid chloroplast genomes from both parents are bipar­ entally transmitted to meiotic progeny. Gillham must therefore be (1963) hypothesized that dominant to for uniparental inheritance. Matagne and Mathieu (1983) extended this analysis by using the PEG-fusion technique to create diploid gametes that were either homozygous or heterozygous for mating type. Uniparental inheritance of the plus chloroplast genome was observed in all crosses in which the minus parent was either haploid or homozygous for mating type: and In contrast, biparental inheritance was observed in all crosses in which the minus parent was heterozygous for mating type: In other words, if a phenotypically minus parent carries the locus, chloroplast genomes from both parents will persist in the zygote (Tsubo and Matsuda, 1984) and will be transmitted to the meiotic progeny. These data suggest locus carries a gene required for protection that the that is not repressed by Mid. Moreover, these results imply that protection occurs prior to zygote formation since the triploid zygotes resulting from the crosses and have identical genotypes but very different chloroplast transmission patterns (Matagne and Mathieu, 1983).

2. The mtl1 Mutation of C. monoica Thus far, the focus of this chapter has been on the transmission of chloroplast DNA by C. reinhardtii. In recent years, important information about chloroplast DNA inheritance has come from work with C. monoica conducted by Van Winkle-Swift and colleagues. Unlike C. reinhardtii, C. monoica is and cells homothallic which means that both are produced in a clonal population. Thus C. monoica can ‘self-mate’ (see for example, van den Ende and VanWinkle-Swift, 1994). A breakthrough in the genetic manipulation of C. monoica came when Van Winkle-Swift and Bauer (1982) and Van WinkleSwift and Burrascano (1983) generated self-sterile and zygote maturation-defective mutants. Although self-sterile, these strains can be outcrossed and thus

102 have greatly facilitated the analysis of interclonal crosses. It should be kept in mind, however, that even when an outcross is performed with C. monoica, the and gametes two parent strains form both and thus each mating includes reciprocal crosses. Once self-sterile mutants were available, an analysis of chloroplast genetics became feasible. When a C. monoica strain carrying a chloroplast mutation that confers resistance to erythromycin, ery-u-1, is outcrossed to an erythromycin-sensitive strain, two types of tetrads are obtained: those in which all four meiotic progeny are resistant to erythromycin and those in which all four meiotic progeny are sensitive to erythromycin (VanWinkle-Swift and Aubert, 1983). This version of chloroplast gene transmission has been termed bidirectional uniparental inheritance and is hypothesized to result from the fact that half gametes and half the the ery-u-1 cells mate as gametes. When the ery-u-1 ery-u-1 cells mate as parent, the resistancemutation is carried by the conferring mutation is transmitted to all the meiotic progeny, but when the ery-u-1 mutation is carried by parent, the chloroplast DNA from the sensitive the strain is transmitted to all the meiotic progeny (VanWinkle-Swift and Aubert, 1983). To identify mating type-specific genes required for chloroplast genome transmission, VanWinkleSwift and Hahn (1986) screened for mutants that create a shift from a bidirectional to a unidirectional transmission of the ery-u-1 mutation. The mtl1 mutation (for mating type limited) created just such a phenotype. If an mtl1 strain resistant to erythromycin is outcrossed to an erythromycin-sensitive strain, only erythromycin-sensitive progeny are obtained, mtl1 ery-u-1 parent is no longer able to as if the transmit chloroplast DNA. Moreover, 50% of the zygotes resulting from this outcross do not germinate. If instead, an mtl1 mutant is allowed to self-fertilize, none of the resulting zygotes germinate (VanWinkleSwift and Hahn, 1986). A careful analysis of the behavior of this mutant in both self-matings and outcrosses indicated that the mtl1 phenotype is parent. The expressed only when carried by the zygote-lethal phenotype was traced to the fact that parent carries the mtl1 mutation, all the when the chloroplast DNA in the resulting zygote is eliminated, leading VanWinkle-Swift and Salinger (1988) to hypothesize that the mtl1 mutant is unable to protect plus chloroplast DNA. Importantly, this degradation of all chloroplast DNA occurs relatively synchronously and long before the two chloroplasts

E. Virginia Armbrust fuse, indicating that the responsible nuclease is simultaneously present in both chloroplasts. Thus, a distantly related cousin of C. reinhardtii also appears to utilize a gene controlled by the locus to somehow protect plus chloroplast DNA from degradation in the zygote. No mtl1-like mutations have yet been identified in C. reinhardtii.

B. Is Plus Chloroplast DNA Protected by Methylation? This simple question reflects one of the more controversial topics in the field of Chlamydomonas chloroplast DNA inheritance. This topic has been reviewedrecently (Harris, 1989; Gillham et al., 1991; Kuroiwa, 1991; Gillham, 1994) and will only be summarized here. When Sager and Lane (1972) first specific methylation event postulated that a protected plus chloroplast DNA from destruction in the zygote, the restriction-modification system of bacteria had only recently been described. Restrictionmodification was an excitingnew means of ‘silencing’ DNA that might also explain chloroplast DNA inheritance in C. reinhardtii (e.g., Sager and Kitchin, 1975). Sager and colleagues rapidly accumulated convincing evidence that the chloroplast DNA of gametes contains a high percentage of 5-methyl gametes (Burton et al., 1979; cytosine relative to Royer and Sager, 1979; Sano et al., 1980), perhaps due to the action of a gamete-specific methyl transferase (Sano et al., 1 9 8 1 ) . Moreover, this methylation event was apparently reversible, as would be expected for protection (Sano et al., 1984). The problem was how to lest the effect of such a global event. Initially, Sager described a mutation mat1 that locus; the was believed to be tightly linked to the chloroplast DNA of the mat1 strain was methylated and the presence of the mutation resulted in a biparental transmission of chloroplast genomes (Sager and Ramanis, 1974; Sager et al., 1981). This ‘methylation mutant’ was later shown to be a diploid cell that had been generated inadvertently during a genetic cross (Sager and Grabowy, 1985). These data nonetheless suggest that in diploids, the chloroplast DNA is methylated and protected. A series of papers from Gillham and colleagues subsequently argued that methylation of plus chloroplast DNA could not explain protection adequately. Bolen et al. (1982) isolated a nuclear mutant me1 that constitutively methylales chloroplast and vegetative DNA to high levels in both

Chapter 6

Chloroplast DNA Inheritance

c e l l s . When e i t h e r or gametes w i t h hypermethylated chloroplast DNA were used for crosses, normal uniparental inheritance patterns were observed, prompting Bolen et al. (1982) to hypothesize that the methylation of chloroplast DNA described by Sager and colleagues could not adequately explain protection. Sager and Grabowy ( 1 9 8 3 ) countered this conclusion w i t h t h e i r observation that additional methylation occurred , but not cells during gametogenesis of carrying the me1 mutation. One year later, Feng and Chiang (1984) presented evidence that treatment of cells with the methylation inhibitors, L-Ethionine and 5-azacytidine, resulted in a hypomethylation of gamete chloroplast DNA. The hypomethylation of chloroplast DNA from either parent had no effect on the subsequent uniparental inheritance of plus chloroplast DNA, prompting Feng and Chiang (1984) to also conclude that protection was not conferred by methylation. In recent years, only one other paper has addressed the possible connection between methylation and protection. Kuroiwa and colleagues (reviewed in Kuroiwa, 1991) have used fluorescent microscopy to document that the elimination of minus nucleoids occurs rapidly and apparently in the absence of any nucleoid swelling, regardless of nucleoid size (Ikehara et al., 1996). Kuroiwa (1991) argues that this absence of swelling indicates that a nuclease similar to DNase I that can rapidly degrade DNA, rather than a restriction enzyme, eliminates the minus chloroplast DNA from early zygotes. The evidence accumulated over the years, then, argues against a global methylation event as a means of conferring protection to plus chloroplast DNA. However, it should be kept in mind that none of these studies eliminates the possibility that protection is conferred by a specific methylation event unaffected by either the me1 mutation or the methylation inhibitors.

V. Zygote Specific Elimination of Minus Chloroplast DNA The simplest explanation for the selective elimination of minus chloroplast DNA is that a nuclease is either activated (Matagne, 1987; Kuroiwa, 1991) or else expressed specifically during early zygote develop­ ment and subsequently degrades unprotected minus chloroplast DNA (Gillham et al., 1991; Gillham, 1994). Over the years, a number of endo- and exo­ nucleases, have been isolated from whole-cell extracts

103 of C. reinhardtii vegetative cells (Burton et al., 1977; Tait and Harris, 1977a,b; Frost and Small, 1984). Presumably, however, these enzymes play a role in DNA repair since each was isolated from vegetative cells (Small, 1987). In the mid 1980s, Kuroiwa and colleagues set out to identify nucleases specifically required for the uniparental inheritance of chloroplast DNA. Rather than use whole-cell extracts from vegetative cells as had been done before, Ogawa and Kuroiwa (1985b) began with 3 h zygotes. They -dependent nucleases that identified a class of displayed both endo- and exo-nucleolytic activity. The extracts were composed ofsix small polypeptides, and this preparation was collectively referred to as nuclease C (Ogawa and Kuroiwa, 1985a,b, 1986a,b, 1987). It had previously been shown (reviewed in Kuroiwa, 1985) that incubation of zygotes with the calcium chelator, EGTA, inhibited the specific elimination of minus chloroplast DNA. Thus, Ogawa and Kuroiwa (1985b) were hopeful initially that they had identified the critical factor. Unfortunately, nuclease C activity was found to be present throughout and the entire life cycle — in zygotes, vegetative cells, and and gametes. This finding prompted Kurowia (1991) to conclude that nuclease C is not specific to chloroplast DNA inheritance. Thus, the identification of a nucleus-encoded enzyme with a detectable nuclease activity that is directed to the chloroplast during early zygote formation still remains elusive. There are, however, two general ways to prevent zygote-specific elimination of minus chloroplast DNA in the gamete prior to zygote —UV irradiation of the mating and a reduction of the chloroplast DNA gamete. As more data are content of the accumulated through experiments involving the use of these two ‘inhibitors’, it appears increasingly likely that multiple gene products, and not just a single nuclease, are involved in the specific elimination of minus chloroplast DNA during early zygote development.

A. Dependence on a UV Sensitive, Specific Factor 1. Brief UV Irradiation of but not Gametes Enhances Biparental Inheritance Sager and Ramanis (1967) first reported and others have since verified (e.g., Gillham, 1969; Nakamura et al., 1988; Uchida et al., 1992a; Armbrust et al.,

104

1993) that brief UV-irradiation of gametes just prior to mating prevents the selective elimination of minus chloroplast DNA in the zygote and thus enhances the frequency of biparental inheritance. gametes prior to mating Comparable treatment of has no effect on the elimination of minus chloroplast DNA in the zygotes; as in wild-type crosses, the resulting meiotic progeny inherit chloroplast DNA parent only (Fig. 4). Furthermore, the from the number of zygotes that transmit minus chloroplast DNA to meiotic progeny is dependent on UV dose; gamete is exposed to UV, the the longer the greater the number of exceptional zygotes that are recovered. The UV effect is also photoreactivable; if gametes are allowed to recover in the irradiated

E. Virginia Armbrust

light prior to mating, the frequency of exceptional zygotes decreases, suggesting that at least one target of UV irradiation is DNA. Sager and Ramanis (1967) hypothesized that UV-irradiation blocked the specific gene product that is synthesis of a necessary for the elimination of minus chloroplast DNA in the zygote. Some twenty years later, Nakamura et al. (1988) presented evidence that the expression of at least six zygote polypeptides is inhibited when the , but not the parent is exposed to brief UV-irradiation just prior to mating. While it was not shown whether any of these UVsensitive gene products are directly involved in minus chloroplast DNA elimination, this work was the first gametes can clear evidence that UV treatment of

Chapter 6 Chloroplast DNA Inheritance inhibit specifically the expression of a subset of genes in the zygote.

2. The Ezy1 Gene The Ezy1 gene (for early zygote) was found in a differential screen designed to identify zygotespecific messages (Ferris and Goodenough, 1987). Ezy1 is tandemly repeated 7–8 times at both the loci (Ferris and Goodenough, 1987), and and the at least three copies of the gene are transcribed within minutes of zygote formation (Armbrust et al., 1993). The negatively charged Ezy1 protein localizes specifically to chloroplast nucleoids during the interval of minus chloroplast DNA degradation. Both uniparental inheritance of chloroplast DNA and transcription of Ezy1 are differentially inhibited by brief UV irradiation of gametes just prior to gametes prior mating; comparable treatment of to mating has no effect on either Ezy1 transcription or inheritance patterns. Importantly, transcription of a zygote-specific wall gene (Woessner and Goode­ nough, 1989) does not display differential sensitivity gametes. Thus, when Ezy1 to UV irradiation of is not expressed, minus chloroplast DNA is not destroyed. The Ezy1 gene has therefore been hypothesized to be involved in the selective elimination of minus chloroplast DNA (Armbrust et al., 1993). Detailed biochemical work with Ezy1 is just beginning so the role played by this protein is not yet known. gamete How could UV irradiation of the possibly affect transcription in the zygote? A common consequence of UV-irradiation is the generation of pyrimidine dimers in DNA; the presence ofunrepaired dimers can completely inhibit expression of a transciptional unit (reviewed in Britt, 1996). One possible explanation, discussed in Armbrust et al. (1993), is that UV exposure damages the irradiated nuclear DNA which in turn prevents subsequent transcription of Ezy1 in the zygote. This explanation copies of Ezy1 are never assumes that the transcribed. Preliminary data now indicates, however, and copies of Ezy1 are expressed that both the (Armbrust, unpublished). The unirradiated copies of Ezy1 should, therefore, be unharmed and available for transcription in the zygote. This result suggests that UV irradiation may affect non-DNA targets that are also necessary for Ezy1 transcription. Polypeptide 4, originally identified by Nakamura et al. (1988) as a zygote polypeptide whose expression is sensitive

105 parent, likely corresponds to UV irradiation of the to Ezy1. Thus, two different groups have indepen­ dently accumulated data that suggest that transcription of Ezy1 and perhaps additional genes may be specific UV-sensitive factor, a dependent on a dependence that may ensure that destruction ofminus chloroplast DNA is prevented when the plus chloroplast DNA is potentially UV-damaged. If this hypothesis is true, then it should be possible specific mutants that are unable to to isolate prevent transcription of zygote-specific genes when UV-irradiated prior to mating. The zygotes resulting mutant from the mating of such a UV-irradiated parent would be predicted to and a wild-type eliminate minus chloroplast DNA and thus would transmit the potentially damaged plus chloroplast DNA to meiotic progeny. In 1974, Sager and Ramanis described the isolation of a mutant with just such a phenotype. The mat2 mutation, which unfortunately has since been lost, was reportedly linked to the locus. When a parent carrying the mat2 mutation was exposed to relatively high levels ofUV irradiation prior to mating, the plus chloroplast genome was still inherited uniparentally (Sager and Ramanis, 1974), implying that destruction of the minus chloroplast genome had not been prevented in the mat2 zygote. cells and wild-type cells Interestingly, mat2 displayed identical UV survival curves, suggesting that the Mat2 gene was not required for DNA repair. Thus, the mat2 mutation may have identified a gene necessary for inactivating transcription of a subset of genes in the zygote when the plus parent is UVirradiated and its chloroplast DNA potentially damaged.

3. The uvsE1 Mutation The uvsE1 mutant was isolated by Rosen and Ebersold (1972) as a strain that is particularly sensitive to UV irradiation, perhaps due to a defect in either excision or recombination-mediated repair. The uvsE1 mutation is unlinked to mating type. Zygotes homozygous for uvsE1 display a reduced frequency of recombination of nuclear genes during meiosis (Rosen and Ebersold, 1972). Rosen et al. (1991) later examined whether the uvsE1 mutation also decreased the frequency of recombination of chloroplast genes as might be expected for a recombination defective mutant. Chloroplast DNA recombination was unaffected by uvsE1, but Rosen et al. (1991) found and the unexpectedly that when both the

E. Virginia Armbrust

106 parent carried uvsE1, as many as 20% of the zygotes displayed biparental inheritance, a dramatic increase from the negligible levels displayed by control crosses. It is provocative that a mutation in a gene that leads to UV-sensitivity, presumably due to a defect in an enzyme required for excision repair of nuclear genes, gamete prior to mating and UV irradiation of the both result in an increased number of exceptional zygotes. The connection between these two phenomena remains unclear, though. Obviously, however, the simple model of a single nuclease acting alone to accomplish the selective elimination of minus chloroplast DNA needs to be modified.

4. Involvement of RecA? In E. coli, the RecA protein is essential for homologous recombination and for a variety of responses to DNA damage. It now appears that a RecA-mediated recombination process in C. reinhardtii chloroplasts may be involved in the repair of chloroplast DNA when damaged by photooxidation, UV irradiation, or other environmental stresses (Cerutti et al., 1995). If a dominant negative mutant form of the E. coli RecA protein is expressed in C. reinhardtii chloroplasts, the transformed cells display reduced survival rates when exposed to a number of DNA damaging agents, and reduced repair and recombination of chloroplast DNA. The most likely explanation for this result is that Chlamydomonas chloroplasts contain a RecA homolog whose activity is inhibited by the dominant negative mutant form of the E. coli RecA protein (Cerutti et al., 1995). Thirty years ago, Sager and Ramanis (1967) hypothesized and Gillham et al. (1987) reiterated gametes prior to that the UV irradiation of mating might activate a RecA-mediated response that included inactivation of a factor necessary for the specific elimination of minus chloroplast DNA in the zygote. Whether this inactivation occurred in the chloroplast or the nucleus was not addressed. However, it should now be possible to determine if the RecA-like protein present in the chloroplast is involved in the uniparental inheritance of chloroplast DNA.

B. Dependence on the Chloroplast DNA Content of the Parent 1. Treatment with an Inhibitor of Chloroplast DNA Replication, 5-fluorodeoxyuridine

The chloroplast genome is present in about 80–90 copies per vegetative cell and accounts for approximately 10–15% of the total DNA (Gillham, 1978). During gametogenesis, the amount of chloroplast DNA per cell decreases to about half that of vegetative cells (Chiang and Sueoka, 1967). If vegetative cells are grown in the presence of 5fluorodeoxyuridine (FdUr), a specific inhibitor of chloroplast DNA replication, the amount of chloroplast DNA in the resulting gametes is reduced dramatically, perhaps as low as about 1% of the total DNA (Wurtz et al., 1977). When FdUr-treated gametes with a reduced chloroplast DNA content are gametes, the number of zygotes mated to untreated parent that transmit chloroplast DNA from the increases dramatically, suggesting that the specific elimination of minus chloroplast DNA has somehow been disrupted (Wurtz et al., 1977). In a manner reminiscent of UV-irradiation, comparable FdUrgametes has no effect on the treatment of subsequent uniparental inheritance of the plus chloroplast genome (Wurtz et al., 1977). Matagne and Beckers (1983) extended the FdUr treatments to include the generation of both diploid and triploid zygotes from parents with reduced chloroplast DNA contents. They too concluded that the relative amount gametes of chloroplast DNA contributed by the influences the resulting inheritance patterns. These early studies did not address how a reduction of plus chloroplast DNA could lead to an increased number of exceptional zygotes. Was the selective elimination of minus chloroplast DNA turned off in these zygotes or was the elimination process simply inefficient, with a decrease in the input of intact plus chloroplast genomes increasing the likelihood that a minus chloroplast DNA molecule would ‘slip through’ to be replicated prior to meiosis? Wurtz et al. (1977) found no increase in zygote lethality as might be expected if minus chloroplast DNA was mostly eliminated in a zygote with very little plus chloroplast DNA.

2. The mat3 Mutation New insight into how the chloroplast DNA content of the parent influences the zygote-specific elimination of minus chloroplast DNA has recently been obtained from an unexpected source. The mat3 mutation was isolated in a screen designed to identify mt+ specific genes required for the uniparental inheritance of the chloroplast genome (Gillham et

Chapter 6 Chloroplast DNA Inheritance al., 1987). The mat3 mutation is tightly linked to the locus and when present in the parent, prevents the elimination of minus chloroplast DNA in the zygote (Gillham et al., 1987; Munaut et al., 1990; Rosen et al., 1991). Thus, meiotic progeny inherit chloroplast genomes from both parents. Interestingly, the presence of mat3 has no effect on the inheritance of mitochondrial DNA (Gillham et al., 1987). It therefore seemed likely that wild-type Mat3 was specific and encoded either the zygote-specific nuclease itself or else a regulator of the nuclease required for minus chloroplast DNA elimination (Gillham et al., 1987). Recent work suggests, however, that Mat3 may play a more indirect role in chloroplast genome transmission (Armbrust et al., 1995). A recently described phenotype associated with the vegetative mat3 mutation is the generation of cells and gametes that are remarkably small (Fig. 5) and contain very little chloroplast DNA, comparable to those levels obtained with Fdur-treatment. The cell-size defect displayed by mat3 cells does not appear to be due to their reduced chloroplast DNA content. For example, a comparable reduction in the amount of chloroplast DNA by treatment with FdUr has no effect on cell size (Hosler et al., 1989; Armbrust et al., 1995). The primary defect of the mat3 mutation instead appears to be a disruption of cell-size control. An understanding of the relation between cell-size control and chloroplast DNA content awaits the cloning of Mat3. Similar to FdUr-treatment, the mat3 mutation is hypothesized to prevent the degradation of minus chloroplast DNA in the zygote as a secondary consequence of the reduced amount of chloroplast parent DNA contributed to the zygote by the (Armbrust et al., 1995). This suggests that Chlamy­ domonas may be able to count intact chloroplast genomes and inhibit minus chloroplast DNA elimination when the amount of chloroplast DNA parent is below a threshold contributed by the level. The fact that C. monoica destroys all chloroplast DNA in the zygote when plus chloroplast DNA is not protected (VanWinkle-Swift and Salinger, 1988) suggests that although zygotes can detect the amount parent, of chloroplast DNA contributed by the they may not be able to determine whether this DNA is protected. Moreover, since the biparental inheritance phenotype associated with mat3 appears to be variable (Gillham et al., 1987; Armbrust et al., 1995), it may be that the inhibition of destruction is also variable; the less chloroplast DNA contributed

107

by the parent, the more completely destruction is prevented in the zygote. Interestingly, when a mat3 zygote is also homozygous for uvsE1, an even higher level of biparental inheritance is observed (Rosen et al., 1991). How might the elimination of minus chloroplast DNA be prevented in zygotes with a reduced amount of plus chloroplast DNA? Ezy1 is expressed at wildtype levels in zygotes resulting from the mating of a and a wild-type parent (Armbrust et al., mat3 1995). Furthermore, the six UV-sensitive poly­ peptides identified by Nakamura et al. (1988) are expressed in zygotes resulting from a mating between gamete and an untreated an FdUr-treated gamete (Nakamura and Kuroiwa, 1989). Nonetheless, in both instances the selective destruction of minus chloroplast DNA is inhibited. These observations suggest that chloroplast DNA inheritance may be regulated by at least two different means: one that prevents the genes required for destruction from being transcribed, as occurs in UV irradiated cells, and one that somehow inactivates the already synthesized proteins involved in destruction, as may occur in strains that have either been treated with FdUr or else carry the mat3 mutation.

C. The Ezy2 Gene As described in an earlier section, the ability to and homozygous generate homozygous

E. Virginia Armbrust

108 sexual zygotes that are able to undergo zygote differentiation and meiosis now allows a direct analysis of the involvement of mating type-specific genes in chloroplast DNA inheritance. For example, despite the fact that ‘minus/minus’ zygotes are locus, their most missing all genes specific to the obvious phenotype is that chloroplast DNA from both parents is transmitted to meiotic progeny (Ferris et al., 1996). The simplest explanation for this biparental inheritance phenotype is that a gene specific locus, and thus absent from these zygotes, to the is required for minus chloroplast DNA elimination in the zygote. An obvious structural difference between the two mating-type loci is a 16 kb segment of DNA repeated locus and present at the 6–8 times at the locus as a single repeat, split in two (Fig. 3). A single zygote-specific gene of about 6 kb, now referred to as Ezy2 has been found within the repeat unit. The mt copy of Ezy2 appears to be a pseudogene as Ezy2 message is transcribed from the locus only within minutes of zygote formation. Preliminary sequence data suggests that the Ezy2 polypeptide has a basic pI and possesses a chloroplast transit peptide (Armbrust and Ferris, unpublished). The role of Ezy2 is still unknown, but it is intriguing that the gene product of a second, multicopy, zygote-specific locus may also be targeted gene located within the to the chloroplast during early zygote development.

a loss-of-function. VanWinkle-Swift et al. (1994) have hypothesized that the wild-type Sup1 gene encodes a nuclease expressed in the zygote from both parental genomes that is able to degrade unprotected chloroplast DNA. Thus, when the zygotespecific nuclease that would normally eliminate minus chloroplast DNA is disabled by the sup1 mutation, the presence or absence of protection of plus chloroplast DNA has no affect on subsequent inheritance patterns. These very simple hypotheses of a specific protector gene and a zygote-specific nuclease fit the original model presented for the uniparental inheritance of chloroplast DNA in C. reinhardtii. Why does uniparental inheritance in C. reinhardtii seem to be so complicated with multiple loci potentially involved, but so simple in C. monoica? Perhaps the apparent differences in the control of chloroplast genome inheritance simply reflect the different evolutionary histories of the two species (VanWinkle-Swift et al., 1994). C. monoica is in the same lineage as C. eugametos, and the C. eugametos lineage diverged from the C. reinhardtii lineage around 350 million years ago (Buchheim et al., 1990; Larson et al., 1992).

D. The sup1 Mutation

This chapter began with a simple model ofchloroplast DNA inheritance that entailed two distinct steps: protection of plus chloroplast DNA presumably due specific event occurring prior to zygote to a formation and destruction of unprotected minus chloroplast DNA during early zygote development. As more is learned about chloroplast DNA inheritance in C. reinhardtii, the number of steps and the number of genes potentially involved appears to keep increasing. I summarize here a slightly more complicated model for the regulation of chloroplast DNA inheritance in C. reinhardtii.

While the control of chloroplast DNA inheritance in C. reinhardtii seems to become more complicated with each newly collected piece of data, the control of uniparental inheritance in C. monoica appears to be remarkably simple. As described previously, VanWinkle-Swift and Salinger (1988) isolated the mtl1 mutant as a strain that is apparently unable to protect plus chloroplast DNA from elimination in the zygote, an inability that is lethal to the zygote. VanWinkle-Swift et al. (1994) have recently described the isolation of an extragenic nuclear mutation, sup1 (suppressor of uniparental inheritance) that sup­ presses this zygote lethality. The sup1 mutation appears to be recessive and unlinked to mtl1. When a zygote is homozygous for sup1, in the presence or absence of the mtl1 mutation, none of the chloroplast nucleoids are eliminated from the zygote and thus chloroplast genomes are transmitted biparentally. The recessive nature of sup1 suggests that it represents

VI. Regulation of Chloroplast DNA Inheritance

A. Regulation of Protection Although there is still nothing known about the molecular basis of protection, it has seemed clear since Gillham’s early studies (Gillham, 1963) that specific protection was due at least in part to a event that is not repressed by Mid. Furthermore, the evidence suggests that protection is reversible. A

Chapter 6

Chloroplast DNA Inheritance

simple way to accomplish this reversibility would be to initiate protection in response to the nitrogen starvation that accompanies gametogenesis. Pro­ tection could then be erased by dilution during the chloroplast DNA synthesis that occurs prior to meiosis and during vegetative growth. At this time, there is no direct way to test this hypothesis. The only potential protector mutation is in C. monoica. No comparable protector mutations exist in C. reinhardtii and there are no known treatments that prevent protection. This inability to disrupt protection has clearly hampered our ability to study its molecular basis. A more detailed understanding of protection must await the cloning of a protector gene.

B. Regulation of Destruction A number of potential components involved in destruction have now been identified. In C. monoica, the Sup1 gene is expressed in the zygote from both parental genomes. In C. reinhardtii, the UvsE1 gene is unlinked to mating type and is also expressed in the zygote from both genomes; the Ezy2 gene is present at the locus as a pseudo gene and at the locus as a functional gene and thus is expressed locus only; and the Ezy1 in the zygote from the and gene is both found at and expressed from the loci in the early zygote. Interestingly, Ezy1 is the and loci, while Ezy2 is multicopy at both the multicopy only at the locus; the Ezy2 pseudogene is present as a single copy. The evidence for the role played by any one of these polypeptides remains circumstantial. However, the activity of one or more enzymes able to completely destroy minus chloroplast DNA must obviously be tightly regulated. An unregulated nuclease that completely degrades minus chloroplast DNA even when the plus chloroplast DNA present in the zygote is damaged or otherwise deficient would likely be lethal. In fact, it appears that Chlamydomonas controls tightly those situations in which the elimination of minus chloroplast DNA is allowed to proceed: if the parent is either UV-irradiated or else possesses a reduced complement of chloroplast DNA, the zygotespecific degradation of minus chloroplast DNA is prevented. UV-irradiation of the parent prior to mating likely initiates a number of responses including the activation of DNA repair enzymes. The UvsE1 gene appears to encode an enzyme required perhaps for excision repair and recombination of the nuclear

109 genome, but is also somehow involved in minus chloroplast DNA elimination in the zygote (Rosen et al., 1991). It is not yet clear whether UvsE1 is actually localized to chloroplast DNA or whether the enzyme plays a more indirect role in uniparental inheritance. The chloroplast localized RecA-like protein also appears to be activated by UV irradiation (Cerutti et al., 1995), but the possible role of this protein in uniparental inheritance has not yet been examined directly. Chlamydomonas apparently also initiates additional responses to UV irradiation of gamete besides DNA repair, since the zygotethe specific expression of at least six polypeptides, including Ezy 1, is inhibited only when the parent is exposed to UV prior to mating (Nakamura et al., 1988; Armbrust et al., 1993). Chlamydomonas appears to respond to the potential damage of plus chloroplast DNA by UV by preventing the zygotespecific transcription of critical components that are necessary for minus chloroplast DNA elimination. Thus, meiotic progeny are assured of inheriting at least some undamaged chloroplast DNA originating parent. It is tempting to imagine that the from the mat2 mutation (Sager and Ramanis, 1974) identified a gene involved in the signaling pathway that leads to an inactivation of transcription in the zygote. The elimination of minus chloroplast DNA is also parent contains a reduced amount prevented if the of chloroplast DNA. However, this inhibition appears to occur after the presumed components of the destruction ‘machinery’ have already been synthe­ sized. Both Nakamura and Kuroiwa (1989) and Armbrust et al. (1995) found that the UV-sensitive zygote-specific polypeptides are expressed at wildtype levels when the elimination of the minus chloroplast DNA is inhibited due to a reduced parent. Since chloroplast DNA content of the minus chloroplast DNA elimination normally begins within the first hour of zygote development, these zygote-specific gene products must be quickly inactivated, perhaps due to a post-translational modification by pre-existing factors. It is intriguing to imagine that a system that may turn offdestruction once the elimination of minus chloroplast DNA is completed in wild-type zygotes, may also prevent destruction from ever beginning in zygotes whose parent either carries mat3 or else has been FdUr­ treated. If Chlamydomonas is truly able to measure the amount of chloroplast DNA contributed to the parent, then it should be possible to zygote by the identify mutants unable to monitor chloroplast DNA

E. Virginia Armbrust

110 content. These mutants would be predicted to destroy minus chloroplast DNA even when the amount of plus chloroplast DNA is below the threshold level, thereby creating a shift from biparental inheritance to either uniparental inheritance of plus chloroplast DNA or zygote lethality.

VI. Evolution of the Uniparental Inheritance of Organelle Genomes The uniparental inheritance of organelle genomes has evolved independently numerous times, and astonishingly diverse methods exist for accomplishing this feat. Yet the driving force for the evolution ofthis nearly universal phenomenon remains mysterious (reviewed in Birky, 1995). Some have hypothesized that the elimination of organelles or organelle genomes from one parent provides a means for preventing (or at least decreasing) the transmission of deleterious cytoplasmic factors (e.g. Coleman, 1982; Law and Huston, 1992). Others have hypothesized that the elaborate steps undertaken to accomplish uniparental inheritance represent a form of detente originally devised to prevent selfish organelle genomes from engaging in potentially lethal conflict for control of the cytoplasm (Cosmides and Tooby, 1981). This need to minimize ‘organelle warfare’ has even been suggested as a means for explaining why most eukaryotes possess only two sexes (Anderson, 1992; Hurst, 1992). Recently, Sears and VanWinkle-Swift (1994) have suggested that the selective elimination of minus chloroplast DNA in Chlamydomonas spp. may have evolved as a way of salvaging nucleotides from the multicopy organelle genome during the starvation that accompanies gametogenesis and zygote maturation. Birky (1995) suggests that, in fact, there may not be a single hypothesis that can adequately explain the myriad of patterns of organelle inheritance that have been described. But Birky also emphasizes the importance of identifying and evaluating the genes involved in uniparental inheritance, a process now well underway in Chlamydomonas.

Acknowledgments I thank Malcolm Campbell, Patrick Ferris, Ursula Goodenough, and Jim Umen for their many helpful comments on an earlier version of this chapter. I am particularly grateful to Ursula and Patrick for

introducing me to and helping me navigate my way through the wonderful world of the Chlamydomonas life cycle and its control by the mating-type locus.

References Anderson A (1992) The evolution of sexes. Science 257: 324– 326 Armbrust EV, Ferris PJ and Goodenough UW (1993) A mating type-linked gene cluster expressed in Chlamydomonas zygotes participates in the uniparental inheritance of the chloroplast genome. Cell 74: 801–811 Armbrust EV, Ibrahim A and Goodenough UW (1995) A mating type-linked mutation that disrupts the uniparental inheritance of the chloroplast genome also disrupts cell size control in Chlamydomonas. Mol Biol Cell 6: 1807–1818 Beck CF and Haring MA (1996) Gametic differentiation of Chlamydomonas. Int Rev Cytol 168: 259–302 Birky CW, Jr (1983) Relaxed cellular controls and organelle heredity. Science 222: 468–475 Birky CW, Jr (1995) Uniparental inheritance of mitochondrial and chloroplast genes: Mechanisms and evolution. Proc Natl Acad Sci USA 92: 1 1 3 3 1 – 1 1 3 3 8 Bolen PL, Grant DM, Swinton D, Boynton JE and Gillham NW (1982) Extensive methylation of chloroplast DNA by a nuclear gene mutation docs not affect chloroplast gene transmission in Chlamydomonas. Cell 28: 335–343 Boynton JE, Gillham NW, Newman SM and Harris EH (1991) Organelle genetics and transformation of Chlamydomonas. In: Herrmann RG (eds) Advances in Plant Gene Research: Cell Organelles, Vol 6, pp 3–64. Springer-Verlag, New York Braten T ( 1 9 7 1 ) The ultrastructure of fertilization and zygote formation in the green alga Ulva mutabilis. J Cell Sci 9: 621– 635 Braten T (1973) Autoradiographic evidence for the rapid disintegration of one chloroplast in the zygote of the green alga Ulva mutabilis. J Cell Sci 12: 385–389 Britt AB (1996) DNA damage and repair in plants. Ann Rev Plant Physiol Plant Mol Biol 47: 75–100 Buchheim MA, Tunnel M.Zimmer EA and Chapman RL(1990) Phylogeny of Chlamydomonas (Chlorophyta) based on cladistic analysis of nuclear 18S rRNA sequence data. J Phycol 26: 689–699 Burton WG, Roberts RJ, Myers PA and Sager R (1977) A sitespecific single-strand endonuclease from the eukaryote Chlamydomonas. Proc Natl Acad Sci USA 7: 2687–2691 Burton WG, Grabowy CT and Sager R (1979) Role ofmethylation in the modification and restriction of chloroplast DNA in Chlamydomonas. Proc Nat1 Acad Sci USA 76: 1390–1394 Campbell AM, Rayala HJ and Goodenough UW (1995) The isol gene of Chlamydomonas is involved in sex determination. Mol Biol Cell 6: 87–95 Cavalier-Smith T (1970) Electron microscopic evidence for chloroplast fusion in zygotes of Chlamydomonas reinhardi. Nature 228: 333–335 Cerutti H, Johnson AM, Boynton JE and Gillham NW (1995) Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA. Mol Cell Biol 15: 3003–3011

Chapter 6

Chloroplast DNA Inheritance

Chiang K-S and Sueoka N (1967) Replication of chloroplast DNA in Chlamydomonas reinhardi during vegetative cell cycle: Its mode and regulation. Proc Natl Acad Sci USA 1506– 1513: Chun EHL, Vaughan MH, Jr. and Rich A (1963) The isolation and characterization of DNA associated with chloroplast preparations. J Mol Biol 7: 130–141 Coleman AW (1982) Sex is dangerous in a world of potential symbionts or the basis of selection of uniparental inheritance. J Theor Biol 97: 367–369 Coleman AW (1984) The fate of chloroplast DNA during cell fusion, zygote maturation, and zygote germination in Chlamydomonas reinhardi as revealed by DAPI staining. Exp Cell Res 152:528–540 Coleman AW and Maguire MJ (1983) Cytological detection of the basis of uniparental inheritance of plastid DNA in Chlamydomonas moewusii. Curr Genet 7: 211–218 Cosmides LM and Tooby J (1981) Cytoplasmic inheritance and intragenomic conflict. J Theor Biol 89: 83–129 Dron M, Rahire M, Rochaix J-D and Mets L (1983) First DNA sequence of a chloroplast mutation: A missense alteration in the ribulosebisphosphate carboxylase large subunit gene. Plasmid 9: 321–324 Ebersold WT (1967) Chlamydomonas reinhardi: Heterozygous diploid strains. Science 157: 447–449 Ebersold WT, Levine RP, Levine EE and Olmsted MA (1962) Linkage maps in Chlamydomonas reinhardi. Genetics 47: 531–543 Feng T–Y and Chiang K–S (1984) The persistence of maternal inheritance in Chlamydomonas despite hypomethylation of chloroplast DNA induced by inhibitors. Proc Natl Acad Sci USA 81: 3438–3442 Ferris PJ (1995) Localization of the nic-7, ac-29 and thi-10 genes within the mating-type locus of Chlamydomonas reinhardtii. Genetics 141: 543–549 Ferris PJ and Goodenough UW (1987) Transcription of novel genes, including a gene linked to the mating-type locus, induced by Chlamydomonas fertilization. Mol Cell Biol 7: 2360–2366 Ferris PJ and Goodenough UW (1994) The mating type locus of Chlamydomonas reinhardtii contains highly rearranged DNA sequences. Cell 76: 1135–1145 Ferris PJ and Goodenough UW (1997) Mating type in Chlamydomonas is specified by mid, the minus-dominance gene. Genetics 146:859–870 Ferris PJ, Woessner JP and Goodenough UW (1996) A sex recognition glycoprotein is encoded by the plus mating-type gene fus 1 of Chlamydomonas reinhardtii. Mol Biol Cell 7: 1235–1248 Frost BF and Small GD (1984) Partial purification and c h a r a c t e r i z a t i o n of the major AP endonuclease from Chlamydomonas reinhardi. Biochim Biophys Acta 782:170– 176 Galloway RE and Goodenough UW (1985) Genetic analysis of mating locus linked mutations in Chlamydomonas reinhardtii. Genetics 111:447–461 Gillham NW (1963) Transmission and segregation of a non­ chromosomal factor controlling streptomycin resistance in diploid Chlamydomonas. Nature 200: 294 G i l l h a m NW (1965) Induction of chromosomal and non­ chromosomal mutations in Chlamydomonas reinhardi with Nmethyl-N´-nitro-N-nirosoguanidine. Genetics 52: 529–537 Gillham NW (1969) Uniparental inheritance in Chlamydomonas

111 reinhardi. Am Nat 103: 355–388 Gillham NW (1978) Organelle Heredity. Raven Press, New York Gillham NW (1994) Organelle Genes and Genomes. Oxford University Press, New York Gillham NW and Fifer W (1968) Recombination of nonchromo­ somal mutations: A three-point cross in the green alga Chlamydomonas reinhardi. Science 162: 683–684 Gillham NW, Boynton JE and Lee RW (1974) Segregation and recombination of non-Mendelian genes in Chlamydomonas. Genetics 78: 439–457 Gillham NW, Boynton JE, Johnson AM and Burkhart BD (1987) Mating type linked mutations which disrupt the uniparental transmission of chloroplast genes in Chlamydomonas. Genetics 115: 677–684 Gillham NW, Boynton JE and Harris EH (1991) Transmission of plastid genes. In: Bogorad L and Vasil IK (eds) Cell Culture and Somatic Cell Genetics, Vol 7A, pp 55–92. Academic Press, Inc., New York Goodenough UW and Ferris PJ (1987) Genetic regulation of development in Chlamydomonas. I n : Loomis W (eds) Genetic Regulation of Development, pp 171–189. Alan R. Liss, Inc., New York Goodenough UW, Hwang C and Martin H (1976) Isolation and genetic analysis ofmutant strains of Chlamydomonas reinhardi defective in gametic differentiation. Genetics 82: 169–186 Goodenough UW, Detmers PA and Hwang C (1982) Activation for cell fusion in Chlamydomonas: Analysis of wild-type gametes and nonfusing mutants. J Cell Biol 92: 378–386 Goodenough UW, Armbrust EV, Campbell AM and Ferris PJ (1995) Molecular genetics of sexuality in Chlamydomonas. Ann Rev Plant Phys Plant Mol Biol 46: 21–44 Grant DM, Gillham NW and Boynton JE (1980) Inheritance of chloroplast DNA in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 77: 6067–6071 Harris EH (1989) The Chlamydomonas Sourcebook. Academic Press, San Diego Harris EH, Burkhart BD, Gillham NW and Boynton JE (1989) Antibiotic resistance mutations in the chloroplast 16S and 23S rRNA genes of Chlamydomonas reinhardtii: correlation of genetic and physical maps of the chloroplast genome. Genetics 123:281–292 Hoeh WR, Sterwart DT, Sutherland BW and Zouros E (1996) Multiple origins of gender-associated mitochondrial DNA lineages in bivalves (Mollusca:Bivalvia). Evolution 50: 2276– 2286 Hosler JP, Wurtz EA, Harris EH, Gillham NW and Boynton JE (1989) Relationship between gene dosage and gene expression in the chloroplast of Chlamydomonas reinhardtii. Plant Physiol 91: 648–655 Hurst LD (1992) Intragenomic conflict as an evolutionary force. Proc R Soc Lond (B) 248: 135–140 Ikehara T, Uchida H, Suzuki L and Nakamura S (1996) Chloroplast nucleoids in large number and large DNA amount with regard to maternal inheritance in Chlamydomonas reinhardtii. Protoplasma 194: 1 1 – 1 7 Kuroiwa T (1985) Mechanisms of maternal inheritance of chloroplast DNA: An active digestion hypothesis. Microbiol Sci 2: 267–270 Kuroiwa T( 1991) The replication, differentiation, and inheritance of plastids with emphasis on the concept of organelle nuclei. Int Rev Cytol 128: 1–62

112 Kuroiwa T, Kawano S and Nishibayashi S (1982) Epifluorescent microscopic evidence for maternal inheritance of chloroplast DNA. Nature 298: 481–483 Kuroiwa T, Kawano S and Sato C (1983a) Mechanisms of maternal inheritance. I. Protein synthesis involved in preferential destruction of chloroplast DNA of male origin. Proc Jpn Acad (B) 59: 177–181 Kuroiwa T, Kawano S and Sato C (1983b) Mechanisms of maternal inheritance. II. RNA synthesis involved in preferential destruction of chloroplast DNA of male origin. Proc Jpn Acad ( B ) 59: 182–185 Larson A, Kirk MM and Kirk DL(1992) Molecular phylogeny of the Volvocine flagellates. Mol Biol Evol 9: 85–105 Law R and Huston V (1992) Intracellular symbionts and the evolution of uniparental cytoplasmic inheritance. Proc R Soc Lond B 248: 69–77 Lemieux C, Turmel M, Seligy VL and Lee RW (1984) Chloroplast DNA recombination in interspecific hybrids of Chlamy­ domonas: Linkage between a nonmendelian locus for streptomycin resistance and restricion fragments coding for 16S rRNA. Proc Natl Acad Sci USA 81: 1164–1168 L i u X–Q, Gillham NW and Boynton JE (1989) Chloroplast ribosomal protein gene rps12 of Chlamydomonas reinhardtii. J Biol Chem 264: 16100–16108 Matagne RF (1981) Transmission ofchloroplast alleles in somatic fuison products obtained from vegetative cells and/or ‘gametes’ of Chlamydomonas reinhardi. Curr Genet 3: 31–36 Matagne RF (1987) Chloroplast gene transmission in Chlamy­ domonas reinhardtii. A model for its control by the matingtype locus. Curr Genet 12: 251–256 Matagne RF and Beckers M-C (1983) Perturbation of chloroplast gene t r a n s m i s s i o n in d i p l o i d and t r i p l o i d zygotes of Chlamydomonas reinhardi by 5-fluorodeoxyuridine. Curr Genet 7: 335–338 Matagne RF and Hermesse M-P (1980) Chloroplast gene inheritance studied by somatic fusion in Chlamydomonas reinhardtii, Curr Genet 1: 127–131 Matagne RF and Mathieu D (1983) Transmission of chloroplast genes in triploid and tetraploid zygospores of Chlamydomonas reinhardtii: Roles of mating-type gene dosage and gametic chloroplast DNA content. Proc Natl Acad Sci USA 80: 4780– 4783 Matagne RF, Deltour R and Ledoux L (1979) Somatic fusion between cell wall mutants of Chlamydomonas reinhardi. Nature 278: 344–346 Matters GL and Goodenough UW (1992) A gene/pseudogene tandem duplication encodes a cysteine-rich protein expressed during zygote development in Chlamydomonas reinhardtii. Mol Gen Genet 232: 81–88 McBride AC and McBride JC (1975) Uniparental inheritance in Chlamydomonas eugametos (chlorophyceae). J Phycol 11: 343–344 Meland S, Johansen S, Johansen T, Haugli K and Haugli F (1991) Rapid disappearance of one mitochondrial genotype after isogamous mating in the myxomycete Physarum polycephalum. Curr Genet 19: 55–60 Mets LJ and Geist LJ (1983) Linkage of a known chloroplast gene mutation to the uniparental genome of Chlamydomonas reinhardii. Genetics 105: 559–579 M u n a u t C, Dombrowicz D and Matagne RF (1990) Detection of chloroplast DNA by using fluorescent monoclonal anti­ bromodeoxyuridine antibody and analysis of its fate during

E. Virginia Armbrust zygote formation in Chlamydomonas reinhardtii. Curr Genet 18: 259–263 Nakamura S and Kuroiwa T (1989) Selective elimination of chloroplast DNA by 5-fluorodeoxyuridine causing no effect on preferential digestion of male chloroplast nucleoids in Chlamydomonas. Eur J Cell Biol 48: 165–173 Nakamura S, Sato C and Kuroiwa T (1988) Polypeptides related to preferential digestion of male chloroplast nucleoids in Chlamydomonas. Plant Sci 56: 129–136 Neale DB, Marshall KA and Sederoff RR (1989) Chloroplast and mitochondrial DNA are paternally inherited in Sequoia sempervirens D. Don Endl. Proc Natl Acad Sci USA 86: 9347– 9349 Ogawa K and Kuroiwa T (1985a) Destruction of chloroplast nuclei of the male gamete by calcium and nuclease C in a cell model of Chlamydomonas reinhardtii. Plant Cell Physiol 26: 493–503 Ogawa K and Kuroiwa T (1985b) Nuclease C polymorphism of calcium-dependent nucleases in Chlamydomonas reinhardtii. Plant Cell Physiol 26: 481–491 Ogawa K and K u r o i w a T ( 1 9 8 6 a ) Base-specific endo­ exonucleolytic activity of Chlamydomonas nuclease C1 & 2. Plant Cell Physiol 27: 701–710 Ogawa K and Kuroiwa T (1986b) Purification of major isozymes of nuclease C and production of active fragments by trypsin. Plant Cell Physiol 26: 1473–1484 Ogawa K and Kuroiwa T (1987) Preferential resistance of phosphodiester bonds to deoxycytidine and 5´-adjacent bases to Chlamydomonas nuclease C. Plant Cell Physiol 28: 323– 332 Ris H and Plaut W (1962) Ultrastructurc of DNA-containing areas in the chloroplast of Chlamydomonas. J Cell Biol 13: 383–391 Rochaix J-D ( 1 9 9 5 ) Chlamydomonas reinhardtii as the photosynthetic yeast. Ann Rev Genet 29: 209–230 Rosen H and Ebersold WT (1972) Recombination in relation to ultraviolet sensitivity in Chlamydomonas reinhardi. Genetics 71: 247–253 Rosen H, Newman SM, Boynton JE and Gillham NW ( 1 9 9 1 ) A nuclear mutant of Chlamydomonas that exhibits increased sensitivity to UV irradiation, reduced recombination of nuclear genes, and altered transmission of chloroplast genes. Curr Genet 19: 35–41 Royer H-D and Sager R (1979) Methylation ofchloroplast DNAs in the life cycle of Chlamydomonas. Proc Natl Acad Sci USA 76: 5794–5798 Sager R (1954) Mendelian and non-Mendelian inheritance of streptomycin resistance in Chlamydomonas reinhardi. Proc Natl Acad Sci USA 40: 356–363 Sager R (1965) Mendelian and non-Mendelian heredity: A reappraisal. Proc Royal Soc Lond Ser B 164: 290–297 Sager R and Grabowy C (1983) Differential methylation of chloroplast DNA regulates maternal inheritance in a methylated mutant of Chlamydomonas. Proc Natl Acad Sci USA 80: 3025–3029 Sager R and Grabowy C (1985) Sex in Chlamydomonas: Sex and the single chloroplast. In: Halvorson HO and Monroy A (eds) The Origin and Evolution of Sex, pp 1 1 3 – 1 2 1 . Alan R. Liss, New York Sager R and Granick S (1954) Nutritional control of sexuality in Chlamydomonas reinhardi. J Gen Physiol 37: 729–742 Sager R and Ishida MR (1963) Chloroplast DNA in Chlamy­

Chapter 6 Chloroplast DNA Inheritance domonas. Proc Natl Acad Sci USA 50: 725–730 Sager R and Kitchin R (1975) Selective silencing of eukaryotic DNA. Science 189: 426–432 Sager R and Lane R (1972) Molecular basis of maternal inheritance. Proc Natl Acad Sci USA 69: 2410–2413 Sager R and Ramanis Z (1963) The particulate nature of nonchromosomal genes in Chlamydomonas. Proc Natl Acad Sci USA 50: 260–268 Sager R and Ramanis Z (1965) Recombination of nonchromo­ somal genes in Chlamydomonas. Proc Natl Acad Sci USA 53: 1053–1060 Sager R and Ramanis Z (1967) Biparental inheritance of nonchromosomal genes induced by ultraviolet irradiation. Proc Natl Acad Sci USA 58: 931–937 Sager R and Ramanis Z (1973) The mechanism of maternal inheritance in Chlamydomonas: Biochemical and genetic studies. Theoret Appl Genet 43: 101–108 Sager R and Ramanis Z (1974) Mutations that alter the transmission of chloroplast genes in Chlamydomonas. Proc Natl Acad Sci USA 71: 4698–4702 Sager R, Grabowy C and Sano H (1981) The mat-1 gene in Chlamydomonas regulates DNA methylation during gameto­ genesis. Cell 24: 41–47 Sano H, Grabowy C and Sager R (1981) Differential activity of DNA methyltransferase in the life cycle of Chlamydomonas reinhardi. Proc Natl Acad Sci USA 78: 3118–3122 Sano H, Grabowy C and Sager R (1984) Loss of chloroplast DNA methylation during dedifferentiation of Chlamydomonas reinhardi gametes. Mol Cell Biol 4: 2103–2108 Sano H, Royer H–D and Sager R (1980) Identification of 5­ methylcytosine in DNA fragments immobilized on nitro­ cellulose paper. Proc Natl Acad Sci USA 77: 3581–3585 Sears BA (1980) Elimination of plastids during spermatogenesis and fertilization in the plant kingdom. Plasmid 4: 233–255 Sears BB and VanWinkle-Swift KP (1994) The salvage/turnover/ repair (STOR) model for uniparental inheritance in Chlamydomonas: DNA as a source of sustenance. J Hered 85: 366–376 Shepherd HS, Boynton JE and Gillham NW (1979) Mutations in nine chloroplast loci of Chlamydomonas affecting different photosynthetic functions. Proc Natl Acad Sci USA 76: 1353– 1357 Small GD (1987) Repair systems for nuclear and chloroplast DNA in Chlamydomonas reinhardtii. Mut Res 181: 31–35 Smith GM and Regnery DC (1950) Inheritance of sexuality in Chlamydomonas reinhardi. Proc Natl Acad Sci USA 36: 246– 258 Spreitzer RJ and Mets LJ (1980) Non-mendelian mutation affecting ribulose-1,5-bisphosphate carboxylase structure and activity. Nature 285: 114–115 Tait GCL and Harris WJ (1977a) A deoxyribonuclease from Chlamydomonas reinhardii. 1. Purification and properties. Eur J Biochem 75: 357–364 Tait GCL and Harris WJ (1977b) A deoxyribonuclease from Chlamydomonas reinhardii. 2. Substrate specificity, mode of action and products. Eur J Biochem 75: 365–371 Tsubo Y and Matsuda Y (1984) Transmission of chloroplast genes in crosses between Chlamydomonas diploids: Correlation

113 with chloroplast nucleoid behavior in young zygotes. Curr Genet 8: 223–229 Uchida H, Kawano S, Nakamura S and Kuroiwa T (1992a) A study on the effects of UV on preferential digestion of chloroplast nuclei in young zygotes of Chlamydomonas reinhardtii. Cytologia 57: 395–399 Uchida H, Nozue F, Kuroiwa H, Osafune T, Sumida A, Ehara T and Kuroiwa T (1992b) Evidence for preferential digestion of male-derived chloroplast DNA in young zygotes of Chlamydomonas reinhardtii by histochemical immunogold electron microscopy. Cytologia 57: 463–470 Uchida H, Kawano S, Sato N and Kuroiwa T (1993) Isolation and characterization of novel genes which are expressed during the very early stage of zygote formation in Chlamydomonas reinhardtii. Curr Genet 24: 296–300 van den Ende H and VanWinkle–Swift KP (1994) Mating-type differentiation and mate selection in the homothallic Chlamydomonas monoica. Curr Genet 25: 209–216 VanWinkle-Swift KP (1978) Uniparental inheritance ispromoted by delayed division of the zygote in Chlamydomonas. Nature 275: 749–751 VanWinkle-Swift KP and Aubert B (1983) Uniparental inheritance in a homothallic alga. Nature 303: 167–169 VanWinkle-Swift KP and Bauer JC (1982) Self-sterile and maturation-defective mutants of the homothallic alga, Chlamydomonas monoica (Chlorophyceae). J Phycol 18: 312– 317 Van Winkle-Swift KP and Burrascano CG (1983) Comple­ mentation and preliminary linkage analysis ofzygote maturation mutants of the homothallic alga, Chlamydomonas monoica. Genetics 103: 429–445 VanWinkle-Swift KP and Halm J-H (1986) The search for mating-type-limited genes in the homothallic alga Chlamy­ domonas monoica. Genetics 113: 601–619 VanWinkle-Swift KP and Salinger AP (1988) Loss of mt+derived zygotic chloroplast DNA is associated with a lethal allele in Chlamydomonas monoica. Curr Genet 13: 331–337 VanWinkle-Swift K, Hoffman R, Shi L and Parker S (1994) A suppressor of a mating-type limited zygotic lethal allele also suppresses uniparental chloroplast gene transmission in Chlamydomonas monoica. Genetics 136: 867–877 Wegener D and Beck C (1991) Identification of novel genes specifically expressed in Chlamydomonas reinhardtii zygotes. Plant Mol Biol 16: 937–946 Whatley JM (1982) Ultrastructure of plastid inheritance: Green algae to angiosperms. Biol Rev 57: 527–569 Woessner JP and Goodenough UW (1989) Molecular charac­ terization of a zygote wall protein: An extensin-like molecule in Chlamydomonas reinhardtii. Plant Cell 1: 901–911 Wurtz EA, Boynton JE and Gillham NW (1977) Perturbation of chloroplast DNA amounts and chloroplast gene transmission in Chlamydomonas reinhardtii by 5-fluorodeoxyuridine. Proc Natl Acad Sci USA 74 (10): 4522–4556 Yaneda H, Hayashi J-I, Takahama S, Taya C, Lindahl K and Yonekawa H (1995) Elimination of paternal mitochondrial DNA in intraspecific crosses during early mouse embryo­ genesis. Proc Natl Acad Sci USA 92: 4542–4546

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Chapter 7 Replication, Recombination, and Repair in the Chloroplast Genetic System of Chlamydomonas Barbara B. Sears

Department of Botany & Plant Pathology, Michigan State University,

East Lansing, Michigan 48824-1312 U.S.A.

Summary I. Introduction II. Replication A. Replication of cpDNA during cell cycle B. Nucleoids as Indicators of cpDNA Abundance C. cpDNA Degradation and Nucleotide Recycling D. Origins of cpDNA Replication E. Enzymes of cpDNA Replication III. Recombination A. Genetic Analysis of Recombination 1. Assessment of Chloroplast Gene Recombination Frequencies in Meiotic Progeny 2. Assessment of Chloroplast Gene Recombination Frequencies Through the

Analysis of Biparental Zygospore Clones 3. Assessment of Chloroplast Gene Recombination Frequencies After Paternal

Marker Selection 4. Assessment of Recombination Frequencies in Vegetative Diploid Zygotes B. Factors that Affect Recombination Frequency C. Recombination Within the Inverted Repeat D. Recombination Hotspots E. Gene Conversion F. Intron Homing IV. Repair A. UV-Damage and Photoreactivation B. Specific Impact of FdUrd on cpDNA C. Other Mutagens D. The Interrelated Processes of Recombination/Repair V. Perspectives and Conclusions Acknowledgments References

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Summary Chloroplast DNA replicates throughout the Chlamydomonas cell cycle, with individual DNA molecules being randomly chosen for replication. Mosaic labeling of the cpDNA in density transfer experiments may indicate that replication is dispersive, rather than semi- conservative, but it also may result from multiple rounds of recombination between the highly polyploid cpDNA molecules. Repair of spontaneous DNA damage may also contribute to dispersive labeling, and cpDNA turnover and nucleotide recycling may comprise a further contributing factor. Two origins of replication have been identified on the cpDNA molecule, and their activity has been studied in vitro. Exposure of cells to a gyrase inhibitor initially inhibits replication, and then stimulates J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 115–138. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

Barbara B . Sears

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a cryptic origin, near a site identified as a recombination hot spot in other studies. Transformation experiments point to the possible existence of at least one additional origin. For cpDNA, recombination is not limited to the sexual cycle, but occurs during vegetative cell growth as well, probably because enzymes that function in recombination, including a RecA-like protein, play an essential role in the repair of DNA damage and hence are present throughout the cell cycle. In mitotic cells, copy correction and ‘flip-flop’ recombination between the two copies ofthe inverted repeat are a consequence ofthese activities. From sexual crosses, biparental zygotes have been recovered in which recombination and gene conversion have been examined using chloroplast genetic markers and/or restriction fragment length polymorphisms (RFLPs). These procedures, combined with chloroplast transformation, have identified a recombination hotspot in the inverted repeat of C. reinhardtii. Other hotspots of recombination have been characterized in crosses of C. eugametos and C. moewusii, as well as C. reinhardtii, where several DNA segments act as ‘selfish DNAs’, inserting themselves into the target site in the plastome contributed by the other parent, and thus proliferating among the progeny. Several of these segments contain homing introns, which encode endonucleases essential for their movement. Several aspects of repair have been characterized biochemically, including the direct reversal of UV-damage by photolyase, and the apparent absence of methyltransferases for reversal of alkylation damage. The removal of pyrimidine dimers in the dark may occur through excision repair, recombinational repair or a combination of the two. When cells are treated with 5-fluoro-deoxyuridine, the cpDNA of C. reinhardtii becomes reduced in abundance and is highly mutable. Thus, this thymidine analog is a potent and specific mutagen, and has been effectively combined with other mutagenic treatments for the induction of non-Mendelian mutations in C. reinhardtii. Future investigations doubtlessly will utilize the ability to transform both the nuclear and chloroplast genomes with the classical genetic approach of inducing and analyzing mutations, and biochemical procedures for in vitro analysis. Heterologous probes and E. coli mutants deficient in specific aspects of replication, recombination and repair will serve as useful resources for the identification ofgenes that affect those processes in the chloroplast genetic system of Chlamydomonas.

I. Introduction The chloroplast genetic system depends upon replication and repair for the perpetuation and preservation of its DNA. In prokaryotes and eukaryotes, some enzymes essential for replication and repair also are utilized for recombination, a complex process in the Chlamydomonas chloroplast, and a process that is not yet fully understood. This chapter reviews genetic, biochemical, and molecular investigations that have yielded insight into these aspects of chloroplast DNA metabolism. Instances are noted where features of replication, recom­ bination, and repair in the chloroplast genetic system of Chlamydomonas differ from higher plants. In this review, the recent literature has received more attention than the older literature because several thorough reviews from previous decades are available. Abbreviations: cpDNA – chloroplast DNA; DAPI – 4´,6diamidino-2-phenylindole; FdUrd – 5-fluoro-deoxyuridine; IPTG MMS – methylmeth­ anesulfonate; mt – mating-type; plastome – plastid genome; RFLP – restriction fragment length polymorphism

Of particular value are reviews that cover cpDNA replication (Keller and Ho, 1981) and repair in Chlamydomonas (Small, 1987), chloroplast recom­ bination, genetic mapping and mobile introns (Gillham, 1978, 1994), cytological observations of chloroplast nucleoids (Kuroiwa, 1991), and the Chlamydomonas Sourcebook (Harris, 1989), which encompasses all ofthese topics. This chapter will not consider the topic of chloroplast gene inheritance; for that, the reader is referred to the chapter by Armbrust.

II. Replication Studies of chloroplast DNA (cpDNA) replication in Chlamydomonas have been facilitated by two important traits that are not general features of higher plant chloroplasts. First, cpDNA of Chlamydomonas has a much higher A-T content than does nuclear DNA, allowing the two classes of DNA to be separated in CsCl buoyant density gradients, even without the addition of ethidium bromide or bisbenzimide (Sager

Chapter 7

Chloroplast Replication, Recombination and Repair

and Ishida, 1963). In those gradients, the major band while the second most represents nuclear or prominent band is composed of cpDNA, also referred to as the (Fig. 1). The cpDNA composes 8– 18% of the total cellular DNA in logarithmically growing cells, and about 3–8% in the gametes (Sager and Ishida, 1963; Chiang and Sueoka, 1967; Chiang, 1971; Sears et al., 1980; Turmel et al., 1980). The second useful trait is the fact that exogenous thymidine is incorporated only into cpDNA due to the presence of a chloroplast-specific thymidine kinase (Swinton and Hanawalt, 1972; Chiang et al., 1975). Since adenine will be incorporated into both nuclear and cpDNA, when cells are provided with two differ­ entially labeled nitrogenous bases (i.e., their differential incorporation and can be used to distinguish the replication patterns of nuclear and cpDNA.

A. Replication of cpDNA during cell cycle An overview of the cell cycle is shown in Fig. 2. When cells are grown under phototrophic conditions in liquid culture with alternating periods of 12 h light, 12 h dark, chloroplast and cell divisions occur

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fairly synchronously during the dark phase (reviewed by Harris, 1989). In the initial experiments designed to examine replication in C. reinhardtii, synchronized cells were grown in and then transferred just after cell division (Chiang and to Sueoka, 1967). When cells were harvested during the next 24 h, shifts in buoyant densities of the peak indicated that nuclear DNA underwent two rounds of semiconservative replication during the dark period, resulting in a band of hybrid density (labeled with ) and a less dense, more abundant band (labeled exclusively with ). Similarly, the peak showed density shifts that indicated cpDNA replicated in a semi-synchronized and semiconservative fashion during the 12-h light period. When the experiments were repeated, the timing of nuclear DNA replication was in agreement with the previous studies, but cpDNA was found to replicate in both the light and dark periods (Chiang, 1971; Lee and Jones, 1973; Turmel et al., 1980, 1981). Furthermore, after cells were transferred to the buoyant density of the broadened, with the peak shifting gradually, a result that was interpreted to be due to random selection of cpDNA molecules for replication (Turmel et al.,

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1980, 1981). Further analysis focused on isolated from cells after one 24-h period of growth in (Turmel et al., 1981). The cpDNA was denatured to examine the buoyant density distri­ butions of the single-strands, and no discrete heavy strand was apparent. This result indicates that nucleotides were incorporated into both strands, suggesting that cpDNA replication may be dispersive rather than semi-conservative. However, Turmel et al. (1981) pointed out that the dispersive labeling could have been caused by frequent recombination among the polyploid plastomes, some that had replicated, and some that had not yet replicated. Moreover, Woefle et al. (1993) have noted that recombination could act to initiate DNA replication and thus yield a cpDNA having a mosaicism of incorporated label. As described in more detail in

Barbara B . Sears

later sections, other studies with mitotically growing cells have demonstrated frequent intramolecular recombination between the inverted repeats (Aldrich et al., 1985) and intra- or intermolecular recom­ bination between duplications created through biolistic transformation (Cerutti et al., 1995), indicating that the enzymes for recombination are indeed present in vegetative cells.

B. Nucleoids as Indicators of cpDNA Abundance The abundance and distribution of cpDNA has been examined cytologically through the use of 4',6diamidino-2-phenylindole (DAPI) to visualize nucleoids, which represent aggregations of cpDNA molecules. The initial application by Coleman (1978) of this fluorochrome to study the Chlamydomonas

Chapter 7

Chloroplast Replication, Recombination and Repair

chloroplast was followed by Kuroiwa, Birky, and others to track cpDNA during the sexual cycle (e.g., Kuroiwa et al., 1982; Birky et al., 1984; Coleman, 1984; Nakamura et al., 1991). These important contributions are described in the chapter by Armbrust. Kuroiwa and coworkers examined changes in number and shape of nucleoids in synchronized liquid cultures of C. reinhardtii (Kuroiwa et al., 1981) and cells growing on solid media (Nakamura et al., 1986). They observed increases in nucleoid size and intensity of DNA staining as the chloroplasts and cells enlarged. Shortly before chloroplast division in the synchronous cultures, the nucleoids became dumbbell-shaped and divided semi-synchronously, with the daughter nucleoids segregating into the four daughter chloroplasts. A subsequent investigation by Ehara et al. (1990) refined the fixation procedures, and reinvestigated the changes in nucleoid shape and distribution during the cell cycle. They found that when cells are grown synchronously, early in the light phase, most of the cpDNA is clustered near the pyrenoid (Fig. 2). Just before the dark phase and prior to division, the cpDNA becomes dispersed throughout each chloroplast. The dispersed cpDNA was found to aggregate if high concentrations of glutaraldehyde (0.5%) were used during the fixation procedures. A functional basis for the dispersion of the cpDNA nucleoids during the dark phase is unclear, since the biochemical characterizations described in the previous section indicate that cpDNA replicates throughout the cell cycle. However, as discussed in Section I.D, the location of cpDNA near the pyrenoid may be meaningful.

C. cpDNA Degradation and Nucleotide Recycling Several observations indicate that cpDNA is not as stable nor as persistent as nuclear DNA. The dispersive labeling observed in cpDNA of vegetative cells was also reported for young zygotes shortly after gamete fusion and in meiotic progeny, and was interpreted to indicate extensive recombination had occurred between the and cpDNAs (Chiang, 1971). However, these experiments are open to criticism since the procedures required to break open the thick walled zygospores caused shearing of the DNA, and hence, the peaks were poorly resolved in the CsCl gradients. Furthermore, since chloroplast markers are inherited predominantly from the parent, and nucleoids disappear shortly after gamete fusion, cpDNA molecules are few doubt that most

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degraded soon after gamete fusion (reviewed in Chapter 6, Armbrust). A shift in the buoyant density of in gametes (see Fig. 1b) was interpreted by Ruth Sager and colleagues to indicate that the cpDNA becomes methylated during gametogenesis and hence protected from an endonuclease that was chloroplast hypothesized to be activated in the (Sager and Lane, 1972). Subsequent studies using methylation-sensitive restriction enzymes (Royer and Sager, 1979) and HPLC characterization ofthe DNA showed that cytosines become extensively methylated gametes (Burton et al., 1979). The cytidine in target was verified by Feng and Chiang (1984) who used 5-azacytidine and L-ethionine to inhibit methylation. A nuclear mutation was isolated that results in hyper-methylation of the cpDNA at all stages of the cell cycle, regardless of mating type (Bolen et al., 1982). For a discussion of the correlations between methylation and uniparental inheritance of chloroplast markers, the reader is referred to Chapter 6 (Armbrust). Sears and VanWinkle-Swift (1994) suggested that cpDNA could have a the degradation of the selective advantage, especially under the conditions of nitrogen starvation which induce gametogenesis and zygospore dormancy. The salvage/turnover/repair (STOR) model postulates that the high polyploidy of cpDNA allows it to be used as a cellular resource for carbon, nitrogen, and nucleotides, especially under conditions of nutrient limitation. The STOR model further suggests that if cpDNAs are damaged, they might be turned over rather than repaired, and that a balance between replication and degradation would thus exist in all stages of the cell cycle, including the vegetative growth phase. This model is consistent with the dispersive labeling of cpDNA that was reviewed previously, and the observations of Small and Greimann (1977), who were studying dark repair of UV-induced lesions. In their unirradiated C. reinhardtii control cultures, Small and Greimann found that when the cells were grown in and then incubated in the adenine and dark for 24 h, the nuclear DNA was stably maintained, but about half of the cpDNA was degraded during the dark period. Results from Cerutti et al. (1995) have shown that when the pathway is inhibited, both the integrity and abundance of cpDNA are diminished under conditions of extensive DNA damage. Their interpretation is that excessively damaged cpDNA molecules are probably degraded rather than repaired. On the other hand, since recombination-repair

120 involves partial DNA degradation and resynthesis, if that process is operating in vegetative cells and/or zygotes, it could contribute to the dispersive labeling patterns observed for cpDNA (H. Cerutti, personal communication). In C. moewusii, evidence for cpDNA turnover has density transfer experiments, been found in in which the density of the cpDNA decreased more rapidly than expected from the net amounts of DNA synthesized (M. Tunnel, personal communication). In addition, a linear 6-kb segment of cpDNA that is consistently recovered from this species (Tunnel et al., 1986) appears to represent a specifically-protected remnant of the degraded cpDNA.

D. Origins of cpDNA Replication Initial attempts to locate the origins of plastome replication identified sequences that were capable of

Barbara B . Sears replication after transformation into the yeast nucleus (Loppes and Denis, 1983;Valletetal., 1984). Another study fortuitously discovered that at least four cpDNA segments were capable of autonomous replication in the Chlamydomonas nucleus (Rochaix et al., 1984). Subsequent investigations (Vallet and Rochaix, 1985; Wu et al., 1986) indicated that those sites differed from the location of two replication bubbles or displacement loops (D-loops) on the native cpDNA molecule visualized with electron microscopy (Waddell et al., 1984; Wang et al., 1984), although several were intriguingly close. Two D-loops that were observed by Madeline Wu and colleagues were mapped to adjacent EcoRI fragments contained in the fifth largest Bam HI fragment on the cpDNA molecule (Bam 5, using the fragment nomenclature reviewed by Harris, 1989; Fig. 3). Two observations suggest that a single site probably serves to originate replication on each cpDNA molecule: 1) although

Chapter 7 Chloroplast Replication, Recombination and Repair the two D-loops map to the same Bam fragment, few (if any) Bam fragments contained two D-loops at the same time; 2) as depicted in Fig. 4, the two strands of each D-loop have double-stranded regions at opposite ends, indicating that individual origins initiate bidirectional replication. These observations suggest that initiation of replication in the Chlamydomonas chloroplast may differ from initiation in higher plants, where two origins are thought to be required in order to accomplish bidirectional replication (Kolodner and Tewari, 1975). The initial effort to define more precisely the location of oriA used Southern hybridizations and heteroduplex analyses, with cloned fragments of cpDNA from C. reinhardtii and strain WXM, with the reasoning that a locus as important as the origin ofreplication would be highly conserved among different strains of Chlamydomonas (Wang et al., 1984). However, the homologies thus indicated were probably due to the presence of ribosomal protein genes that were subsequently identified near the most frequent origin of replication, oriA (Wu et al., 1986; Lou et al., 1987), since conserved regions in eubacterial origins are composed of A-T rich motifs of only 11–16 bp (Kornberg and Baker, 1992). Although no further investigations were pursued with strain WXM, oriA of C. reinhardtii was characterized extensively by Wu and colleagues. Cloned DNA fragments containing oriA were three to eight times more active than the control vector sequences in directing incorporation of radioactivelylabeled dTTP when used for in vitro replication studies (Wu et al., 1986; Hsieh et al., 1991). Two ‘back-to-back’ prokaryotic promoters were identified and proposed to function as sites ofprimer synthesis for replication initiation (Wu et al., 1986; Lou et al., 1987; Nie et al., 1987). As Wu and coworkers dissected components involved in cpDNA replication, they found that a 500-bp fragment adjacent to the oriA promoters specifically bound polypeptides of 18, 24, and 26 kDa (Nie et al., 1987). The 18-kDa protein was deduced to be encoded by a cpDNA gene, since incubation of cells in chloramphenicol specifically reduced its abundance. The 18 kDa protein was isolated and purified by Wu et al. (1989) for Nterminal amino acid sequencing, with the surprising result that the protein appeared to be the product of the ndhI (frxB) gene, which encodes an iron-sulfur protein that is a component of a thylakoid-localized NADH dehydrogenase. The amino acid sequence information has enabled degenerate oligonucleotides

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to be constructed as probes, but the ndhI gene has not been located on the cpDNA of Chlamydomonas (J. D. Rochaix, personal communication), suggesting that the protein may be encoded in the nucleus, in spite of the chloramphenicol results. Wu and colleagues speculated that the protein might serve a dual function by responding to redox levels with a conformational change that would affect the protein complex at the origin and thus alter initiation of replication (Wu et al., 1989, 1993). Subsequent immunological analyses showed that the ndhI gene product was located in one or more membrane vesicles near the pyrenoid and closely apposed to a subset of chloroplast nucleoids (Zhang and Wu, 1993). Because a charge differential exists across the vesicular membrane (transmembrane potential), the vesicles probably contain other components of photosynthetic electron transfer. The abundance and distribution of the 18-kD protein generally paralleled that of the nucleoids: increasing in the light, decreasing in the dark, decreasing when cells were grown in the presence of 5-fluoro-deoxyuridine (FdUrd), and chloroplasts during becoming greatly reduced in gamete fusion. The one exception noted by Wu and Zhang was that gametes ofboth mating-types seemed to contain more of the protein than did vegetative cells, whereas cpDNA abundance and nucleoid number have been found to decrease in gametes (Chiang, 1971; Sears et al., 1980; Turmel et al., 1980). In vitro characterizations by Wu and coworkers delimited oriA on the Bam 5 fragment to a 224-bp segment that contains the two promoters and a portion ofthe fragment that shows protein-binding properties

122 (Hsieh et al., 1991). However, others found that the 224-bp oriA segment was unable to sustain independent replication of a small plasmid following biolistic transformation (Suzuki et al., 1997). In contrast, the Bam 10 fragment (which contains the atpB gene and neither oriA nor oriB) becomes highly amplified when a plasmid containing it is transformed into the chloroplast (Boynton et al., 1988; Kindle et al., 1994; Suzuki et al., 1997). The amplifications were found as extrachromosomal tandem arrays, but always in conjunction with rearrangements in the cpDNA, and with the apparent prerequisite that an integrated copy be present (Suzuki et al., 1997). These observations led Suzuki et al. (1997) to propose that the Bam 10 plasmid did not replicate autono­ mously, but rather that tandem arrays of the plasmid were generated after a single cross-over event during transformation created a third copy of the atpB gene and a portion of the inverted repeat in the same orientation as the neighboring segment. After initiation ofreplication of the cpDNA, recombination between the unreplicated third copy and a replicated original copy could occur, leading to rolling circle replication of the segment of the cpDNA delimited by these two copies. A second crossover event could terminate replication or result in the excision of the tandem repeats. Although complex, this model explains nicely the otherwise perplexing observations that the Bam 10 plasmid is recovered as an extra­ chromosomal amplicon in transformation experi­ ments. However, the model is not confirmed by the results of Dürrenberger et al. (1996), who used the Bam 10 fragment as the insertion site for a third copy of a portion of the 23S rRNA gene, in both direct and inverted orientations. As discussed in more detail in Section III.F, the presence of a third copy in direct orientation led to deletions between the two closest copies, without the appearance of extrachromosomal amplifications. This suggests that an attribute intrinsic to the Bam 10 fragment itself may be responsible for the generation of the extrachromosomal arrays. The possibility should be considered that additional replication origins exist on the cpDNA, including the 8-kb Bam 10 fragment, since the original D-loop studies of Waddell et al. (1984) reported that replication bubbles in Bam HI fragments of about 9 and 13-kb occurred at a similar frequency to those denoted as oriB in the 17-kb Bam 5 fragment. Moreover, Mosig and coworkers discovered that a third site becomes active in initiating cpDNA replication when the chloroplast gyrase is inhibited

Barbara B . Sears by novobiocin (Woefle et al., 1993). This cryptic ‘origin’ maps in or near a recombination hotspot characterized by Newman et al. (1992), and initiates unidirectional replication, possibly from recom­ bination intermediates.

E. Enzymes of cpDNA Replication Three research groups have undertaken the isolation and characterization of DNA polymerases from Chlamydomonas reinhardtii, and two of these have focused specifically on characterizing the chloroplast enzyme(s). The initial studies by Ross and Harris (1978a, 1978b) characterized three distinguishable DNA polymerase activities fractionated from sonicated cell extracts as complexes of about 100­ kDa and 200-kDa. Keller and Ho (1981) isolated chloroplasts from a cell wall-less strain, CW-15, and showed that radioactive deoxyribonucleotides were incorporated primarily into the DNA. From such a chloroplast fraction, a 180-kDa protein complex having DNA polymerase activity was isolated. The major component was composed of protein(s) of about 40-kDa, and minor components of 15- and 75-kDa were present. In the same study, another DNA polymerase activity was identified, which may represent the nuclear enzyme. Replication in the isolated chloroplasts was inhibited by low concentrations of ethidium bromide, which had been shown by Flechtner and Sager (1973) to inhibit cpDNA replication in Chlamydomonas, and by nalidixic acid, which inhibits prokaryotic gyrases. Keller and Ho concluded that the replication complex must be anchored in the thylakoid membrane since many chloroplasts were stripped of their envelopes during the isolation procedures. Their preliminary characterizations showed that a DNase activity was purified along with the DNA polymerase, and the 40­ kDa protein with exonuclease activity was proposed to provide proof-reading ability to the cpDNA polymerase. In contrast, Wu and coworkers isolated DNA polymerase activity after breaking open wild-type cells by sonication. Initial investigations used both a thylakoid membrane fraction and soluble proteins (Wu et al., 1986), but later investigations were limited to soluble proteins, since that fraction was the source of the polymerase activity (Wang et al., 1991). Extensive purification yielded a DNA polymerase activity composed of proteins of 116 and 80 kDa, sizes similar to proteins composing the animal

Chapter 7

Chloroplast Replication, Recombination and Repair

mitochondrial polymerase (Wang et al., 1991). Similar to enzymes from chloroplasts of higher plants, the Chlamydomonas DNA polymerase showed a preference for poly (dA).(dT) over calf thymus DNA, and was inhibited by N-ethylmaleimide. The ability to synthesize DNA was sensitive to ethidium bromide, and was not inhibited by aphidicolin, thus resembling the chloroplast enzyme from higher plants (Sala et al., 1980; McKown and Tewari, 1984). Since the DNA polymerase characterized by Wu and coworkers is clearly different from that characterized by Keller and colleagues from isolated chloroplasts, it is conceivable that one of those polymerases may actually be the mitochondrial enzyme, particularly since Chlamydomonas mitochondria are highly sensitive to ethidium bromide mutagenesis (Alex­ ander et al., 1974; Gillham et al., 1987a). Another possibility is that multiple DNA polymerases may function in cpDNA replication and repair, as is the case in eubacteria and mammalian mitochondria. Investigations by Mosig and colleagues have focused on enzymes that affect the superhelicity of cpDNA. In vitro studies have shown that both a gyrase activity (topoisomerase II) and a relaxing activity (topoisomerase I) can be isolated from Chlamydomonas cells, and in vivo studies have shown that the gyrase inhibitors, novobiocin and nalidixic acid, differentially affect synthesis of several chloroplast transcripts (Thompson and Mosig, 1985). Chloroplast DNA replication is also inhibited soon after the addition of novobiocin, which suggests that DNA gyrase activity is required for normal cpDNA synthesis (Woelfle et al., 1993). Other investigations assessed plastome structure in light- and dark-grown cells by measuring the ability of a psoralen compound to intercalate into cpDNA (Thompson and Mosig, 1990). Cells in the light phase yielded a cpDNA that bound less psoralen than did cpDNA from cells in the dark phase, indicating that in the light, the cpDNA either had less superhelicity or more proteins associated with it, which consequently limited its accessibility.

III. Recombination

A. Genetic Analysis of Recombination In sexual crosses of Chlamydomonas cellular fusion of the gametes is followed by nuclear and then chloroplast fusion (Cavalier-Smith, 1970). Thus, the

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cpDNAs have an opportunity to recombine, but this can occur only if cpDNAs from both parents are able to persist in the zygote (biparental zygotes). Consequently, the likelihood of recovering recom­ binant cpDNAs is highest if the frequency of biparental chloroplast gene transmission is high. In hybrid backcrosses following interspecific crosses of C. eugametos × C. moewusii, high frequencies of biparental inheritance of chloroplast markers are observed (Lemieux el al., 1980, 1981; Lee and Lemieux, 1986). The zygotes from the interspecific cross have low viability, producing only about 10% viable progeny (Gowans, 1963), however, the progeny from repeated backcrossing show improved zygote viability (Lee and Lemieux, 1990), and high levels of biparental transmission are still observed (Bussières et al., 1996). In C. reinhardtii, spontaneous recombinants are rare because the chloroplast genes are inherited predominantly from the maternal parent, but exogenous chemicals and gametes prior to irradiation can be applied to the mating to increase the frequency of biparental inheritance (reviewed by Harris, 1989; Sears and VanWinkle-Swift, 1994; Chapter 6, Armbrust).

1. Assessment of Chloroplast Gene Recombination Frequencies in Meiotic Progeny Sager and coworkers were the first to map chloroplast genetic distances by assessing recombination frequencies between markers that showed nonMendelian inheritance in C. reinhardtii (Sager and Ramanis, 1968, 1976). Laborious microdissections of tetrad and octad meiotic products were performed, and the progeny were allowed to produce colonies that were then scored for the presence of both nuclear and chloroplast markers. Because only the first three to four cytoplasmic divisions of the zygote were analyzed, a recombinant molecule had to sort out during the first few cell divisions of the zygote, if the recombination event was to be scored. The cumulative data allowed the markers to be mapped relative to each other, and the initial maps coincided reasonably well with results obtained subsequently by Gillham and Boynton and their coworkers (reviewed by Harris et al., 1989). However, Sager’s genetic data were subjected to a complex mathematical analysis to create a circular map (Singer et al., 1976), and a membrane attachment site was proposed as an anchor point for the genetic map and for the plastid chromosome. The circular diagram was an attractive

124 model because the cpDNA had been shown to be a circular molecule (Behn and Herrmann, 1977). However, as pointed out by Harris (1989), the antibiotic resistance markers, which span about half of the circle of Singer et al. (1976), are in fact located within a 21-kb stretch on the 200-kb cpDNA molecule. Their order on the circular map does not correspond well to the locations that subsequently have been shown to be the sites of mutation in the physical map of the cpDNA (Harris et al., 1989). When the zygotes fulfill the essential condition for recombination analysis of containing chloroplast markers from both parents, one might expect that most of the meiotic progeny would be heteroplasmic (containing a mixture of chloroplast alleles from both parents), and therefore be useless for analyzing recombination. Surprisingly, among the 8-cell progeny resulting from meiosis followed by a single mitotic division, the majority of cells are homoplasmic for at least one locus (Sager and Ramanis, 1968, 1976; Forster et al., 1980). The rapid segregation is probably due to many factors, including the reduction in cpDNA copy number in gametes and possibly in zygotes, destruction ofmost ofthe copies of the plastomes, internal chloroplast membrane structure acting as a barrier to plastome mixing (Mets and Geist, 1983) and the likelihood that nucleoids, rather than individual cpDNA molecules, are the functional genetic units (VanWinkle-Swift and Birky, 1978). Gene conversion may also act to reduce heteroplasmicity, with stochastic events resulting in genetic drift within the population of organelle DNA molecules (Birky and Skavaril, 1976).

2. Assessment of Chloroplast Gene Recom­ bination Frequencies Through the Analysis of Biparental Zygospore Clones In contrast to the analysis of meiotic progeny, the biparental zygospore clone analysis of Gillham, Boynton, Harris, and colleagues assesses chloroplast gene recombination long after it has actually occurred. The procedure is analogous to the assessment of recombination frequencies in phage ‘crosses’, where bacteria are subject to a mixed infection, and recombinant phage are recognized when the progeny phage are diluted for subsequent single infections (Gillham et al., 1974). For the Chlamydomonas analysis, zygospores are allowed to sporulate and form colonies, which are then replica-plated to distinguish those in which chloroplast genes were

Barbara B . Sears inherited from both parents. Fifty or more biparental zygote colonies are selected and taken through dilution-plating on non-selective media so that individual cells give rise to colonies. From each biparental zygote dilution plate, 64 subclones are then routinely transferred to an 8 × 8 grid, replicaplated and scored for chloroplast marker composition. To calculate recombination frequency between two markers, the denominator is taken as the number of total subclones tested, and the numerator is the sum of the number of subclones that displayed a nonparental combination of the two markers. Percentage recombination is then converted to map units. Figure 5 shows a genetic map that was composed from these data. As this figure shows, for the antibiotic-resistance markers in genes that encode components ofthe chloroplast ribosome, the physical map aligns well with the genetic map, wi th approximately 1% recombination per 1000 bases. However, when crosses include other markers, such as those in the nearby psbA gene, recombination occurs at a high frequency between them and the ribosomal antibiotic-resistance markers, making them appear unlinked, since they approach the theoretical limit of 25% recombination according to the phage model, when multiple rounds of recombination and replication occur prior to sampling (Harris et al., 1989). Subsequent studies have shown that a recombination hotspot lies between the psbA and 23S rRNA genes (Newman et al., 1992), as further described in Section III.D. A disadvantage of assessing recombination frequencies by subcloning biparental zygospore clones is that early recombination events are amplified in this scoring system. Although it is true that early events may be multiply counted, the likely occurrence of any recombination event should still be distancedependent. If large numbers of biparental zygospore clones are analyzed, sampling errors should diminish as the recovery ofearly and late recombination events balance each other. One other comment should be made about the analogy between this method of analysis and determination of recombination frequency in phage crosses. Phage crosses are performed with equal titers from the two phage to accomplish double infections, whereas in C. rein­ hardtii, a maternal bias in the contribution of chloroplast markers is usually reflected in the composition of the biparental zygospore colonies (reviewed by Harris et al., 1989).

Chapter 7

Chloroplast Replication, Recombination and Repair

3. Assessment of Chloroplast Gene Recom­ bination Frequencies After Paternal Marker Selection Mets recognized that zygospore clone analysis tended to overcount early recombination events, and proposed an alternative means of mapping chloroplast gene recombination frequencies (Mets and Geist, 1983). The paternal marker selection procedure begins with zygospore colonies, which are scored by replica-plating. From each biparental zygospore colony, a single subclone is initially chosen based on its growth on medium that selects for one of the paternal markers. That subclone is then scored for the presence of other markers. This procedure is modeled after techniques for mapping of bacterial genes by co-transformation or co-transduction, where the denominator is also defined by the selected marker. An additional goal of the procedure was to establish a method that required analysis of fewer progeny, so that it could be used for the assessment of RFLP inheritance in crosses. Since the initial selection for one paternal marker eliminates from consideration all of the non-recombinant maternal chloroplast molecules, recombination events compose a larger

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fraction of the progeny analyzed. Consequently, map distances are larger when assessed in this way. Because only a single product is scored from each biparental zygospore colony, a recombination event that occurs early in a single zygospore has a chance to be counted only once, rather than multiple times. However, recombinant molecules that have the maternal marker at the chosen paternal locus are excluded from consideration. Thus, although this procedure accomplishes its goal of removing a bias due to early recombination events, it has the disadvantage of excluding a subset of the recom­ bination events from consideration. For their analysis, Mets and Geist (1983) used erythromycin- and streptomycin-resistance markers that had been included in the analyses of others, a newly isolated rbcL mutation that results in acetatedependence (A( 10-6C)), a psbA mutation that results in a photosystem II deficiency, and a mutation in the psbA gene that confers resistance to DCMU. Using the paternal marker selection procedure in two crosses, the recombination frequencies did not give map distances that were additive for a linear map, and they also could not be assembled into a clear circular map. In comparing the disparate recom­

126 bination frequencies of their two crosses, Mets and Geist noted that one cross had a much lower recombination frequency than did the other cross. In the first cross, where maternal and paternal plastomes were more abundant among the progeny than were recombinants, Mets and Geist proposed that the normal, internal architecture of the chloroplasts in those strains could have limited the extent of mixing of the plastomes, and thus recombination may have been minimal. For the other cross, more recombinants were recovered than parental plastome types, and Mets and Geist suggested that the disruption of normal thylakoid stacking in the photosystem IIcells would have allowed more mixing deficient of the plastomes to occur in the zygotes. In the latter cross, the cpDNA appeared to have served as the resident DNA molecule, which incorporated segments of a fragmented cpDNA. It was further noted that if one or two of the genetic markers were located in one or both of the single copy regions of cpDNA, and one or more markers were present in the inverted repeats of the chloroplast DNA, it could be difficult to assemble a genetic map. In fact, subsequent cpDNA sequencing showed that those hesitations were well-founded, since rbcL gene was mapped to a single-copy region, and the other three markers were located within the inverted repeat.

4. Assessment of Recombination Frequencies in Vegetative Diploid Zygotes As summarized in the chapter by Armbrust, vegetative diploid zygotes can be selected using appropriate markers, and they transmit chloroplast markers from both parents at a high frequency (Gillham, 1963, 1969; VanWinkkle-Swift, 1978). An analysis of recombination frequencies in vegetative zygotes derived from three-marker crosses of C. reinhardtii showed no consistent correlation in the recovery of reciprocal recombinant progeny within a population of zygotes, nor within individual zygote clones (VanWinkle-Swift and Birky, 1978). In fact these observations provided the basis for an extensive comparison highlighting the non-reciprocality of organelle gene recombination in Chlamydomonas and yeast. In spite of these findings, Girard-Bascou found that she could use the production of wild-type recombinants in vegetative zygotes to estimate map distance between different mutations affecting photosynthesis (Girard-Bascou, 1987). The wildtype recombinants were recognized by their low

Barbara B . Sears fluorescence or by their ability to grow on minimal media. The recombination frequency between each pair of mutations was calculated to be twice the frequency of the wild-type recombinants, divided by a value representing the differential growth rate of the wild-type recombinants. Girard-Bascou used this procedure to distinguish five loci among the eight mutations analyzed (Girard-Bascou, 1987), with the two closest loci lying in one of the single copy regions of cpDNA, separated by 1–4% recombination and, 1910 bp (Girard-Bascou et al., 1987).

B. Factors that Affect Recombination Frequency The most important parameter for homologous recombination is the extent of sequence homology between the two DNAs. The transformation studies of Newman et al. (1990, 1992) and Suzuki et al. (1997) indicate that high levels of homologous recombination in the chloroplast of C. reinhardtii require at least 150–200-bp sequence homology. Most likely, DNAs with smaller segments of homology are also capable of recombination, but at lower frequencies, since eubacterial recombination can involve sites with as little as 25-bp homology (Allgood and Silhavy, 1988). Studies of recombination frequencies in crosses have shown that increasing levels of UV-irradiation applied to gametes raise the proportion of zygospores in which the progeny inherit chloroplast alleles from both parents, but numbers and types of recombinants recovered from each biparental zygospore do not change markedly (Gillham et al., 1974; Sager and Ramanis, 1976b). In contrast, when a single population of C. reinhardtii zygospores was sampled and induced to germinate after longer and longer periods of dormancy, recombination frequency and map distances increased over time (Sears, 1980a). During the extended dormancy, the overall frequency of biparental zygospores actually dropped, while the proportion of both maternal and paternal zygospores increased (Sears, 1980b; Rosen et al., 1991). These results suggest that at least part of the cpDNA can persist in the zygospore for reasonably long periods of time and eventually recombine. Further­ more, in terms of cpDNA metabolism, the zygospore is not in fact quiescent.

C. Recombination Within the Inverted Repeat As has been observed in higher plants, homologous

Chapter 7

Chloroplast Replication, Recombination and Repair

intramolecular recombination occurs between the two copies of the large inverted repeat, resulting in two isomers of cpDNA that have the two single copy regions in opposite orientations (Aldrich et al., 1985; Palmer et al., 1985). To determine if ‘flip-flop’ recombination is a frequent event, Aldrich et al. (1985) assessed the arrangement of the cpDNA isolated from three liquid cultures that had been derived from single vegetative cells. They reasoned that if flip-flop recombination of the cpDNA is infrequent, a single cell and the clonal line derived from it would have predominantly a single isomer. In fact, near-equal stoichiometry of the two arrange­ ments was demonstrated for all three cultures by Southern hybridizations. These studies cannot formally rule out the possibility that a sorting process exists that preserves equimolar amounts of the isomeric genomes from one cell generation to the next, but it is an unlikely explanation in light of other observations of vegetative segregation. Aldrich and colleagues also concluded that cpDNA recombination is not limited to the sexual cycle since their DNA was isolated from vegetative cell cultures. The fact that both copies of the inverted repeat are identical indicates that copy correction must occur between the two repeats. The existence of a copy correction process has been convincingly demon­ strated through the isolation of many symmetrical deletions in the two repeats (Myers et al., 1982), and the observation that transformational replacement within the inverted repeat is never recovered in a single copy (Boynton and Gillham, 1993). Myers et al. (1982) suggested that when the helix is broken within the inverted repeat, one copy of the inverted repeat will serve as the template for recombinational repair, with the end result that both copies will be identical. The occasional instances in which non­ identical copies of the inverted repeat have been recovered are those in which a large deletion extends into a single copy region ofcpDNA and/or an essential gene has been deleted from one copy of the inverted repeat (e.g., Myers et al., 1982; Dürrenberger et al., 1996). In their consideration of other recombination events involving the inverted repeat, Boynton et al. (1992) have posited that flip-flop recombination and copy correction can be viewed as ‘different manifestations of the same mechanism.’

D. Recombination Hotspots Interspecific crosses between C. moewusii and

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C. eugametos (and their or progeny) have shown that recombination occurs more frequently within the inverted repeat than between single copy regions of the cpDNA (Lemieux et al., 1990). The high recombination frequency of the inverted repeat is observed even when the homing introns that are discussed later are neutralized because they are present in the cpDNAs of both parents. The five-fold difference in recombination frequencies of the different segments of cpDNA was proposed to be due to the potential of the two copies of the inverted repeat to recombine with each other, after a donor parent recombinationally allele from the opposite replaces one copy of the allele present in the resident cpDNA. In C. reinhardtii, Newman et al. (1992) found that different intervals within the inverted repeat displayed markedly different levels of recombination. In their investigation, three-point crosses were performed with C. reinhardtii and C. smithii (UTEX strain 1062), which have extensive restriction fragment length polymorphisms (RFLPs), but may in fact be more appropriately classified as a single species (A. Coleman, personal communication.). Through biparental zygospore analysis of genetic recombination frequencies, a hotspot for recombination was identified near the 3´-end of the psbA gene. The hotspot was localized to a 500-bp segment by quantifying preferred sites of homologous recombination in transformation experiments. The hotspot contains an AT-rich segment, but does not house any of the short repeats (Schneider et al., 1985; Rochaix, 1978; Gelvin and Howell, 1979) that were previously hypothesized to play a role in recombination (Palmer et al., 1985; Boynton et al., 1992).

E. Gene Conversion Gene conversion events have been invoked to explain the observations that both copies of the large inverted repeat are always identical. As elaborated in Section III.C, even if alterations are introduced at a single site by mutation or transformation, a ‘copy correction’ process acts quickly to create a mirror image in the second copy of the inverted repeat. Assuming that strand exchange between the repeats results in a heteroduplex, mismatch repair is probably the process that acts to ‘restore’ perfect pairing, resulting in elimination or fixation of a new allele. Gene conversion between molecules could lead to genetic

Barbara B . Sears

128 drift in the population of cpDNA molecules within an individual chloroplast (Birky and Skavaril, 1976), while vegetative segregation would lead to homo­ plasmicity as the chloroplasts and cells divide. No matter what type of analysis has been used to measure frequencies of recombinant progeny from genetic crosses, a disparity has been found in the recovery of reciprocal recombination products (e.g., Gillham, 1965a; Gillham et al., 1974; Boynton et al., 1976; Sager and Ramanis, 1976a; VanWinkle-Swift and Birky, 1978; Forster et al., 1980; Mets and Geist, 1983; Harris et al., 1989). The production of non­ reciprocal recombination products is probably due to biased or directional gene conversion events (Birky and Skavaril, 1976). However, the likelihood that both reciprocal and nonreciprocal recombination events occur has been discussed by VanWinkle-Swift and Birky (1978), who pointed out that multiple exchange events occurring within the organelle may act to obscure the nature of the original event. This article should be consulted by anyone seriously interested in organelle recombination processes, because it considers all aspects that can complicate the recognition and scoring of recombinant progeny, including intercellular growth differences, sample size, and intracellular selection. In addition to these considerations, Galloway and Holden (1985) have pointed out that the occurrence of different recombination products within a single zygospore clone may result coincidentally because circum­ stances such as cytoplasmic mixing, which are necessary for the production of one recombinant type, may also lead to the occurrence of another. Interspecific crosses between C. moewusii and C. eugametos have provided much valuable information about the process of gene conversion. Since these species have many differences in cpDNA physical markers (summarized by Turmel et al., 1987), RFLPs spanning the entire plastome can be examined in recombinant progeny. A subset of progeny were chosen for analysis through paternal marker selection from reciprocal crosses. An initial study ofantibiotic­ resistance genes and physical markers in and near the rRNA operon showed that regardless of the direction of the cross, 100% of the progeny inherited an RFLP in the 23S rRNA of C. eugametos, which was subsequently shown to represent a 955-bp intron (Lemieux and Lee, 1987; Turmel et al., 1991). These observations were noted to resemble the preferential inheritance of the yeast omega factor, which is an intron in the mitochondrial large rRNA gene

(reviewed by Dujon, 1989), where adjacent markers also show a ‘polarity’ of preferential inheritance. Two additional sites also are inherited preferentially by the progeny, and represent insertions of 21-kb in the inverted repeat and 6-kb in one of the single-copy segments of the cpDNA of C. moewusii relative to C. eugametos, (Lemieux et al., 1988,1990;Bussières et al., 1996). As depicted in Fig. 6, most loci are inherited from both parents, but the three sites denoted as c, g, and r (23S rRNA intron, 21-kb segment, and 6-kb segment respectively) show unidirectional inher­ itance. In the case of the 23S rRNA intron and the 21­ kb insert, adjacent markers are preferentially coinherited, but show lesser degrees of polarity (Lemieux and Lee, 1987; Lemieux et al., 1990; Bussières et al., 1996).

F. Intron Homing As reviewed above, C. Lemieux, Turmel and colleagues had observed directional gene conversion involving the intron in the large subunit rRNA gene in progeny of interspecific crosses of C. moewusii and C. eugametos. Those results led them to hypothesize that intron transposition might be mediated by an endonuclease encoded by the 218­ codon open reading frame (ORF) found within the intron (Gauthier et al., 1991; Turmel et al., 1991). Expression of the ORF in E. coli showed that its polypeptide product was required to linearize a plasmid carrying the C. moewusii sequence that lacks the intron (Gauthier et al., 1991). Subsequent investigations characterized the recognition site of the endonuclease to a 15–19-bp non-symmetric, degenerate sequence, with cleavage producing a 4­ base staggered cut at the site of insertion (Marshall and Lemieux, 1991, 1992). These investigations showed that the C. eugametos intron was auto­ mobile, and should be classified as a ‘homing intron’, since its target is the exact site at which the intron inserts into the gene (Dujon et al., 1989). Following the convention for nomenclature (Dujon et al., 1989), the endonuclease encoded by the C. eugametos 23S rRNA intron has been named I-CeuI, for Intronencoded homing endonuclease from C. eugametos (Gauthier et al., 1991). Turmel, C. Lemieux and colleagues have performed similar characterizations on related mobile introns in the 23S rRNA gene of C. humicola (Côté et al., 1993) and C. pallido­ stigmatica (Turmel et al., 1995).

Chapter 7

Chloroplast Replication, Recombination and Repair

The 23S rRNA gene of Chlamydomonas reinhardtii also contains an intron that has an open reading frame. The 163-codon ORF has some resemblance to genes for mitochondrial maturases (Rochaix et al., 1985), however the polypeptide product has no intronprocessing activity (Thompson and Herrin, 1991). Consequently, Dürrenberger and Rochaix (1991) assessed whether the polypeptide product of the ORF was involved in intron mobility. By placing the ORF under the control of an IPTG-inducible promoter, they showed that when the protein is expressed in E. coli, it specifically cleaves a plasmid carrying a cDNA copy of about 620-bp of the 23S rRNA gene, including the splice junction that remains after intron removal from the RNA. The site of double-strand cleavage was at or near the exon-exon junction. This approximate location was more precisely defined by Thompson et al. (1992) and Dürrenberger and Rochaix (1993) to 19–20-bp spanning the exon junction. As is the case with ICeuI, endonucleolytic cleavage was found to leave 4-base staggered 3´-overhangs, with a 3´-OH and a (Thompson et al., 1992). The C. reinhardtii homing endonuclease is known as I-CreI. In their first investigation, Dürrenberger and Rochaix (1991) attempted to examine the endo­ nucleolytic targeting by transformation of C. rein­ hardtii chloroplasts with a copy of the 23S cDNA segment located ectopically on the cpDNA. However, all of the transformants that had comprehensible integration patterns contained copies of the cDNA segment in which the intron already had transposed from the resident 23S rRNA gene! A subsequent collaborative investigation between Rochaix, Herrin, and their coworkers overcame this obstacle through the construction of a strain in which the intron was present in the resident 23S rRNA gene, but the reading frame was disrupted (Dürrenberger et al., 1996). The deletion that disrupted the reading frame also reduced the size of the intron, enabling the copy to be distinguished from the wild-type copy that subsequently would be introduced. In the strain and its derivatives, the 620-bp cDNA segment of the 23S rRNA was introduced by transformation into a site in a single-copy region of the nearby Bam 10 fragment. I-CreI activity was constructs with brought in later by mating the wild-type strains. In those crosses, specific cleavage at the CreI recognition site in the 23S cDNA segment was seen 1.5–2 h after mating. The investigation of Dürrenberger et al. (1996) led

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to a surprising rinding when meiotic progeny were analyzed to assess the frequency of intron movement. In the crosses described above, different results were obtained depending on whether the cDNA copy was placed in an inverted or direct orientation relative to the intron-containing 23S rRNA gene. When the cDNA homing site was initially located in an inverted orientation, about half of the meiotic progeny analyzed (from four out of seven zygospores) intron in its original position contained the as well as in the cDNA site. Thus, the intron had successfully moved into the cDNA target site through the endonuclease activity provided in trans. In both this cross and a control cross between two parents, a similar fraction of the progeny (20–25%) contained cpDNA in which the ectopic cDNA copy was eliminated by replacement with the wild-type cpDNA, indicating that sizable segment from the cpDNA are able to survive gamete pieces of the fusion and be recombinationally active, even in the absence of the endonuclease. In contrast, when the cDNA homing site was initially located in a direct orientation, the only products recovered contained deletions that had occurred between the 23S rRNA intron and the nearby gene containing the cDNA of the 23S rRNA gene. Since the deletion removed the intervening 16S rRNA gene in that copy of the inverted repeat, the deletion product certainly would not have had a selective advantage, and yet all of the progeny contained the deletion. Thus, rather than mobilizing the intron, the double-stranded DNA cleavage caused by the CreI enzyme appeared to initiate recombination between the cut site and the closest region of homology. Several optional cpDNA elements are candidates to contain or be located next to homing introns, including the 21-kb and 6-kb segments of C. moewusii cpDNA represented as RFLPs ‘g’ and ‘r’ in Fig. 6 (Bussières et al., 1996), and the C. reinhardtii ‘Wendy’ element characterized by Mosig and coworkers (Fan et al., 1995). The 21-kb segment appears to be propelled into C. eugametos cpDNA due to its linkage to a homing intron in the nearby psbA gene of C. moewusii. The gene conversion events associated with the 6-kb segment have a less clear basis, although the recovery of cpDNA linearized at the right end of the 6-kb segment indicates that an endonucleolytic target is present (M. Turmel, personal commun­ ication). The Wendy element contains ORFs with some homology to transposases and homing endonucleases. Since it is absent from the cpDNA of

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C. smithii, an assessment of its presence in the progeny of reciprocal crosses should indicate whether it has the properties of a homing intron.

IV. Repair

A. UV-Damage and Photoreactivation Ultraviolet irradiation causes bonds to form between adjacent pyrimidines on the same strand of a DNA helix (Kornberg and Baker, 1992); these pyrimidine dimers are mutagenic because they cannot be ‘read’ by DNA polymerase. Analogous to other biological systems, the Chlamydomonas chloroplast genetic system can respond to UV-damage through both a light-dependent photolyase-mediated repair and a dark repair pathway (Small, 1987), although the enzymes have not yet been isolated. The existence of

Barbara B . Sears

a photolyase was initially suggested by the observation that the impact of UV on chloroplast gene inheritance is greatly reduced if irradiated gametes are exposed to light prior to mating (Sager and Ramanis, 1967). The photoreactivation process was studied in detail by Small and coworkers, as elaborated below. Initial biochemical characterizations failed to find evidence for thymine dimer excision in the dark (Swinton and Hanawalt, 1973a,b), but subsequent investigations by Small and colleagues documented the dark repair of thymine dimers in cpDNA by using lower fluences of ultraviolet radiation and by utilizing a dimer-specific endonuclease from Micrococcus luteus to quantify pyrimidine dimers in the cpDNA (reviewed by Small, 1987). Small and Greimann and exposed them (1977) grew cells in to UV followed by a dark or light period. The DNA was extracted and half of it was treated

Chapter 7

Chloroplast Replication, Recombination and Repair

with the dimer-specific nuclease, while the other untreated halfserved as a control. When more dimers were present, the nuclease cleaved the cpDNA into smaller fragments. The degree of fragmentation was assessed in alkaline sucrose gradients, where smaller molecules do not sediment as readily as do larger ones. The distribution of the tritium label in the gradients thus indicated the prevalence of thymine dimers at each time point. After the cells were exposed to 90 min of light, their cpDNA appeared to be completely normal, whereas little repair occurred in the dark during the corresponding amount of time. After 24 h in the dark, thymine dimers were diminished to 15% of their initial abundance. Although the evidence is not conclusive, it suggests the existence of nucleotide excision repair and/or recombinational repair in chloroplasts. Small and colleagues also characterized several UV-sensitive mutants of C. reinhardtii, but found that their repair defects were specific for nuclear DNA damage (summarized by Small, 1987). However, Rosen et al. (1991) found that one UVsensitive mutant, uvsE1, increased the frequency of transmission of paternal chloroplast markers in sexual crosses, but only if the allele was present in both the and parents. The uvs-E1 allele interacts synergistically with the mat-3 nuclear allele (Gillham et al., 1987b), in promoting transmission of chloroplast genes from the paternal parent. As a mutagenic agent, UV has been used by geneticists for the induction of nuclear mutations in Chlamydomonas (Gillham and Levine, 1962; Girard et al., 1980;VanWinkle-Swift and Burrascano, 1983; and others reviewed by Harris, 1989), but only a few plastome mutations have been isolated after UVirradiation in C. reinhardtii (Hudock et al., 1979) and C. monoica (VanWinkle-Swift and Aubert, 1983; Van Winkle-Swift and Thuerauf, 1986). The fact that cpDNA mutations are induced only rarely in UVmutagenesis experiments seems paradoxical to the finding that UV specifically affects chloroplast gene inheritance when gametes are irradiated (Chapter 6, Armbrust). However, as pointed out by Sager and Ramanis (1967), UV applied to gametes may have a minimal impact on nuclear genes because chrom­ osomal DNA replication and meiosis occur many days after the irradiation, with repair being possible during an extended period of time. Furthermore, if UV-damage to the cpDNA leads to recombinational repair, an effect might not be noticed in vegetative

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cultures, since each cell contains a chloroplast with many copies of the identical DNA molecule that can act as the repair template. In contrast, gametes would have fewer copies of the cpDNA, and in the young zygote, a subset ofthese would have been contributed parent. by the

B. Specific Impact of FdUrd on cpDNA The uridine analog 5-fluoro-deoxyuridine has been found to affect specifically the abundance and integrity of cpDNA in C. reinhardtii, reducing the fraction of cpDNA from 8–18% of the total cellular DNA to 1–2% (Wurtz et al., 1977), and also diminishing the number of chloroplast nucleoids (Matagne and Hermesse, 1981; Nakamura and Kuroiwa, 1989). In bacteria, FdUrd binds to and inhibits thymidylate synthetase, the enzyme that converts dUMP to dTMP (reviewed by Kornberg and Baker, 1992). Assuming that the analogous enzyme is its target in Chlamydomonas, it is noteworthy that FdUrd has little mutagenic impact on the plastome until C. reinhardtii cells enter stationary phase or gametogenesis (Wurtz et al., 1979). This observation suggests that the chloroplast thymidylate synthetase is more active in C. reinhardtii when the cells are starved than during logarithmic growth (Sears and VanWinkle-Swift, 1994). This deduction is consistent with activation of the nucleotide salvage pathway during the massive turnover of ribosomes and rRNA that occurs under conditions of nitrogen starvation (Siersma and Chiang, 1971; Martin et al., 1976). An alternative, and notmutually exclusive explanation, is that cpDNA repair is more efficient in exponentially growing cells. FdUrd has been combined effectively with ethylmethanesulfonate (Spreitzer and Mets, 1980), ICR-191 (Harris et al., 1982) and X-irradiation (Myers et al., 1982) in the production of nonMendelian mutations. In contrast, FdUrd induces nuclear mutations in C. monoica (VanWinkle-Swift and Hahn, 1986; VanWinkle-Swift and Thuerauf, 1991), and the nitrogenous base analog, 5-fluorouracyl, has been used to induce nuclear mutations in C. reinhardtii (Girard et al., 1980). As reviewed in the chapter by Armbrust, the impact of FdUrd on cpDNA has also been observed when cells exposed to the analog are crossed to untreated gametes. Those crosses show reduced trans­ mission of chloroplast markers from the maternal

132 parent, with a dramatically increased frequency of exclusively paternal transmission and moderate levels of biparental transmission (Wurtz et al., 1977, 1979). As reviewed in the chapter by Goldschmidt-Clermont, FdUrd has also been used to reduce cpDNA levels for chloroplast transformation experiments.

C. Other Mutagens In higher plants, cpDNA mutations are induced most effectively through the use of nitroso-methyl-urea and nitroso-guanidine (reviewed by Hagemann, 1976; Börner and Sears, 1986), but in Chlamydomonas these agents have been used primarily to target nuclear genes (reviewed by Harris, 1989). The nitroso­ compounds cause mutations because they alkylate nitrogenous bases, particularly guanine residues, which then mispair during replication (Dodson et al., 1982). In bacteria, the primary lesion, can be reversed by a methyltransferase which transfers the offending methyl group to one of its own cysteine residues (Yarosh, 1985). In higher plants, the nucleus most likely contains methyltransferases, while the chloroplast probably lacks these specific repair enzymes. In contrast, Frost and Small (1987) found that methyltransferase activity is completely absent from extracts of C. reinhardtii, suggesting that this particular repair pathway is not present in either the nucleus or chloroplast of Chlamydomonas. The susceptibility of the two genetic systems to alkylation damage is reflected by the induction of both nuclear and plastome mutations by nitrosoguanidine (Gillham, 1965b). Similarly, both 2-amino-3-phenylbutanoic acid and methylmethane­ sulfonate (MMS) have been used to induce nuclear as well as plastome mutations in several species of Chlamydomonas (McBride and McBride, 1975; Hawks and Lee, 1976; Lee and Lemieux, 1986).

D. The Interrelated Processes of Recombination/Repair In a study designed to investigate the molecular mechanisms of homologous recombination, Cerutti et al. (1995) transformed chloroplasts with constructs that introduced a copy of the wild-type E. coli recA gene into the chloroplast genetic system of C. reinhardtii. Cell growth and survival after treatment with DNA damaging agents appeared to be fairly normal in cells expressing the wild-type E. coli RecA, indicating that chloroplast repair processes

Barbara B . Sears were intact. Recombination frequencies were measured by assessing the deletion of a segment between direct repeats bracketing a selectable marker introduced as a disruption of the chlL gene. The chlL gene is required for light-independent chlorophyll synthesis: when the reading frame is intact, Chlamydomonas cells are green in the dark; when the gene is disrupted, cells are yellow in the dark. Dark-grown cells containing the E. coli RecA showed a larger number ofyellow colonies with green sectors than did control cells, indicating that presence of RecA increased the frequency of recombinational deletion of the chlL disruption. Parallel studies with a control construct having a 24-codon internal deletion that abolishes RecA function showed that expression of the functionless protein did not affect the chloroplast genetic system. A third type of construct had up to 42 codons deleted from the N-terminus, which in E. coli drastically reduces the ability of the protein to form oligomers and thus acts as a dominant negative mutation. The N-terminal truncation product reduced the frequency ofrecombination as measured through the chlL assay, reduced the ability of the cells to repair cpDNA damage incurred by methyl methanesulfonate, and decreased the survival ofcells exposed to FdUrd, MMS, or UV-light. The specific decrease in plastid DNA level caused by FdUrd was also more pronounced in transformants with the Nterminal deletion of RecA than in the other genotypes. Although antibodies for E. coli RecA cross-react only slightly with a protein from wild-type Chlamydomonas the functional assays indicate that a similar protein functions in the algal chloroplast system. The observation of active recombination in vegetatively-growing cells led Cerutti et al. (1995) to suggest that the primary biological role of recom­ bination in the plastids is for the repair of cpDNA damage. Although no experiments have directly addressed the question of whether mismatch repair occurs within the Chlamydomonas chloroplast, much of the evidence reviewed here points to its existence. An active mismatch repair system could provide justification for the extensive methylation of cpDNA observed during gametogenesis and in vegetative cells under certain conditions. In E. coli, hetero­ duplexes resulting from misincorporation during replication are corrected by the mismatch repair enzymes (Friedberg et al., 1995). Those enzymes recognize, cleave, and remove the unmethylated strand bracketing the mismatch. Since methylation

Chapter 7 Chloroplast Replication, Recombination and Repair is a post-replication modification, this repair strategy leaves the parental (template) strand intact, while removing the newly synthesized daughter strand which should contain the replicative error. In other biological systems, transformational replacement of alleles and gene conversion involve mismatch repair of heteroduplexes (Friedberg et al., 1995). Analogous genetic observations have been made with Chlamy­ domonas, where non-reciprocal events typify cpDNA recombination and copy correction occurs between the inverted repeats.

V. Perspectives and Conclusions Both structural and functional features of the chloroplast genetic system reflect its evolutionary derivation from an endosymbiotic, prokaryotic ancestor (reviewed by Gillham, 1994). Given this history, many components of the replication, repair, and recombination machinery are likely to resemble their eubacterial counterparts. Such similarities could allow the isolation of genes involved in cpDNA metabolism by screening of Chlamydomonas clone libraries with heterologous probes from cyano­ bacteria. A functional screen could also be undertaken to search for complementation of defined E. coli mutants using a cDNA expression library generated from Chlamydomonas RNA. This approach has been employed successfully for cloning genes involved in recombination/repair from an Arabidopsis expression library (e.g., Pang et al., 1992, 1993a,b). Reverse genetics could then be pursued to assess the functional contribution to cpDNA metabolism of the genes thus identified. Investigations into the processes of replication, recombination, and repair of cpDNA in Chlamy­ domonas have exploited to a very limited extent the ability to isolate mutations in genes that encode products important for these processes. The isolation and analysis ofmutators that target chloroplast genes will enable a genetic dissection of replication and repair, and will also provide a useful resource for biochemical characterizations. The ability to transform both the chloroplast and nuclear genetic compartments in this alga enables foreign genes to be introduced to antagonize or supplement the endogenous genes, and also provides the option of introducing a selectable target for studying recombination or repair. This technological advance in genetic engineering has enhanced the versatility

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of Chlamydomonas as a model experimental organism for studying chloroplast genetic processes.

Acknowledgments The author is grateful for the constructive critiques and suggestions provided by Heriberto Cerutti and Jean-David Rochaix, and the comments and information provided by Annette Coleman, Lib Harris, Karen Kindle, Gisela Mosig, MoniqueTurmel, Karen VanWinkle-Swift, and Madeline Wu during the preparation of this chapter. Contributors of the figures are also gratefully acknowledged.

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Chapter 8 Chloroplast Transformation and Reverse Genetics Michel Goldschmidt-Clermont Departments of Molecular Biology and of Plant Biology, University of Geneva, Sciences II, 30 quai E. Ansermet, 1211 Geneva 4, Switzerland

Summary I. Introduction II. Delivery of DNA to the Chloroplast III. Selectable Markers and Reporters A. Selectable Markers 1. Photosynthesis 2. Drug Resistance B. Reporters and Foreign Gene Expression IV. Fate of Transforming DNA A. Integration in the Chloroplast Genome B. Extrachromosomal DNA C. Homoplasmic and Heteroplasmic Transformants D. Co-Transformation E. Marker Recycling V. Reverse Genetics A. Gene Inactivation B. Site-Directed Mutagenesis VI. Conclusion and Perspective Acknowledgments References

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Summary For chloroplast transformation, the most efficient method to introduce DNA is particle bombardment. To then select the cells which harbor a transformed plastid, two classes of markers are available. With one class, selection is based on the rescue of a non-photosynthetic mutant with the wild-type chloroplast gene. With the other class, selection is based on a mutation or a foreign gene conferring resistance to an antibiotic or a herbicide. Transforming DNA is integrated by homologous recombination, and only in exceptional cases is it maintained extrachromosomally. The modified and wild-type copies of the highly polyploid plastid genome usually segregate rapidly, although in some circumstances a heteroplasmic mixture of genomes is retained. The available technology and markers readily allow chloroplast gene inactivation and site-directed mutagenesis. These possibilities are enhanced by strategies such as co-transformation or the repeated use of unstable markers.

J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria im Chlamydomonas, pp. 139–149. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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I. Introduction

II. Delivery of DNA to the Chloroplast

Stable transformation of the chloroplast involves three main steps: the introduction of DNA into the organelle, the expression of a marker for selection, and the replication of the introduced DNA. The major challenge has been the first step, the delivery of DNA across the cell wall and three membranes: the plasma membrane and the two chloroplast envelopes. The breakthrough for chloroplast transformation came with the development of microprojectile bombardment for DNA delivery (Klein et al., 1987; Boynton et al., 1988). The transforming DNA has to carry a marker that can be selected, or screened for, to identify the transformants. Wild-type copies of photosynthesis genes were used as the original selectable markers, with the corresponding photosynthesis mutants as hosts. Mutations conferring resistance to translation inhibitors or to photosynthesis inhibitors have also proven useful for selection, and engineered bacterial genes have provided further markers and reporters. The stable propagation of the introduced DNA results from its integration into the plastid genome, a step which is facilitated by the recombination machinery active in the chloroplast. The chloroplast genome is present in approximately eighty copies in the single chloroplast of C. reinhardtii. The high level of ploidy implies that in the initial stages after transformation, the plastid is heteroplasmic: it contains both wildtype and modified copies of the genome. This may hamper the expression of recessive markers, but the problem is alleviated by the rapid segregation that usually occurs. With the appropriate tools available, targeted gene inactivation and site-directed muta­ genesis of chloroplast genes have become routine experiments. In a very short time, an extraordinary amount of knowledge has been gained using this technology, mostly in the fields of photosynthesis and of chloroplast gene expression. The pioneering work on chloroplast transformation in Chlamy­ domonas has established strategies that have also been applied fruitfully for the transformation of tobacco plastids (Svab et al., 1990; Svab and Maliga, 1993).

Ten years after its original description, ‘biolistic’ bombardment with DNA-coated microprojectiles remains the method of choice for chloroplast transformation (Boynton et al., 1988). The alternative is vortexing in the presence of glass beads (Kindle et al., 1991), but this is less efficient and requires cellwall deficient algae, either following treatment with a preparation of autolysin (gamete lytic enzyme) to degrade the cell wall or due to a nuclear cw mutation in the host. Biolistic transformation involves the bombardment of cells with micron-sized microprojectiles carrying the transforming DNA. To reduce drag on the microprojectiles, the procedure is performed in a partly evacuated chamber. Acceler­ ation of the microprojectiles is commonly based on one of two principles. In the first, the microprojec­ tiles are deposited on a macroprojectile (a plastic bullet), or on a disk, which is accelerated and then stopped in flight by a perforated stopping plate or a mesh that allows the microprojectiles to continue their trajectory to the target cells. In the first generation of gene guns, the macroprojectile was accelerated by the explosion of a charge of gunpowder (Klein et al., 1987; Zumbrunn et al., 1989). More recent versions use a burst of compressed air or helium to propel the macroprojectile or a plastic membrane disk (reviewed by Sanford et al., 1993). The alternate principle is to accelerate the microprojectiles directly in a flow or a sudden burst of high-pressure gas, usually helium (Finer et al., 1992; Takeuchi et al., 1992).

Abbreviations: AAD – aminoglycoside 3´´-adenyl transferase ( or aminoglycoside 3´´-adenylyl transferase); GUS – -Glu­ curonidase; PCR – polymerase chain reaction; rDNA – ribosomal RNA gene cluster; RFLP – restriction fragment length poly­ morphism; rRNA – ribosomal RNA

III. Selectable Markers and Reporters

A. Selectable Markers 1. Photosynthesis Several decades of genetic research in C. reinhardtii have provided a rich collection ofchloroplast mutants with defects in photosynthesis. These mutants can be grown in the presence of acetate, but not on minimal medium in the light. Many of the corresponding chloroplast genes have been identified and cloned, providing a source of selectable markers for transformation of the corresponding mutant hosts: transformants can be selected for phototrophy on minimal medium. This is the strategy that was used in the original demonstration of chloroplast

Chapter 8

Chloroplast Transformation

transformation (Boynton et al., 1988). A mutant with a deletion of the atpB gene was rescued by bombardment with a plasmid containing a wild-type fragment covering the deletion, with segments of homology on either side allowing homologous recombination. An advantage of using a deletion mutant as a host is the lack of reversion. A similar strategy has also been used with mutations in a variety of other photosynthesis genes (for thorough reviews see Boynton and Gillham, 1993; Erickson, 1996). A variation on this theme is the use of the tscA gene as a marker in tscA deletion hosts (GoldschmidtClermont et al., 1991; Kindle et al., 1991, Choquet et al., 1992). The tscA RNA is required for transsplicing of the psaA RNA (Chapter 11, Herrin et al.), and thus indirectly for photosynthesis: the lack of tscA prevents the accumulation of PSI, so that selection for phototrophic growth can also be applied with this marker. However some mutants with deletions of the tscA locus, such as H13, have complex rearrangements of the chloroplast DNA (Choquet et al., 1992), and the marker is not simply integrated in replacement of the deletion when vectors such as rbcX or atpX are used (Goldschmidt-Clermont, 1991). Another variation is based on the light sensitivity of certain photosynthesis mutants, like those that are defective in Photosystem I. These mutants fail to grow at high light intensities even when acetate is provided in the medium. Transformants that have regained an active gene loose their light sensitivity and can be selected on acetate medium in the light (Redding et al., 1998; M. Fleischmann, N. Fischer and J. -D. Rochaix, personal communication). The two repeated copies of the psbA gene of C. reinhardtii contain four introns and span 6.5 kb, properties that complicate site-directed mutagenesis. However intron-less derivatives of the psbA gene can be used for transformation, and the resulting strains that lack all the introns have apparently normal phenotypes (Johanningmeier and Heiss, 1993; Minagawa and Crofts, 1994; Mayfield et al., 1994).

141 Kindle et al., 1991; Newman et al., 1991; Roffey et al., 1991). Likewise mutations of the psbA gene that confer resistance to a herbicide such as metribuzin or DCMU can be used for selection (Przibilla et al., 1991; Newman et al., 1992). The obvious advantage of drug resistance over selection for photosynthetic activity is that the host need not carry a mutation. The markers are integrated by homologous recom­ bination, replacing the wild-type (drug sensitive) copies of the corresponding genes. However herbicide resistance and also spectinomycin resistance mutations can have effects on photosynthetic activity that should be taken into account by using appropriate control strains in structure-function studies of the photosynthetic complexes (Lers et al., 1992; Monod et al., 1994; Heifetz et al., 1997; M. Fleischmann and J.-D. Rochaix, personal communication). Another strategy to obtain resistance is to express a gene product that can inactivate an antibiotic. This was achieved by transformation with the bacterial aadA gene, engineered with 5´ and 3´ sequences from Chlamydomonas chloroplast genes, to obtain expression of aminoglycoside 3´´-adenyl transferase (AAD) and thus resistance of the transformants to spectinomycin and streptomycin (GoldschmidtClermont, 1991). For the commonly used atpXAAD construct, the predicted protein product is a fusion ofAAD to the first 25 N-terminal amino acids of AtpA (Leu et al., 1992). In other constructs, the normal AAD protein is expressed by fusion at the level of the translation initiation codon. The aadA cassette provides a dominant marker which is portable since it can be integrated at virtually any site in the chloroplast genome if flanking regions of homology are provided for homologous recombination. For gene inactivation or site-directed mutagenesis, this has the advantage of allowing the marker to be linked to the gene of interest, while the use of resistance mutations in the rDNA generally calls for cotransformation approaches (Section IV.D). New versions of the aadA cassette that allow its excision permit repeated rounds of transformation with the same marker (‘recycling’) as discussed below (Section IV.E; Fischer et al., 1996).

2. Drug Resistance B. Reporters and Foreign Gene Expression Another approach has been to use mutations that confer resistance to various inhibitors. Mutations in the rRNA genes that bestow resistance to spectino­ mycin, streptomycin or erythromycin have been used to transform wild-type cells (Newman et al., 1990;

If regions of homology are provided for homologous recombination, foreign DNA can be stably integrated into the chloroplast genome (Blowers et al., 1989). Chimeric genes with alien DNA downstream of a

142 Chlamydomonas chloroplast promoter are trans­ cribed. The presence or addition of sequences from the 3´ end of a chloroplast gene can provide signals for the formation of a stable RNA with apparently homogeneous 3´ ends as assayed by Northern analysis (Chapter 10, Stern and Drager). Such chimeric transcripts with the sequences of bacterial genes like nptII, uidA, or aadA accumulate in the transformed cells (Blowers et al., 1989; 1990; GoldschmidtClermont, 1991; Klein et al., 1992). Chimeric RNAs with a mitochondrial intron from Scenedesmus obliquus or with the atpF gene of spinach are also expressed in Chlamydomonas chloroplasts (Herden­ berger et al., 1994; Deshpande et al., 1995). The S. obliquus intron is spliced in Chlamydomonas, but not the intron in the spinach atpF gene (Chapter 11, Herrin et al.). Transgenic expression of a foreign gene in the chloroplast to produce a functional protein was first demonstrated in Chlamydomonas with the aadA cassette by demonstrating AAD activity in crude extracts of transformants (Goldschmidt-Clermont, 1991). Using constructs with the uidA gene, glucuronidase (GUS) activity can also be measured in extracts from transformed cells (Sakamoto et al., 1993). These genes can thus be used as reporters of gene expression for the analysis of transcription, RNA processing, RNA stability and translation under the control of a variety of promoters as well as 5´and 3´-untranslated sequences (Sakamoto et al., 1993; Nickelsen et al., 1994; Zerges and Rochaix, 1994; Zerges et al., 1997; Stampacchia et al., 1997). With the aadA cassette, the level of resistance of the transformants to different concentrations of spectinomycin or streptomycin for growth on solid media can provide a rough indication of the expression level of the AAD reporter. The bacterial recA gene from E. coli has also been expressed in the Chlamydomonas chloroplast (Cerutti et al., 1995). Production of the wild type bacterial RecA protein leads to an increase in chloroplast recombination, but expression ofa dominant negative mutant RecA inhibits recombination. These results elegantly show that the transgenic RecA is functional in Chlamydomonas. It is also noteworthy that different truncated mutant forms of transgenic RecA accumulate to reduced levels compared to the wildtype, as assayed by immunoblotting. Because these proteins are expressed under the control of the same elements and the mRNA levels are similar, these

Michel Goldschmidt-Clermont differences in accumulation suggest that the truncated proteins have different degrees of susceptibility to proteolytic degradation in the chloroplast. Thus post­ translational events could also be important for achieving optimal transgenic expression, in addition to factors such as the rate of transcription, the processing and stability of the mRNA, and the efficiency of translation initiation and elongation. For genetic engineering it may be important to investigate the contribution of these different steps, since in many attempts to develop new markers, or to express genes in the chloroplast, the product was poorly expressed or undetectable.

IV. Fate of Transforming DNA

A. Integration in the Chloroplast Genome In most cases, the analysis of stable transformants shows that the introduced DNA has recombined with the chloroplast genome. This process requires homology between the transforming DNA and the recipient. Empirically, it seems that the length of homology has to be in the order of one kilobase for efficient transformation, but shorter segments are known to be sufficient for homologous recombination (Section IV.E). When the selectable marker or mutation is flanked by a region of homology on only one side, integration is the apparent result of a single crossover with a duplication ofthe region ofhomology (Fig. 1; Kindle et al., 1991). This type of integration is reversible and thus genetically unstable, since recombination between the direct repeats will lead to excision ofthe marker (Boynton and Gillham, 1993). When there is homology on both sides ofthe marker, DNA integration is formally the result of a double crossover. The actual mechanism is not known, and could involve exchanges on both sides of the marker or gene conversion. Most transformation experiments have been done with circular plasmid DNA. Transformation with linearized plasmid DNA can be more efficient when there is sequence homology on both sides of the marker (Blowers et al., 1989). Conversely linear DNA is not as effective for transformation when it has homology on only one side ofthe marker (Kindle et al., 1991). The sites of recombination have been mapped in transformation experiments using the genes for

Chapter 8

Chloroplast Transformation

ribosomal RNA marked with multiple drug resistance mutations and restriction fragment length poly­ morphisms (RFLP; Newman et al., 1990). Even though circular plasmid DNA is used for trans­ formation, a bias is observed for recombination events that occur near the junctions of the chloroplast DNA with the plasmid vector, and result in the integration of long segments of transforming DNA. The remaining exchange events are not randomly distributed along the region. Likewise, a segment where recombination occurs preferentially (‘hot­ spot’), both in sexual crosses and in transformation experiments, has been identified in the 3´ part of the psbA gene (Newman et al., 1992). Transformation of tobacco plastids also results in the incorporation of long segments of DNA (Staub and Maliga, 1992). The introduction of chimeric constructs that use chloroplast DNA fragments to direct gene expression generates duplications of these segments, which are usually already present in the host genome. Recombination between the introduced copy and the endogenous one can lead to rearrangements or deletions. A chimeric construct, with sequences of atpA driving the expression of aadA, can be inserted between psaB and rbcL. The introduced atpA segment can then recombine with the nearby atpA gene so that a deletion of around 2 kb is generated and

143

maintained as a minor heteroplasmic component (Bingham and Webber, 1994; heteroplasmic versus homoplasmic transformants are discussed in Section IV.C). Likewise, when an atpA-aadA cassette replaces the psbl gene, the atpA segment recombines with the nearby atpA gene to generate a stable, homoplasmic deletion of around 2kb (Künstner et al., 1995). A duplication of a sequence from psbC introduced at the psaB locus can recombine with the endogenous psbC gene more than 60 kb away, generating a minor population of fragmented genomes (Fischer et al., 1996). Similar alterations have also been described in tobacco chloroplast transformants (Svab and Maliga, 1993). Depending on the relative position and orientation of the duplicated sequences, the distance between them, and the effect of the rearrangements on chloroplast DNA maintenance and gene expression, there may or may not be selective pressure against the rearranged genomes. Thus in some cases a homoplasmic situation will be reached, while in others the altered genome will only be apparent as a component of a heteroplasmic mixture.

B. Extrachromosomal DNA A circular plasmid DNA with the atpB gene as a selectable marker can propagate in its free form after

144

transformation of an atpB deletion host (Boynton et al. 1988). However when the cells are transferred to solid medium, the free plasmid is lost, and only the integrated gene copy is retained. To my knowledge, attempts to create a stable replicating vector for the chloroplast of Chlamydomonas have not yet been successful (e.g., Suzuki et al., 1997). In tobacco plastids, following transformation with a segment from the rDNA locus, small extrachromosomal circular DNAs, constituted of monomers and multimers of a plastid DNA segment, have been observed (Staub and Maliga, 1994). This segment has been used as part of a shuttle vector that can be propagated in E. coli. After transformation of tobacco plastids with the shuttle vector, extrachromosomal plasmids and copies integrated in the genome are present, as well as non-transformed plastid genomes. It is therefore unclear whether the shuttle vector replicates autonomously, or is being continuously generated by excision from the transformed chloroplast genomes. An intriguing phenomenon of gene amplification has been observed in transformation experiments with deleted versions of atpB that lack part of the inverted repeat at the 3´ end of the mRNA (Kindle et al., 1994). Rescue of the photosynthetic defect of an atpB mutant host gene with some of these 3´ end truncations is only partial. The transformants show poor photoautotrophic growth which is attributed to the accumulation of reduced amounts of atpB transcripts which are unstable and ofvariable length, and hence of reduced levels of the subunit ofATP synthase (Chapter 10, Stern and Drager). However ‘robust’ transformants that have better photosyn­ thetic capability and wild-type levels ofthe subunit to In these arise at a frequency of transformants, tandem arrays of fifteen or more copies of the transforming plasmid increase the total copy number of the truncated atpB gene to 10–30 copies per chloroplast genome, and thus probably to thousands per chloroplast. Although these arrays appear to be episomal, a copy of the transforming plasmid is integrated (Suzuki et al., 1997). In addition, there are also complex rearrangements at the atpB locus in the robust transformants. The amplified DNA could be replicating autonomously, or could be continuously generated from the integrated copy. The transformed strains exhibit genomic instability in sexual crosses, and the amplified arrays are lost following new rounds of transformation. These two types of instability limit the potential of the amplified

Michel Goldschmidt-Clermont

units for genetic engineering aimed at the overexpression of gene products in the chloroplast.

C. Homoplasmic and Heteroplasmic Transformants In each C. reinhardtii cell, the genome of the single chloroplast is present in approximately eighty copies. This high degree of ploidy has important conse­ quences for transformation. Following delivery, the initial event is presumably integration of DNA into one copy of the chloroplast genome. Depending on its degree of dominance, a selectable marker will be expressed phenotypically if it is carried by a sufficient fraction of genome copies in the subsequent growth of the host cell or some of its descendants. The segregation process is stochastic but can be influenced by selection or screening of desired markers, it eventually leads to the appearance of segregants that have a uniform genetic content. In most cases, segregation of markers is rapid, suggesting that in addition to assortment of the genomes at each division, gene conversion is involved in the process. If the marker is introduced in the inverted repeat, both copies are identical in the transformants, demonstrating that an active mechanism of copy correction is operating (Newman et al., 1990). When all the copies ofthe chloroplast genome are identical, the transformant is termed homoplasmic. Transformation frequencies can be improved if the host cells are pretreated with FUdR (5­ fluorodeoxyuridine), an inhibitor of chloroplast DNA replication (Newman et al., 1990; Kindle, 1991; Boynton and Gillham, 1993). This treatment reduces the copy number of the chloroplast genomes, and thus probably facilitates the segregation and expression of recessive markers in the first stages of transformation. Because FudR is mutagenic, and because transformation frequencies are not limiting for most experiments, the treatment is not used widely. In some cases, both wild-type and recombinant copies are maintained in the transformants in spite of selection, and a heteroplasmic state persists. The heteroplasmic situation is genetically unstable, and the marker is lost if selective pressure is removed. Persistent heteroplasmy in gene disruption experi­ ments with the aadA cassette has been interpreted as revealing genes that have a vital function for the cell. Selection for spectinomycin resistance requires the maintenance of some copies of the chloroplast genome with the gene disruption and the aadA gene,

Chapter 8

Chloroplast Transformation

while the vital function requires wild-type copies. Persisting heteroplasmic states have been observed with a disruption of ORF472 (GoldschmidtClermont, 1991), but sequencing of the locus has recently shown that ORF 472 is actually a part of the 3119 bp rpoC2 gene (S. Nuotio and S. Purton, personal communication). Indeed disruptions of the genes encoding subunits of RNA polymerase, rpoB1, rpoB2 and rpoC2 remain heteroplasmic, as well as those of rps3 (a ribosomal protein), of clpP (a subunit of ClpP protease), and of an open reading frame (ORF 1995) of unknown function (Liu et al, 1993; Huang et al., 1994; Rochaix, 1995; Boudreau et al., 1997). Presumably these genes are important for the survival of the cell even on acetate-containing medium in the dark, growth conditions where disruptions of genes involved in photosynthesis, or in chlorophyll synthesis, readily become homoplasmic. An alternate explanation, that the gene disruptions remain heteroplasmic because they affect genes required for the expression of the aadA marker itself, appears plausible in some cases but less likely for clpP (Chapter 10, Stern and Drager). A somewhat different situation prevails in tobacco, where a disruption mutant of the rpoB gene becomes homoplamic and is viable in culture on sucrose-containing medium, although it is not competent for photosynthesis (Allison et al., 1996).

D. Co-Transformation When two different markers are introduced on separate vector molecules, there is a high frequency of events (up to 80%) where both are incorporated in the same transformant (reviewed by Boynton and Gillham, 1993; Webber et al., 1995; Erickson, 1996). This suggests that a limiting step for transformation is delivery of DNA to the chloroplasts rather than its integration and segregation. It is not known whether all the cells and their single chloroplasts in a population are competent for transformation. The high frequency of co-transformation is useful for directed mutagenesis of the chloroplast genome, since it allows strategies where the selectable marker and the mutation of interest are in different loci (Kindle et al., 1991; Newman et al., 1991; Roffey et al., 1991). Cells are bombarded with a mixture of two separate DNA molecules, one with a marker conferring resistance to an antibiotic and the other with the gene carrying the mutation of interest. The antibiotic resistance marker is usually a mutation in

145 the rDNA or the insertion of an aadA cassette in a genetically silent position of the genome. Transformants are selected on medium containing the antibiotic, and co-transformants which have also incorporated the second mutation in at least some copies of the genome are identified by testing their genotype (colony hybridization, Southern blotting, PCR), or when readily apparent, their phenotype. Subculturing of the co-transformants and testing then leads to the identification of strains that are homoplasmic for the unselected mutation.

E. Marker Recycling For some applications, it is useful to introduce several modifications in the chloroplast genome in successive rounds of transformation. When photosynthesis markers cannot be used because the genes under study are also involved in photosynthesis, there is currently a scarcity of suitable selectable markers: the most convenient rDNA marker confers resistance to spectinomycin, like the aadA cassette. This limitation can be overcome by using strategies where the selectable cassette is introduced in a genetically unstable form, and can thus be lost from the transformed strains before being re-used in the next round of transformation and selection (Fischer et al., 1996; Redding et al., 1998). Two such strategies, dubbed ‘marker recycling,’ have been developed. One relies on recombination between direct repeats, the other on heteroplasmic transformants. The first approach is to use an aadA cassette flanked by direct repeats for transformation, with selection for spectinomycin resistance (Fig. 2). When selective pressure is removed, recombination between the direct repeats leads to excision and loss of the marker, so that a spectinomycin sensitive strain can be obtained for the next step of transformation. While a 483 bp repeat (bacterial plasmid DNA) leads to efficient excision, a repeat of230 bp from the atpA promoter is not sufficient. A direct repeat of 216 bp flanking an aadA cassette inserted in the chlN gene does allow recombination and the appearance of green cells that have regained the ability to synthesize chlorophyll in the dark (Cerutti et al., 1995), so the precise length required may depend on the sequence used. Another factor which may influence how readily genomes with the excision are segregated and recovered is the recessive versus dominant nature of the marker being scored: spectinomycin sensitivity versus chlorophyll synthesis respectively in the

146

examples above. This is because spectinomycin sensitivity will not become phenotypically apparent until most cells are nearly homoplasmic, while chlorophyll synthesis in a sector of a colony can probably be scored even if the cells are still heteroplasmic. The second approach is to co-transform with the construct of interest and with the aadA marker inserted in a gene where it remains heteroplasmic (Fig. 3). Appropriate screening and subculturing leads to strains which are homoplasmic for the desired mutation, but heteroplasmic for the aadA cassette. On removal of selection for spectinomycin resistance, the cassette is rapidly lost, and a spectinomycin sensitive yet mutant strain can be obtained for the next round of transformation.

Michel Goldschmidt-Clermont

Chapter 8

Chloroplast Transformation

The recombination method is experimentally more direct: selection for the marker which is closely linked to the mutation of interest rapidly leads to strains that are homoplasmic for the mutation. However after excision, one copy of the direct repeat remains in the mutant genome (Fig. 2). The second method requires co-transformation, and thus calls for more laborious screening to obtain a homoplasmic mutant, but avoids the presence of any extraneous DNA in the mutant genome.

V. Reverse Genetics

147 advantages for subsequent site-directed mutagenesis: one is that there is no need to repeatedly subculture the transformants to obtain homoplasmic strains lacking any wild-type copy. The other is that there is no risk of obtaining recombinants that carry the selectable marker but not the desired mutation. If a selectable marker can be placed in close proximity to the gene of interest without affecting the phenotype, then the selection of the mutated strains is direct and efficient. Elegant vectors with engineered restriction sites, that facilitate mutagenesis and mutant analysis, and with the aadA cassette for selection have been constructed for psbA, psaA and psaB (Minagawa and Crofts, 1994; Redding et al., 1998).

A. Gene Inactivation The functions of the products from numerous chloroplast genes and open reading frames have been investigated by transformation-mediated disruption (see Rochaix, 1997, for a recent compilation). These disruptions are obtained with two main strategies: one is co-transformation of a plasmid carrying the altered gene, flanked by regions of homology for recombination on both sides, together with a plasmid carrying the selectable marker. The other is to insert within the gene a selectable aadA cassette, or to substitute the gene with a cassette. Variations ofboth ofthese strategies to allow recycling of the marker have been used to generate deletions of photosynthesis genes (Section IV.E). If the gene that is targeted has a non-essential or conditional phenotype, such as acetate requirement or lightsensitivity, homoplasmic transformants are obtained that allow a detailed analysis. However when the disruption remains heteroplasmic, little information can be derived other than inferring that the gene may have an essential function (Section IV.C).

B. Site-Directed Mutagenesis The development of efficient tools for chloroplast transformation allows one to obtain routinely sitedirected mutations in virtually any gene of interest. Structure-function analysis of many components of the photosynthetic complexes and of Rubisco are described in the corresponding chapters of this book. The most efficient strategy is probably to first generate a complete deletion of the gene of interest using the recyclable marker approach, unless, as is the case for psbA, a classical deletion mutant is available. The use of a deletion mutant as the host has two main

VI. Conclusion and Perspective With the available bombardment technology and selectable markers, chloroplast transformation frequencies are more than adequate for most experiments: hundreds of transformed colonies are usually recovered quite readily. Homologous recombination and gene conversion greatly enhance the usefulness of chloroplast transformation because they lead to predictable insertion of the introduced DNA at sites of homology, and because they contribute to the rapid segregation of transformed genomes. With these properties C. reinhardtii is a very useful organism to investigate the chloroplast genome, the expression of chloroplast genes, as well as the role of the proteins encoded and the relations of their structure and function. Indeed research projects that use C. reinhardtii chloroplast trans­ formation have flourished, and a large number of reports citing these approaches are in the published literature. Nevertheless, despite this bright perspective, there is still room for technical improvement. It would be very useful to develop an inducible or repressible gene expression system for the chloroplast. This would be of help to analyze the function of essential genes, since conditional mutants could be obtained. Homoplasmic cell lines would be viable under inducing conditions, but the gene could be turned off or repressed to investigate the resulting phenotype. There is also a need for additional selectable markers, even though marker recycling alleviates the problem. Ideally new markers should be unrelated to photosynthesis, dominant, and portable to any site in the plastid genome. There are still unresolved

148 problems with the production of foreign proteins in the chloroplast. This may be due to suboptimal gene expression, and perhaps also to post-translational events, including protein degradation. To date, only foreign proteins of bacterial origin (AAD, GUS and RecA) have been expressed successfully in the chloroplast of C. reinhardtii (Section III.B). To my knowledge, there are no reports of the introduction of nuclear genes in the chloroplast resulting in the accumulation of their protein products, although a chimeric protein consisting of plastocyanin fused to AAD did accumulate as assayed by immunoblotting (N. Rolland and J.-D. Rochaix, personal communi­ cation). A better understanding of chloroplast gene expression and post-translational events will be of prime interest in fundamental research, but also for applications in biotechnology. Transformation will be a basic tool in this endeavor, and in turn the technique will benefit from its results.

Acknowledgments I thank Nicolas Roggli for his expert help in preparing the figures. Experimental work was supported by the Swiss National Fund for Scientific Research (3134014.92).

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Chapter 8

Chloroplast Transformation

Lers A, Heifetz PB, Boynton JE, Gillham NW and Osmond, CB (1992) The carboxyl-terminal extension of the D1 protein of Photosystem II is not required for optimal photosynthetic performance under and light-saturated growth conditions. J Biol Chem 267: 17494–17497 Leu S, Schlesinger J, Michaels A and Shavit N (1992) Complete DNA sequence of the Chlamydomonas reinhardtii chloroplast atpA gene. Plant Mol Biol 18: 613–616 Liu X-Q, Huang C and Xu H (1993) The unusual rps3-like orf712 is functionally essential and structurally conserved in Chlamydomonas. FEBS Lett 336: 225–230 Mayfield SP, Cohen A, Danon A and Yohn CB (1994) Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5´ untranslated region. J Cell Biol 127: 1537–1545 Minagawa J and Crofts AR (1994) A robust protocol for sitedirected mutagenesis of the D1 protein in Chlamydomonas reinhardtii: A PCR-spliced gene in a plasmid conferring spectinomycin resistance was introduced into a psbA deletion strain. Photosynth Res 42: 121–131 Monod C, Takahashi Y, Goldschmidt-Clermont M and Rochaix JD (1994) The chloroplast ycf8 open reading frame encodes a Photosystem II polypeptide which maintains photosynthetic activity under adverse growth conditions. EMBO J 13: 2747– 2754 Newman S, Boynton JE, Gillham NW, Randorph-Anderson BL, Johnson AM and Harris EH (1990) Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: Molecular and genetic characterization of integration events. Genetics 126: 875–888 Newman S, Gillham NW, Harris EH, Johnson AM and Boynton JE (1991) Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii. Mol Gen Genet 230: 65–74 Newman S, Harris EH, Johnson AM, Boynton JE and Gillham NW (1992) Nonrandom distribution of chloroplast recom­ bination events in Chlamydomonas reinhardtii: Evidence for a hotspot and an adjacent cold region. Genetics 132: 413–429 Nickelsen J, van Dillewijn J, Rahire M and Rochaix JD (1994) Determinants for stability of the chloroplast psbD RNA are located within its short leader region in Chlamydomonas reinhardtii. EMBO J 13: 3182–3191 Przibilla E, Heiss S, Johanningmeier U and Trebst A (1991) Sitespecific mutagenesis of the D1 subunit of Photosystem II in wild-type Chlamydomonas. Plant Cell 3: 169–174 Redding K, MacMillan F, Leibl W, Brettel K,Hanley J, Rutherford AW, Breton J and Rochaix J-D (1998) A systematic survey of conserved histidines in the core subunits of Photosystem I by site-directed mutagenesis reveals the likely axial ligands of P700. EMBO J 17: 50–60 Rochaix J-D (1995) Chlamydomonas reinhardtii as the

149 photosynthetic yeast. Annu Rev Genet 29: 209–230 Rochaix J-D (1997) Chloroplast reverse genetics; New insights into the function of plastid genes. Trends Plant Sci 2: 419–425 Roffey RA, Goldbeck JH, Hille CR and Sayre RT (1991) Photosynthetic electron transport in genetically altered Photosystem II reaction centers of chloroplasts. Proc Natl Acad Sci USA 88: 9122–9126 Sakamoto W, Kindle K and Stern DB (1993) In vivo analysis of Chlamydomonas chloroplast petD gene expression using stable transformation of beta-glucuronidase translational fusions. Proc Natl Acad Sci USA 90: 497–501 Sanford JC, Smith FD and Russell JA (1993) Optimizing the biolistic process for different biological applications. Methods in Enzymol 217: 483–509 Stampacchia O, Girard-Bascou J, Zanasco J-L, Zerges W, Bennoun P and Rochaix J-D( 1997) A nuclear-encoded function essential for translation of the chloroplast psaB mRNA in Chlamydomonas. Plant Cell 9: 773–782 Staub JM and Maliga P (1992) Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation. Plant Cell 4: 39–15 Staub JM and Maliga P (1994) Extrachromosomal elements in tobacco plastids. Proc Natl Acad Sci USA 91: 7468–7472 Suzuki H, Ingersoll J, Stern DB and Kindle KL (1997) Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts. Plant J 11: 635–648 Svab Z and Maliga P (1993) High-frequency plastid trans­ formation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90: 913–917 Svab Z, Hajdukiewicz P and Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87: 8526–8530 Takeuchi Y, Dotson M and Keen NT (1992) Plant transformation: A simple particle bombardment device based on flowing helium. Plant Mol Biol 18: 835–839 Webber AN, Bingham SE and Lee H (1995) Genetic engineering of thylakoid protein complexes by chloroplast transformation in Chlamydomonas reinhardtii. Photosynth Res 44: 191–205 Zerges W and Rochaix JD (1994) The 5´ leader of a chloroplast mRNA mediates the translational requirements for two nucleusencoded functions in Chlamydomonas reinhardtii. Mol Cell Biol 14: 5268–77 Zerges W, Girard-Bascou J and Rochaix J-D (1997) Translation of the chloroplast psbC mRNA is controlled by interactions between its 5´ leader and the nuclear loci TBC1 and TBC3 in Chlamydomonas reinhardtii. Mol Cell Biol 17: 3440–3448 Zumbrunn G, Schneider M and Rochaix J-D (1989) A simple particle gun for DNA-mediated cell transformation. Technique 1: 204–216

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Chapter 9 Chloroplast RNA Stability Jörg Nickelsen

Allgemeine Botanik, Ruhr-Universität Bochum,

Universitätsstr. 150, D-44780 Bochum, Germany

Summary I. Introduction II. Cell Cycle Dependent Regulation of Chloroplast RNA Stability III. Nuclear Mutants Affected in Chloroplast RNA Stability IV. Towards a Molecular Model of Chloroplast RNA Stabilization/Degradation A. Cis-acting Elements 1. The 3´ Untranslated Regions 2. The 5´ Untranslated Regions B. Trans-acting Factors C. Chloroplast Ribonucleases D. RNA Stability and Translation V. Conclusions and Perspectives Acknowledgments References

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Summary The potential importance of RNA stability to the regulation of chloroplast gene expression is documented by recent work on the molecular aspects of chloroplast biogenesis. In Chlamydomonas reinhardtii, two lines of evidence support the idea that gene-specific regulated RNA decay plays a crucial role in determining the different steady-state levels of single transcripts. First, during the cell cycle, a light-dependent and/or circadian control of RNA accumulation takes place that appears to involve both transcriptional and posttranscriptional mechanisms. Secondly, numerous well-characterized photosynthetic nuclear mutants exhibit defects in the stabilization of specific Chloroplast transcripts. This further substantiates the significance of gene-specific aspects of chloroplast RNA metabolism. The recently developed chloroplast and nuclear transformation techniques, combined with appropriate in vitro RNA degradation and RNA binding assays, now allow the identification of the cis-acting RNA elements involved and the associated trans-acting factors. Recent evidence suggests that both 3´ and 5´ untranslated regions contain determinants for mRNA stability. The cloning of nuclear genes affecting chloroplast RNA turn­ over has led to a molecular model of multisubunit complexes mediating RNA stabilization. Furthermore, this work provides the platform for molecular links to subsequent steps of gene expression, such as transcript 5´ processing and translation. The overall picture emerging is that of a complex molecular network in which a number of gene-specific regulatory mechanisms are involved.

J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 151–163. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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I. Introduction The coordinated expression of both nuclear and organellar genes results in the constitution of a functional chloroplast capable of performing photosynthesis. The different levels of gene expression that could serve as targets mediating this coordinate regulation are comprised oftranscriptional as well as posttranscriptional steps within the two cell compartments. In higher plants, the expression of nuclear genes for plastid proteins is mainly controlled at the transcriptional level (Kuhlemeier, 1992); however, recent evidence indicates that regulated RNA turn­ over also contributes to the establishment of particular RNA steady-state levels (Abler and Green, 1996). In contrast, within the plastid, regulation of gene expression has been shown to be mostly dependent on posttranscriptional processes like RNA stabili­ zation, translation or even posttranslational modifica­ tions (Gruissem and Tonkyn, 1993; Sugita and Sugiura, 1996). In Chlamydomonas reinhardtii, as compared to higher plants, relatively little is known concerning the control of nuclear gene expression. Thus, although transcriptional regulation has been demonstrated for Cab or RbcS gene expression for instance, posttrans­ criptional events also appear to contribute to overall regulation of gene expression, reminiscent of the situation found in higher plants (Gagne and Guertin, 1992; Hwang and Herrin, 1994; Jacobshagen et al., 1996). Furthermore, recent genetic evidence supports the idea that differential RNA stabilization might play a crucial role at least for the expression of some nuclear genes encoding chloroplast polypeptides (Hahn et al., 1996). Within the C. reinhardtii chloroplast there is a clear preference for posttranscriptional processes acting as control points for gene expression levels (Rochaix, 1996). The aim of this chapter is to present and discuss the genetic and molecular aspects of chloroplast RNA stabilization that have arisen to date. Accordingly, I will first summarize what is known about the significance of RNA stabilization throughout the cell cycle of C. reinhardtii. Then well-characterized nuclear mutants of chloroplast RNA metabolism will be presented that have led to Abbreviations: GUS – glucuronidase; TPR – tetratricopeptide repeat; UTR – untranslated region

Jörg Nickelsen recent molecular models of how RNA can be stabilized/degraded in a regulated fashion.

II. Cell Cycle Dependent Regulation of Chloroplast RNA Stability When C. reinhardtii cultures are grown photo­ autotrophically under 12 h light/12 h dark regimes, cells divide synchronously into two to eight daughter cells at the end of the dark phase giving rise to a stepwise two- to three-fold increase in cell number. The synthesis of photosynthetic components is maximal in the middle of the light phase indicating a tight control of gene expression by light and/or an endogenous circadian mechanism (Harris, 1989). In order to dissect the regulatory checkpoints where this control is mediated in the chloroplast of C. reinhardtii, thorough work has been done on analyzing the transcription rates, mRNA levels, and polypeptide synthesis rates during the cell cycle. To quantitate processes of RNA metabolism, in general, transcription rates of particular genes are compared to the steady-state levels of the corres­ ponding transcripts. While the RNA levels are determined by Northern analysis, transcription can be followed by in vitro run-on transcription assays. For these assays, cells are permeabilized by either toluene treatment (Guertin and Bellemare, 1979) or repeated freeze and thaw cycles (Gagne and Guertin, 1992) and, subsequently, preinitiated transcripts are elongated for a limited time in the presence of radioactively labeled ribonucleotides. Resulting RNAs are visualized by hybridization with filterimmobilized DNA fragments of the gene of interest. Alternatively, transcripts may be pulse-labeled in vivo by short incubations of phosphate-depleted cells with radioactive orthophosphate (Herrin et al., 1986). When these pulse-labeled cells are subsequently kept in light or darkness for defined times, the halflives of individual transcripts under different light conditions can be followed (Salvador et al., 1993). Table 1 summarizes some of these data. Despite the large differences with regard to particular values that might have been caused by the different techniques (transcription analysis in permeabilized cells versus in vivo pulse-labeling, Sieburth et al. 1991) or strains used, some important principles are revealed. The steady-state levels of RNAs can vary to some extent during the cell cycle. While the

Chapter 9

Chloroplast RNA Stability

maximum/minimum ratio of transcript levels for some highly expressed genes like psbA, psbD, rbcL, and rrnL range only from 1.3 to 2.3/2.8, the levels of transcripts corresponding to other genes fluctuate much more, with tufA RNA representing the most striking example. This suggests that gene-specific mechanisms regulate the abundance of individual transcripts within the chloroplast of C. reinhardtii. Recent work indicates that, in some cases, the different transcript levels are not related to the cell cycle directly, but rather, are light dependent (psaB, psbA, and rbcL) or follow an endogenous circadian control (atpA, atpB, and tufA; Salvador et al., 1993). When transcription rates of the these genes are analyzed in parallel, one finds that they are maximum in the very early light phase (20 min) with a significant decline toward the end of the light period (7 h) and a subsequent increase during the next late dark phase. For tufA, rrnL, atpA, and atpB, max/min ratios of transcription rates are quite high, pointing to an important role for transcriptional control of chloroplast gene expression. However, the corres­ ponding steady-state RNA levels do not fluctuate as much, suggesting that additional processes of differential RNA stabilization are superimposed on a general effect of transcriptional control. Overall, RNA half-lives vary remarkably between the genes analyzed but are generally greater in the dark. For instance, the most stable chloroplast transcript known is the 16S rRNA, which exhibits a half-life of roughly 7 h in the light and 30 h in the dark. In contrast, tufA transcripts have a half-life of just 30 min in the light-grown versus 1.3 h in dark-

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grown cells. For rbcL RNA a six-fold increase in mRNA stability (3.5 h in light as compared to 21 h in dark) was found which represents the highest light/ dark ratio reported. Thus, enhanced RNA stabilization during the dark phase of the cell cycle appears to compensate for the drop in transcriptional activity within this period thereby keeping the steady-state levels of some highly expressed genes like psbA, psbD, and rbcL relatively constant. Interestingly, inhibition of chloroplast transcription by rifampicin treatment at the beginning of the light phase results in the subsequent loss of synthesis of the psbA and psbD gene products D1 and D2, respectively, while translation of rbcL mRNA continues (Herrin et al., 1986). This suggests that residual rbcL transcripts from the preceding cycle remain competent for translation, in contrast to psbA and psbD mRNAs, which have to be newly synthesized. The molecular basis for this difference in gene expression is not clear, but it is possible that different 5´ modifications on these mRNAs might be involved in discrimination between translatable and non-translatable chloroplast messengers (Section IV.A). For other transcripts, like tufA, probably due to their short half-lives, a compensation of reduced transcription in the dark does not take place, resulting in a diurnal fluctuation of transcript levels during the cell cycle. In conclusion, evidence obtained to date suggests a complex regulatory network determining individual transcript levels involving processes of differential transcription as well as controlled RNA stabilization. These effects are apparently superimposed upon a general increase in the transcription rate in the early

154 light phase and enhanced RNA stability in the dark.

III. Nuclear Mutants Affected in Chloroplast RNA Stability As photosynthetic function is dispensable in C. reinhardtii when cells are grown in the presence of acetate, it has been fairly easy to isolate photosynthetic mutants of C. reinhardtii (Rochaix, 1995). Among these are a number of nuclear mutants that exhibit defects in the stability of single chloroplast transcripts (Table 2). None of these mutations appear to be analogous to the recently characterized Arabidopsis mutation hcf109, which reduces the RNA accumulation of particular segments from four different plastid polycistronic transcription units (Meurer et al., 1996). However, in C. reinhardtii, gene-specific defects seem to be a general phenom­ enon of nuclear mutations affecting not only RNA stabilization, but also other steps of chloroplast gene expression (see Chapter 10, Stern and Drager; Chapter 11, Herrin et al.; Chapter 12, Hauser et al.). When the phenotypes of the C. reinhardtii mutants are analyzed in more detail, subtle differences between them become apparent. While most of the mutants fail to accumulate any RNA from the affected chloroplast genes, mutants and ncc1 still accumulate 5 and 10% of wild-type levels of petB and atpA transcripts, respectively. Interestingly, is deficient in photosynthetic activity, but in ncc1 about half of the affected ATP synthase complex assembles allowing wild-type growth rates under phototrophic growth conditions (Drapier et al., 1992; Gumpel et al., 1996). Another difference concerns the effects on polycistronic chloroplast mRNAs. For instance, in the mutants 222E and GE2.10, besides psbB RNA, additional smaller transcripts—encoded further downstream of psbB and probably cotranscribed with it—are absent, pointing to a more pleiotropic effect in this case. In contrast, psaA exon 2 sequences that are cotranscribed with the upstream psbD gene are apparently not degraded in the psbD RNA stability mutant nac2-26, since normal accumulation of mature psaA message occurs. Interestingly, the same RNA is degraded when it remains connected to psbD sequences in double mutants carrying, in addition to nac2-26, a mutation which blocks psaA RNA maturation/splicing from this dicistronic transcript. This suggests that psbD sequences can target these splicing intermediates for

Jörg Nickelsen rapid degradation in the mutant nuclear background (Kuchka et al., 1989). All of the mutants listed in Table 2 have been shown to transcribe the affected genes to nearly wild-type levels by run-on and/or in vivo pulse labeling assays. One exception is a recently described mutant called 76-5EN that accumulates only very low amounts of rbcL mRNA. Apparently, in 76-5EN, the rbcL gene is only weakly transcribed in 10 min pulse-labeling experiments but the resulting mRNA appears to remain stable for at least 1 h (Hong and Spreitzer, 1994). Whereas this mutant would represent the first example of a nuclear mutation affecting the transcription of a chloroplast gene, these results are still a matter of debate (Rochaix, 1996). It remains possible that, in 76-5EN, the rbcL mRNA was rapidly degraded and thereby escaped detection during the relatively long 10 min pulse, as has been reported for psbB transcripts in the mutant 222E (Monod et al., 1992). Since the RNA stability assays of Hong and Spreitzer (1994) were not strand-specific, one cannot exclude the possibility that the stable low abundance transcripts that were detected were generated from the opposite DNA strand. A similar situation has been found for petD gene transcription (Sturm et al., 1994). To date, it is still unknown how many nuclear genes can be involved in the stabilization of a particular chloroplast transcript. It is unlikely that mutagenesis of the nuclear genome has been performed to saturation, suggesting that there might be additional loci to be discovered. However, for the nac2-26 mutation affecting psbD RNA accumulation, a second allele has been found by analyzing the (S. Purton, unpub­ photosynthetic mutant lished). The mutants GE2.10 and 222E both exhibit a defect in psbB transcript stabilization; it is unknown yet whether they represent different alleles of the same gene.

IV. Towards a Molecular Model of Chloroplast RNA Stabilization/Degradation The combined analysis of both nuclear mutants with defects in chloroplast RNA stability and chloroplast transformants carrying reporter genes fused to putative regulatory regions now represents a powerful approach for addressing questions of the molecular basis of RNA decay in the chloroplast of C. reinhardtii. First, the target sites within chloroplast

Chapter 9

Chloroplast RNA Stability

transcripts recognized by the nuclear factors can be identified and other cis-acting determinants of RNA stability can be tested in vivo. Secondly, the cloning of the mutated nuclear genes together with recently developed in vitro RNA stabilization and RNA binding assays provide the tools for detailed characterization of the factors involved. These data can help to elucidate putative molecular connections between processes of RNA stabilization and translation (Gillham et al., 1994; Nickelsen and Rochaix, 1994).

A. Cis-acting Elements The identification of RNA elements responsible for determining the half-lives of their corresponding transcripts has been one major goal of research in chloroplast molecular biology during the last years. To date, no common motifs have been identified, but accumulating evidence suggests that almost all regions of chloroplast transcripts—namely the 5´ and 3´ untranslated regions as well as coding sequences—can be involved in RNA stabilization/ degradation.

1. The 3´ Untranslated Regions Initial work on higher plants has suggested that stem-loop structures within the 3´ untranslated regions (3´ UTR) of plastid mRNAs serve as barriers against exonucleolytic degradation of upstream sequences in vitro (Stern and Gruissem, 1987) and in organello (Adams and Stern, 1990). Furthermore, they appear to mediate correct 3´ end formation, and thus, a close

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relationship between 3´ maturation and RNA stability has been proposed. With the availability of a biolistic chloroplast transformation system for C. reinhardtii (Boynton et al., 1988) this hypothesis became testable in vivo also. One of the best characterized examples is the atpB 3´ UTR (Stern et al., 1991). Successive deletion of the atpB stem-loop structure leads to a loss of up to 80% of the atpB mRNA, indicating a significant role of this secondary structure element for RNA stabilization. In addition, the residual transcripts are heterogenous with regard to the length of their 3´ ends, indicating a role for stem-loops in RNA maturation. Interestingly, a 3´ UTR from the spinach petD gene can substitute functionally for the C. reinhardtii 3´ region, raising the possibility that common structural motifs exist in the 3´ UTRs of higher plants and algae (Stern et al., 1991). Even more surprising is the finding that a sequence of 18 guanosine nucleotides replacing the atpB stem-loop allows wild-type levels of atpB mRNA to accumulate (Drager et al., 1996). This is reminiscent of the situation in Saccharomyces cerevisiae, where polyG tracts can impede 5´–3´ exonucleases. While a significant impact of the atpB 3´ UTR stem-loop on the level of its mRNA accumulation has been documented, results obtained with chimeric GUS genes containing different 3´ regions do not support the idea that 3´ UTRs, in general, are involved in determining RNA half-lives. In these experiments, constructs consisting of the atpB 5´ region and the bacterial uidA gene, were fused to either the rbcL or the psaB 3´ flanking regions and, subsequently, introduced into the chloroplast genome. Although it

156 could be shown that the 3´ UTRs are required for correct 3´ end formation of the chimeric transcripts, the measurement of RNA half-lives after pulse labeling of transformant cells with phosphate revealed no differences between constructs harboring a 3´ region or not (Blowers et al., 1993). Contradictory results, as far as the psaB 3´ UTR is concerned, have been obtained after removal of the 3´ stem-loop structure from the endogenous psaB gene, which resulted in a 75% reduction in the halflife of its mRNA (Lee et al., 1996). It is unknown what the reason for these different findings is but one might speculate that the coding region and/or the 5´ untranslated portion of psaB, and perhaps also of other genes, affect the function of the 3´ UTR by direct and/or indirect interactions. In other words, if the rate limiting step for RNA degradation, in the case of the psaB transcript, is mediated by the 5´ region and/or coding sequences, this can be compensated for by an intact 3´ stemloop, but not by a deleted version. In this way, the chimeric mRNA might not be dependent on an appropriate 3´ end for stabilization. A role for proteincoding sequences in RNA stabilization has been suggested for psbA transcripts in spinach (Klaff 1995) and also for the rbcL gene in C. reinhardtii (Salvador et al., 1993). Moreover, recent genetic and molecular evidence indicate that the 5´ UTRs play key roles in determining the stability of at least some chloroplast RNAs.

2. The 5´ Untranslated Regions Although mRNA stability has been shown to be affected by alterations of cis-elements within the 3´ UTR, a complete loss of RNA has never been observed. This might be due to the use of experimental approaches that are based on the selection of chloroplast transformants by photoautotrophic growth, thereby selecting for at least partial functional activity of the transcripts analyzed. Alternatively, incomplete RNA degradation might be an inherent feature of 3´ UTR-mediated RNA destabilization. This might be further supported by the finding that in the nuclear mutant ncc1, which still accumulates 10% of wild-type atpA mRNA amounts (Table 2), RNA degradation initiates from the 3´ end of the message (Drapier et al., 1992). However, most of the other mutants analyzed (Table 2) do not accumulate any transcripts from the affected genes and, consequently, cannot grow

Jörg Nickelsen

photoautotrophically. Among these the nuclear mutant nac2-26, which exhibits defects in psbD RNA stabilization, has been analyzed in detail (Fig. 1). Chimeric reporter genes containing either the 5´ or 3´ flanking regions of the psbD gene fused to the bacterial aadA gene (conferring spectinomycin resistance; Chapter 8, Goldschmidt-Clermont) were inserted into the chloroplast genome by biolistic transformation. After crossing of the resulting transformants with nac2-26 the analysis of tetrads demonstrated a cosegregation of Photo­ system II deficiency and spectinomycin sensitivity in case of 5´ psbD-aadA fusions. Subsequent analysis of RNA levels revealed that both psbD and the chimeric transcripts are destabilized in the mutant nuclear background. In contrast, an aadA-3´ psbD chimera remained stable independent of the genetic nuclear background. This indicates that the 5´ leader region but not the 3´ UTR of the psbD transcript is sufficient to mediate accelerated RNA degradation in the absence of NAC2 function. The degradation of psbD leader RNA has also been followed in lysed

Chapter 9 Chloroplast RNA Stability chloroplasts in vitro pointing to a pathway involving at least three endonucleolytic cuts (Nickelsen et al, 1994). This resembles the situation found in bacteria, where the significance of 5´ leader regions for RNA stabilization/degradation events is widely accepted and endonucleolytic 5´ degradation has been demonstrated to occur (Bouvet and Belasco, 1992; Hansen et al., 1994). Using a similar approach, evidence has been obtained recently that in mutant 222E, in which psbB transcript stability is affected, the psbB 5´ flanking region also contains the target site for the nucleusencoded factor (F. Vaistij, M. Goldschmidt-Clermont, and J.-D. Rochaix, unpublished). Interestingly, psbB transcripts are very similar to psbD RNAs with regard to their 5´ ends. In both cases two different 5´ ends can be detected that correspond to a longer less abundant form and a shorter predominant version (Nickelsen et al., 1994; J. Nickelsen, unpublished). Although it has not yet been demonstrated directly, preliminary indirect evidence (see below) suggests that, at least in case of the psbD gene, the shorter form represents a 5´ processed form (J. Nickelsen, unpublished results). As psbA RNA messages reveal similar 5´ heterogeneity (Nickelsen et al., 1994), these three mRNAs appear to represent a special group of chloroplast transcripts. The concerted synthesis of their products and the assembly of an intermediate complex structure comprising D1, D2, and P5 proteins has been observed during Photo­ system II biogenesis (De Vitry et al., 1989). Thus, a model has been proposed in which obligate mRNA 5´ end maturation reflects a coordinate step during gene expression of psbA, psbD, and psbB, respectively (Rochaix, 1996). In order to further test this hypothesis, chloroplast transformants have been generated which contain altered psbD 5´ UTRs. Preliminary data suggest that deletion of the region from position –74 (long mRNA 5´ end) to –47 (mature mRNA 5´ end), removing all sequences unique to the larger RNA, results in loss of psbD transcripts. The psbD transcription rate in this mutant is similar to wild-type indicating the absence of putative promoter elements, within the –74 to –47 region, that might have driven the transcription of the short psbD RNA (see above) and thus, the results confirm the significance of this region for RNA processing and stabilization. Further analyses narrow down the crucial stability element to the first 12 nt of the psbD leader (J. Nickelsen and J.-D. Rochaix, unpublished). This result is somewhat

157 surprising since the processed mature form starting at position –47, which does not contain this element, accumulates as the predominant transcript. Appar­ ently, during psbD gene expression, there is a strict requirement for a 5´ processing reaction, which is impaired in the mutated versions, that stabilizes the message. This might occur, for example, if the maturation reaction is closely coupled to subsequent steps of factor assembly on the mature mRNA. Mutations introduced into the 5´ part of the endogenous psbA gene, in general, affect RNA accumulation only slightly. Two exceptions are a deletion removing the putative Shine-Dalgarno sequence, GGAG, at position –28, which results in only 20% of wild-type RNA accumulation, and a substitution of nucleotides from position –56 to –60 by GCCTC, which then allows base pairing with the GGAG motif and results in only 2% of wild-type mRNA levels (Mayfield et al., 1994). It is not clear whether these decreases in mRNA steady-state levels are a consequence of reduced translatability of the molecules. The same also applies to the psbD mutants mentioned above and the petD 5´ deletions that have been tested in vivo (Sakamoto et al., 1994). The possible connection between translation and RNA stabilization will be discussed in Section IV.D. Additional data supporting the idea of 5´ regions playing a key role in RNA degradation have been obtained when 5´ rbcL-reporter gene fusions were analyzed in vivo. Reporter gene transcripts are degraded rapidly in cells grown in the light, but are stabilized after addition of 5´ sequences from the rbcL protein-coding region (Salvador et al., 1993), again pointing to long range interactions over the whole molecule. Finally, recent work based on the analysis of reporter gene constructs revealed that in the nuclear mutant F16 (Table 2) at least one determinant for petD mRNA stabilization is also located within its 5´ leader region (Drager et al., 1998). Taken together, recent reports about the ciselements mediating the regulation of RNA accumu­ lation suggest that almost all regions of chloroplast transcripts can contain positive or negative deter­ minants of RNA stabilization. One of the key issues for the future will be to elucidate those elements which control the rate limiting steps. The 5´ regions might include some of them since mutations in these regions can lead to a complete absence of the mRNA. Furthermore, in the nuclear mutants nac2-26, 222E and F16, that mediate their RNA destabilization

158 effects via chloroplast leader regions, no transcripts from the affected genes are detectable. From a functional point of view, degradation initiated from the 5´ end might result in an immediate inactivation of translation capacity avoiding the waste of energy for the synthesis of incomplete polypeptides. In addition, such incomplete polypeptides might be deleterious when they are assembled into inactive complexes because they could sequester other subunits. It is interesting to note that in higher plants, like in E. coli, polyadenylation of chloroplast transcripts appears to accelerate degradation of RNA molecules (Kudla et al. 1996). It is not clear yet whether similar processes also occur in C. reinhardtii, but it adds an exciting novel aspect to what is known to date about RNA stabilization in chloroplasts. However, in order to understand these processes more precisely, the trans-acting factors that interact with these RNA elements will also have to be characterized.

B. Trans-acting Factors To characterize and isolate the factors mediating the differential regulation of RNA stability in chloro­ plasts, biochemical as well as genetic approaches have been initiated. In higher plant plastids, a number of different RNA binding proteins have been identified that interact specifically with the 3´ flanking regions of various chloroplast transcripts in vitro. For some of these, functions have been assigned (for a review, see Sugita and Sugiura, 1996). While RNA protein interactions within the 3´ flanking regions of higher plant plastid transcripts have been studied intensively, so far, in vitro work on C. reinhardtii has mainly focused on analyzing 5´ leader regions in RNA binding experiments (Danon and Mayfield, 1991; Zerges and Rochaix, 1994; Hauser et al., 1996). The initial goal has been to identify protein factors involved in translational regulation of chloroplast mRNAs, but as mentioned above, these regions can also serve as determinants of RNA stability, at least in C. reinhardtii. Hence, additional genetic data and/or appropriate in vitro assays are required to distinguish whether a particular RNA binding activity is involved in either of these processes. Using wild-type chloroplast lysates, the psbD leader RNA, that serves as the target site of the nucleus-encoded factor affected in the mutant nac2-26 (Table 2), was shown to interact with a 47

Jörg Nickelsen kDa protein as monitored by UV-light mediated crosslinking of RNA-protein complexes (Nickelsen et al., 1994). Both the stability of the RNA probe and the binding activity were lower when chloroplast lysates from nac2-26 were analyzed. Interestingly, this factor only bound to a probe comprising the long leader from position –74 to +1. An in vitro transcript, that corresponds to the mature form (–47 to +1) did not form a stable complex indicating that the binding site of the 47 kDa protein is not within the short leader. Because deletion of positions –74 to –47 of the psbD leader resulted in RNA destabilization in vivo (compare Section IV.A.2), confirming the significance of this region for RNA stability, the 47 kDa protein represents a good candidate for a factor involved in RNA stabilization or subsequent steps of gene expression, such as 5´ processing or translation. Binding of proteins, with molecular weights in the range of 47 kDa, to 5´ regions of various chloroplast mRNAs in C. reinhardtii has been reported (Danon and Mayfield, 1991; Zerges and Rochaix, 1994; Hauser et al., 1996; Chapter 12, Hauser et al.) raising the question of whether these binding activities are transcript-specific or not. Although only the isolation of these factors and/or their respective genes will allow one to resolve this problem, the picture emerging from competition experiments is that numerous proteins in this size range are capable of interacting with RNA molecules. Depending on the sample preparation procedure used, one might enrich for particular proteins. For example, some of the methods involve whole cell extracts further purified on heparin-affinity columns (Danon and Mayfield, 1991; Hauser et al., 1996) while others start from purified chloroplasts lysed with (Nickelsen et al., 1994) or without (Zerges and Rochaix, 1994) solubilization of membrane proteins. In addition to a biochemical approach to identify factors regulating RNA stability, the isolation of nuclear genes from mutants listed in Table 2 is underway. The Nac2 cDNA has recently been cloned by using a strategy that involves the complementation of a photosynthetic mutant with a cosmid library and subsequent isolation of the introduced DNA by plasmid rescue (J. Nickelsen and J.-D. Rochaix, unpublished). The primary structure of the cDNA revealed a polypeptide with some interesting features. The main portion of the protein consists of 9 TPR characteristic, 34 amino-acid (tetratricopeptide) motif repeats (Goebl and Yanagida,

Chapter 9

Chloroplast RNA Stability

1991). TPR domains have been found in a variety of unrelated proteins and have been proposed to mediate protein-protein interactions (Lamb et al., 1995) suggesting that the Nac2 factor could be part of a multisubunit complex. The C-terminus contains a hydrophobic stretch capable of forming a single putative membrane spanning helix, and thus this factor is tentatively classified as a peripheral membrane protein. Based on the structure of the Nac2 factor as well as on the in vivo and in vitro data concerning the psbD leader analyses, the following model could be proposed on how the stabilization of psbD RNA might be achieved at the molecular level (Fig. 2). It is assumed that the nascent 5´ end of the psbD mRNA is bound by the 47 kDa protein, which could interact either directly or indirectly with the Nac2 factor and other proteins. As a consequence the whole RNAprotein complex would be anchored in a membrane system, e.g. the envelope or the thylakoids, by the hydrophobic tail of Nac2. Data obtained so far do not allow a precise localization of the Nac2 factor, but in view of information supporting the idea of a close relationship of these two major chloroplast membrane systems, with thylakoids emerging from the envelope membrane (Hoober et al., 1994; Chapter 19, Hoober et al.), both could potentially be involved in the biogenesis of Photosystem II. The RNA binding could result in targeting of the psbD transcript to its final destination for translation following the model of cotranslational membrane insertion of D1 protein in higher plants (Kim et al., 1991). Then 5´ processing and/or ribosomal loading would take place, with 5´ maturation possibly having the function of allowing correct binding of ribosomes that might otherwise be sterically blocked by the Nac2 complex. In addition, 5´ processing might fulfill the function of regenerating active Nac2 complex that is ready for binding to another psbD mRNA molecule. The idea of a membrane-associated stabilization complex is further supported by the observation that the RNA binding activity of the 47 kDa protein can only be detected in the presence of anionic detergents like Triton X-100 (Nickelsen et al., 1994). This hypothesis, which remains to be tested, takes into account the relationship between the different processes occurring on the 74 nt psbD leader, which have to follow a highly coordinated program in terms of spatial and temporal regulation. The ongoing isolation of additional genes involved in transcript-specific RNA stabilization in C. rein­

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hardtii will add more information on the molecular aspects of this process and will allow us to elucidate whether the mechanisms involved have common features. The psbB RNA stability mutant 222E (Table 2) has recently been complemented by using an indexed cosmid library (Zhang et al., 1994) and based on the genomic subclone a cDNA clone has been isolated that complements the mutation (F. Vaistij, M. Goldschmidt-Clermont and J.-D. Rochaix, personal communication). Also, the exhibiting a defect in petA insertional mutant mRNA accumulation, has been complemented by a 18 kb wild-type genomic fragment (Gumpel et al., 1995).

C. Chloroplast Ribonucleases The current models of differential mRNA stabili­ zation in chloroplasts predict distinct RNA elements that interact with trans-acting factors to protect the RNA against nucleolytic attack. Thus, an additional determinant of transcript decay that has to be considered is the chloroplast RNA degradation machinery. Work on higher plants has recently provided insights into these ribonucleases and their molecular organization. A 54 kDa protein in mustard and a 41 kDa from spinach have been shown to have intrinsic endonucleolytic activities that could be involved in 3´ end maturation and/or RNA degradation (Nickelsen and Link, 1993; Yang et al., 1996). Furthermore, a model has been proposed in which 3´ end maturation is mediated by a high molecular weight complex containing ancillary RNA binding proteins and endo- as well as 3´–5´ exonucleases resembling RNase E and polyribonucleotide phosphorylase from E. coli, respectively (Hayes et al., 1996). Much less is known about the presence and nature

160 of ribonucleases in chloroplasts of C. reinhardtii, although it has been shown that the 3´ end formation of atpB transcripts is mediated by an endonucleolytic cut downstream of the mature 3´ terminus near an essential stem-loop structure (Section IVA.1) and subsequent 3´–5´ exonucleolytic trimming of the last 10 nt (Stern and Kindle, 1993). The activity of one or more endonucleases in chloroplast lysates has also been detected when the pathway of psbD leader RNA degradation was analyzed in vitro (Section IVA.2). No 5´–3´ exonucleolytic activities have been detected in chloroplasts to date. But, recently, the in vivo analysis of reporter genes in C. reinhardtii demonstrated that poly G tracts—that are known to impede the activity of 5´–3´ exonucleases in yeast (Vreken and Raue, 1992; Section IVA.1)—can stabilize chloroplast transcripts when they are introduced into 5´ leader regions (Drager et al., 1998), This suggests that 5´–3´ exonucleases might also exist in chloroplasts, though other expanations for the observed effects are possible. Hence, identification and characterization of chloroplast ribonucleases in C. reinhardtii represents one of the challenges for future molecular research work.

D. RNA Stability and Translation Besides the putative existence of particular RNA stabilizing protein factors interacting with their cognate target sites on chloroplast transcripts, another aspect concerning RNA turn-over is the effect of ribosome assembly on the stability of translated mRNAs. A complex relationship between these two processes has been documented in both prokaryotes and eukaryotes (Petersen, 1993; Jacobson and Peltz, 1996). Recent data on this subject from C. reinhardtii are summarized below. Impairment of translation by mutations in the psbA (Mayfield et al., 1994) and petD (Sakamoto et al., 1994) 5´ untranslated regions was accompanied by a significant decrease in RNA levels. However, as already mentioned (IVA.2), it is possible that these cis-acting mutations affect RNA stability directly. Another example is the chloroplast mutant Fud47, which harbors a 42 bp direct repeat within the psbD gene (Erickson et al., 1986) and accumulates reduced levels of psbD mRNA, suggesting a stabilization effect of polysomal assembly. Also in this case, it is possible that the direct repeat in Fud47 forms a cryptic signal leading to enhanced degradation by

Jörg Nickelsen plastid ribonucleases. A second class of translational mutants exhibits no drastic changes in the levels of the transcripts. Among these are the nuclear mutants F35 and F34 affected in translation of psbA (Girard-Bascou et al., 1992) and psbC (Rochaix et al., 1989), respectively. Also psbD ATG-initiation codon mutants that have been introduced into the chloroplast genome (J.-D. Rochaix, unpublished results) accumulate wild-type levels of psbD mRNA. Likewise, only a slight decrease in petD RNA levels was observed after changing the start codon to AUU or AUC although this led to a five- to ten-fold reduction in the trans­ lation rate of subunit IV of the cytochrome complex (Chen et al., 1993). Finally, nuclear mutants deficient in atpA and psbD translation contain increased levels of the corresponding transcripts (Drapier et al., 1992; Kuchka et al., 1988). In addition, a psaB frame shift mutation also leads to a more than twofold increase in psaB mRNA, suggesting that reduced translation can stabilize chloroplast transcripts (Xu et al., 1993). A negative effect on psaB RNA accumulation was observed when the stop codon was moved further downstream, thereby extending the translated region of the psaB protein coding region. This supports the idea of cotranslational mRNA destabilization in chloroplasts of C. reinhardtii (Lee et al., 1996). To test this hypothesis, the impact of translation inhibitors like chloramphenicol and lincomycin on the accumulation of several chloroplast transcripts was examined. Interestingly, a significant stabilization effect of both chemicals was observed for psaA and psaB RNAs, while other RNAs, e.g. rbcL and atpB, remained unaffected. This points to gene-specific mechanisms of RNA degradation that can be linked to active translation in the case of the psaA and psaB transcripts. Secondly, the data show that in these two cases, polysomal assembly does not play an important role for RNA stabilization, since chloramphenicol inhibits peptidyl-transferase activity and blocks the ribosomes on the mRNA molecules. In contrast, lincomycin acts on the formation of the first peptide bond of newly initiated polypeptides leading to polysome run-off that forces the mRNA into a non­ polysomal state. Taken together, no general relationship between RNA stability and translation appears to exist. Nevertheless, gene-specific effects suggest that, in some cases, active translation can influence the halflife of the RNA. Assuming that defined cis-acting

Chapter 9

Chloroplast RNA Stability

RNA elements mark hotspots for the regulation of RNA stability by being exceptionally accessible or, alternatively, exceptionally protected, one might speculate that the ribosome moving across these sites can increase or decrease the degradation process. This could explain why particular transcripts behave differently when they are not translated and, depending on the location of the cis-element, blocking of translation at the level of initiation, elongation, or termination could result in different effects. Thus, gene-specific RNA stabilization after antibiotic treatment of cells could be a consequence of special RNA stability determinants within psaA and psaB mRNAs.

V. Conclusions and Perspectives The role of differential RNA synthesis and degradation as factors contributing to the process of regulated gene expression has been documented in almost all organisms examined to date. In chloroplasts as well as in mitochondria (Margossian and Butow, 1996) mRNA stabilization appears to play a key role in determining the levels of organellar transcripts. Analyses of nuclear and chloroplast mutants in C. reinhardtii have revealed that mRNA decay is not a random process in which non-specific nucleases degrade RNA based only on target size or ribosomal association. Instead, a picture of gene-specific stabilization effects is emerging in which defined cis-acting elements and their cognate trans-acting protein factors allow precise determination of particular RNA steady-state levels. Apparently, both 5´ and 3´ untranslated regions of chloroplast transcripts carry these cis-elements, and in the future, one of the most important questions will be to determine the rate limiting steps of RNA decay that cause the irreversible functional inactivation of an RNA molecule (Petersen 1992). As the 5´ leader mediates degradation of psbD, psbB and petD mRNAs in at least three nuclear mutants (nac2-26, 222E and F16, respectively), 5´ regions appear to play an important role in the control of these initial events. Consequently, the algal cell would benefit from avoiding partial translation of transcripts degraded from the 3´ end. However, it should be taken into account that a mutated nuclear or chloroplast background represents an artificial situation that might lead to misinterpretation of the results.

161 The available techniques for nuclear as well as chloroplast transformation will allow the identi­ fication of more trans-acting factors mediating genespecific RNA stabilization. The TPR domain on the Nac2 protein suggests that multisubunit complexes are involved, and possibly that these complexes are, at least in some cases, associated with the chloroplast membrane system. Together with the available in vitro systems for monitoring RNA degradation and RNA-protein complex formation, it should be feasible to characterize the molecular mechanics of RNA stability in more detail. One emphasis will be to elucidate the interrelationship ofprocesses involving 5´ leader regions; namely RNA stabilization, RNA targeting, 5´ processing, and translation. Furthermore, it will be exciting to test whether processes recently discovered in higher plants like RNA polyadenylation also occur in C. reinhardtii and to study their role in RNA metabolism. Finally, with the identification of factors involved in higher plant plastid RNA stabilization (Meurer et al., 1996), similarities and differences observed will provide ideas of how mechanisms of regulated RNA decay developed during evolution.

Acknowledgments I want to thank J.-D. Rochaix for his support and many stimulating discussions. In addition, I wish to thank those who kindly provided unpublished data of their work and E. Schmidt for critical reading of the manuscript. Furthermore, I would like to thank U. Kück for providing basic support and laboratory space. This work is supported by the Deutsche Forschungsgemeinschaft.

References Abler ML and Green PJ (1996) Control of mRNA stability in higher plants. Plant Molec Biol 32: 63–78 Blowers A, Klein U, Ellmore GS and Bogorad L (1993) Functional in vivo analyses of the 3´ flanking sequences of the Chlamydomonas chloroplast rbcL and psaB genes. Mol Gen Genet 238: 339–349 Bouvet P and Belasco JG (1992) Control of RNase E-mediated RNA degradation by 5´-terminal base pairing in E. coli. Nature 360: 488–491 Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AR, Jones BL, Randolph-Anderson, Robertson TM, Klein KB, Shark B and Sanford J (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240: 1534–1538

162 Chen X, Kindle K and Stern DB (1993) Initiation codon mutations in the Chlamydomonas chloroplast petD gene result in temperature-sensitive photosynthetic growth. EMBO J 12: 3627–3635 Danon A and Mayfield SP (1991) Light-regulated translational activators: identification of chloroplast gene-specific mRNA binding proteins. EMBO J 10: 3993–4002 De Vitry C, Olive J, Drapier D, Recouvreur M and Wollman F­ A (1989) Posttranslational events leading to the assembly of Photosystem II complex: a study using photosynthesis mutants from Chlamydomonas reinhardtii. J Cell Biol 109: 991–1006 Drager RG, Zeidler M, Simpson CL and Stern DB (1996) A chloroplast transcript lacking the 3´ inverted repeat is degraded by 3´–5´ exoribonuclease activity. RNA 2: 652–663 Drager RG, Girard-Bascou J, Choquet Y, Kindle KL and Stern DB (1997) In vivo evidence for 5´–3´ exoribonuclease degradation of an unstable chloroplast mRNA. Plant J 13: 85–96 Drapier D, Girard-Bascou J and Wollman F-A (1992) Evidence for nuclear control of the expression of the atpA and atpB chloroplast genes in Chlamydomonas. Plant Cell 4: 283–295 Gagne G and Guertin M (1992) The early genetic response to light in the green unicellular alga Chlamydomonas eugametos grown under light/dark cycles involves genes that represent direct responses to light and photosynthesis. Plant Mol Biol 18: 429–445 Gillham NW, Boynton JE and Hauser CR (1994) Translational regulation of gene expression in chloroplast and mitochondria. Annu Rev Genet 28: 71–93 Goebl M and Yanagida M (1991) The TPR snap helix: A novel protein repeat motif from mitosis to transcription. Trends Biochem Sci 16: 173–177 Gruissem W and Tonkyn JC (1993) Control mechanisms of plastid gene expression. Crit Rev Plant Sci 12:19–55 Guertin M and Bellemare G (1979) Synthesis of chloroplast ribonucleic acid in Chlamydomonas reinhardtii toluene-treated cells. Eur J Biochem 96: 125–129 Gumpel NJ, Ralley L, Girard-Bascou J, Wollman F-A, Nugent JHA and Purton S (1995) Nuclear mutants of Chlamydomonas reinhardtii defective in the biogenesis of the cytochrome complex. Plant Molec Biol 29: 921–932 Hahn D, Bennoun P and Kück U (1996) Altered expression of nuclear genes encoding chloroplast polypeptides in nonphotosynthetic mutants of Chlamydomonas reinhardtii: evidence for post-transcriptional regulation. Mol Gen Genet 252: 362–370 Hansen MJ, Chen L, Fejzo MLS and Belasco JG (1994) The ompA 5´ untranslated region impedes a major pathway for mRNA degradation in Escherichia coli. Molec Microbiol 12: 707–716 Harris EH (1989) The Chlamydomonas source book. Academic Press, San Diego Hauser CR, Gillham NW and Boynton JE (1996) Translational regulation of chloroplast genes: proteins binding to the 5´ UTRs of chloroplast mRNAs in Chlamydomonas reinhardtii. J Biol Chem 271: 1486–1497 Hayes R, Kudla J, Schuster G, Gabay L, Maliga P and Gruissem W (1996) Chloroplast mRNA 3´ end processing by a high molecular weight protein complex is regulated by nuclear encoded RNA binding proteins. EMBO J 15:1132–1141 Herrin DL, Michaels AS and Paul A-L (1986) Regulation of

Jörg Nickelsen genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase and the Photosystem II polypeptides D1 and D2 during the cell cycle of Chlamydomonas reinhardtii. J Cell Biol 103: 1837–1845 Hong S and Spreitzer RJ (1994) Nuclear mutation inhibits expression of the chloroplast gene that encodes the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Plant Physiol 106: 673–678 Hoober JK, White RA, Marks DB and Gabriel JL (1994) Biogenesis of thylakoid membranes with emphasis on the process in Chlamydomonas. Photosynth Res 39: 15–31 Hwang S and Herrin DL (1994) Control of the gene transcription by the circadian clock in Chlamydomonas reinhardtii. Plant Molec Biol 26: 557–569 Jacobshagen S, Kindle KL and Johnson CM (1996) Transcription of CABII is regulated by the biological clock in Chlamydomonas reinhardtii. Plant Molec Biol 31: 1173–1184 Jacobson A and Peltz SW (1996) Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu Rev Biochem 65: 693–739 Kim J, Gamble-Klein P and Mullet JE (1991) Ribosomes pause at specific sites during synthesis of membrane bound chloroplast reaction center protein D1. J Biol Chem 266: 14931–14938 Klaff P (1995) mRNA decay in spinach chloroplasts: psbA mRNA degradation is initiated by endonucleolytic cleavages within the coding region. Nucl Acids Res 23: 4885–4892 Kuchka MR, Goldschmidt-Clermont M, van Dillewijn J and Rochaix J-D (1989) Mutation at the Chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PS II. Cell 58: 869–876 Kudla J, Hayes R and Gruissem W (1996) Polyadenylation accelerates degradation of chloroplast mRNA. EMBO J 15: 7137–7146 Kuhlemeier C (1992) Transcriptional and post-transcriptional regulation of gene expression in higher plants. Plant Molec Biol 19: 1–14 Lamb JR, Tugendreich S and Hieter P(1995)Tetratrico peptide repeat interactions: to TPR or not to TPR. Trends Biochem Sci 20: 257–259 Lee H, Bingham SE and Webber AN (1996) Function of 3´ noncoding sequences and stop codon usage in expression of the chloroplast psaB gene in Chlamydomonas reinhardtii. Plant Mol Biol 31: 337–354 Leu S, White D and Michaels A (1990) Cell cycle-dependent transcriptional andpost-transcriptionalregulation ofchloroplast gene expression in Chlamydomonas reinhardtii. Biochem Biophys Acta 1049: 311–317 Margossian SP and Butow RA (1996) RNA turnover and the control of mitochondrial gene expression. Trends Biochem Sci 21: 392–396 Mayfield SP, Cohen A, Danon A and Yohn CB (1994) Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured element contained within the 5´ untranslated region. J Cell Biol 127: 1537–1545 Meurer J, Berger A and Westhoff P (1996) A nuclear mutant of Arabidopsis with impaired stability on distinct transcripts of the plastid psbB , psbD/C, ndhH and ndhC operons. Plant Cell 8: 1193–1207 Monod C, Goldschmidt-Clermont M and Rochaix J-D (1992)

Chapter 9 Chloroplast RNA Stability Accumulation of chloroplast psbB RNA requires a nuclear factor in Chlamydomonas reinhardtii. Mol Gen Genet 231: 449–459 Nickelsen J and Link G (1993) The 54 kDa RNA binding protein from mustard chloroplasts mediates endonucleolytic transcript 3´ end formation in vitro. Plant J 3: 537–544 Nickelsen J and Rochaix J-D (1994) Regulation of the synthesis of D1 and D2 proteins of Photosystem II. In: Baker NR and Bowyer JR (eds) Photoinhibition of photosynthesis: From Molecular Mechanisms to the Field, pp 179–194. Information Press Ltd, Oxford Nickelsen J, van Dillewijn J, Rahire M and Rochaix J-D (1994) Determinants for stability of the chloroplast psbD RNA are located within its short leader region in Chlamydomonas reinhardtii. EM BO J 13: 3182–3191 Petersen C (1992) Control of functional mRNA stability in bacteria: Multiple mechanisms of nucleolytic and non­ nucleolytic inactivation. Mol Microbiol 6: 277–282 Purton S and Rochaix J-D (1994) Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library. Plant Mol Biol 24: 533–537 Rochaix J-D (1995) Chlamydomonas reinhardtii as the photosynthetic yeast. Annu Rev Genet 29: 209–230 Rochaix J-D (1996) Post-transcriptional regulation of chloroplast gene expression in Chlamydomonas reinhardtii. Plant Mol Biol 32: 327–341 Sakamoto W, Kindle KL and Stern DB (1993) In vivo analysis of Chlamydomonas chloroplast petD gene expression using stable transformation of beta-glucuronidase translational fusions. Proc Natl Sci USA 90: 497–501 Sakamoto W, Chen X, Kindle KL and Stern DB (1994) Function of the Chlamydomonas reinhardtii petD 5´ untranslated region in regulating the accumulation of subunit IV of the cytochrome complex. Plant J 6: 503–512 Salvador ML, Klein U and Bogorad L (1993a) Light regulated and endogenous fluctuations of chloroplast transcript levels in Chlamydomonas. Regulation by transcription and RNA degradation. Plant J 3: 213–219

163 Salvador ML, Klein U and Bogorad L (1993b) 5´ sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts. Proc Natl Acad Sci USA 90: 1556–1560 Sieburth LE, Berry-Lowe S and Schmidt G W (1991) Chloroplast RNA stability in Chlamydomonas: rapid degradation of psbB and psbC transcripts in two nuclear mutants. Plant Cell 3: 175– 189 Stern DB and Kindle KL (1993) 3´ end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a twostep process. Molec Cell Biol 13: 2277–2285 Stern DB, Radwanski ER and Kindle KL (1991) A 3´ stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo. Plant Cell 3: 285–297 Sugita M and Sugiura M (1996) Regulation of gene expression in chloroplasts of higher plants. Plant Molec Biol 32: 315–326 Vreken P and Raue HA (1992) The rate limiting step in yeast PGK1 mRNA degradation is an endonucleolytic cleavage in the 3´-terminal part of the coding region. Mol Cell Biol 12: 2986–2996 Xu R, Bingham SE and Webber AN (1993) Increased mRNA accumulation in a psaB frame-shift mutant of Chlamydomonas reinhardtii suggests a role for translation in psaB mRNA stability. Plant Molec Biol 22: 465–474 Yang J, Schuster G and Stern DB (1996) CSP41, a sequencespecific chloroplast mRNA binding protein, is an endo­ ribonuclease. Plant Cell 8: 1409–1420 Zerges W and Rochaix J-D (1994) The 5´ leader of a chloroplast mRNA mediates the translational requirements for two nucleusencoded functions in Chlamydomonas reinhardtii. Mol Cell Biol 14: 5268–5277 Zhang H, Herman PL and Weeks DP (1994) Gene isolation through genomic complementation using an indexed library of Chlamydomonas reinhardtii DNA. Plant Mol Biol 24: 663– 672

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Chapter 10 Chloroplast RNA Synthesis and Processing David B. Stern and Robert G. Drager

Boyce Thompson Institute for Plant Research at Cornell University,

Tower Road, Ithaca, NY 14853, U.S.A.

Summary I. Transcription of Chloroplast Genes A. General Considerations B. RNA Polymerase 1. Plastid-Encoded rpo Genes 2. Nucleus-Encoded Factors 3. Evidence for a Nucleus-Encoded Chloroplast RNA Polymerase C. Promoters 1. Prokaryotic-Like –10/–35 Promoters 2. Other Promoter Types 3. Transcriptional Regulation by DNA Topology D. Transcription Termination 1. Inverted Repeats Are Inefficient Transcription Terminators 2. Consequences of Transcriptional Read-Through II. Processing of Chloroplast mRNAs A. General Considerations B. 5´-End Processing 1. The Extent of 5´-End Processing 2. Possible Mechanisms for 5´-End Processing 3. The Relationship Between 5´-End Processing and mRNA Accumulation C. 3´ End Processing 1. Examples of 3´-End Processed Transcripts 2. Mechanisms of 3´-End Processing 3. Nuclear Mutations Affecting Chloroplast mRNA 3´-End Formation 4. Does the 3´ UTR Regulate mRNA Stability? Acknowledgments References

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Summary Transcription and RNA maturation are two essential steps in gene expression. In chloroplasts, transcription is carried out by at least two biochemically and genetically separable activities, which may participate in establishing different basal expression rates for ribosomal RNAs, transfer RNAs and protein-coding genes. Because chloroplast RNA polymerases do not generally terminate transcription at sites corresponding to the 3´ termini of mature transcripts, these termini must be formed by RNA processing events. In Chlamydomonas reinhardtii chloroplasts, it appears that most or all transcript 5´-ends are also formed by RNA processing rather than by transcription initiation. Thus, RNA processing converts primary transcripts of generally unknown dimensions to the mature, accumulating transcripts. Molecular, genetic and biochemical approaches have been

J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 165–181. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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used to unravel the chloroplast transcription and RNA processing machinery, with the most information gained to date from the analysis of chimeric reporter genes introduced into chloroplasts by biolistic transformation. The picture painted by these data reveals both similarities and differences between these processes in Chlamydomonas and land plants. However, some perceived differences, particularly based on the phenotypes of nuclear mutants which affect chloroplast mRNA metabolism, may reflect selection or screening procedures and thus may mask an overall congruity between gene expression mechanisms in the chloroplasts of all organisms.

I. Transcription of Chloroplast Genes

B. RNA Polymerase

A. General Considerations

1. Plastid-Encoded rpo Genes

While most genes in land plant cpDNA are organized into clusters or operons, Chlamydomonas chloroplast genes are somewhat dispersed. These organizational differences are reflected in the patterns of accumu­ lating transcripts, which are often very complex in land plants, but usually simple in Chlamydomonas. However, Chlamydomonas cpDNA contains some conserved gene clusters, such as the ribosomal protein genes which resemble the E. coli S10 and spc operons (Boudreau et al, 1994), and co-transcription of nonconserved groups of genes does occur (Levy et al., 1997). As discussed in the second part of this chapter, rapid RNA processing can also lead to the illusion of independent transcription of two genes that are in fact co-transcribed. The enzymes that carry out chloroplast trans­ cription are poorly characterized in Chlamydomonas, with somewhat more information being available for land plants and Euglena. In this chapter, therefore, data for other organisms are also discussed. One reason for the lack of information regarding Chlamydomonas chloroplast RNA polymerases is that Chlamydomonas chloroplasts are somewhat difficult to isolate in large quantities, which has limited the amount of biochemical work that has been carried out to date. Also, because transcription initiation is not generally considered to be an important control point for chloroplast gene expression in Chlamydomonas, less experimental emphasis has been put on this area than on post­ transcriptional controls.

Like the chloroplast genomes of land plants (Ohyama et al., 1986; Shinozaki et al., 1986; Hiratsuka et al., 1989; Maier et al., 1995), Euglena gracilis (Hallick et al., 1993), and Porphyra purpurea (Reith and Munholland, 1993), Chlamydomonas spp. cpDNAs contains open reading frames encoding potential proteins with homology to subunits of E. coli RNA polymerase. While plants contain the genes rpoA, rpoB, rpoC1 and rpoC2, encoding putative subunits, in Chlamydomonas spp. cpDNA and only rpoB1, rpoB2 and rpoC2 have been located (Boudreau et al., 1994). In Chlamydomonas, the subunit has been split into two proteins, while only the C-terminal half of the E. subunit is encoded in cpDNA. Since the sequence of the Chlamydomonas chloroplast genome has not been completed, whether the remaining genes are present is unknown. Biochemical evidence from maize (Hu and Bogorad, 1990; Hu et al., 1991) and pea (Little and Hallick, 1988) suggests that the chloroplast-encoded RNA polymerase subunits are present in vivo and form part of the RNA polymerase. In addition, when the rpoB gene was deleted from tobacco cpDNA by biolistic transformation, transcription of many chloroplast genes was abolished (Allison et al., 1996). Supporting data were also obtained from a barley mutant, albostrians, which was reported to lack chloroplast ribosomes and also was deficient in transcription of a class of chloroplast genes (Hess et al., 1993). When similar deletions were attempted in Chlamydomonas chloroplasts, however, the cells remained heteroplasmic, suggesting that the rpoB1, rpoB2 and rpoC2 genes performed essential functions (Fischer et al., 1996). Since the deletions were performed by replacement of the coding regions with a selectable marker cassette which included the aadA coding region driven by the atpA promoter, it is possible that the rpo genes are not essential for cell

Abbreviations: cpDNA – chloroplast DNA; cpRNA – chloroplast RNA; IR – inverted repeat; LRP – light-regulated promoters; NEP – nucleus-encoded RNA polymerase; Rubisco – ribulosel,5-bisphosphate carboxylase/oxygenase; TAC – transcriptionally active chromosome; UTR – untranslated region

Chapter 10 Chloroplast Transcription viability per se, but rather for the expression of the selectable marker. In the case of the tobacco rpoB deletion, the aadA cassette was driven by a modified rRNA promoter; rRNA genes are thought to be transcribed by a nucleus-encoded chloroplast RNA polymerase (see below). The lingering hetero­ plasmicity of genomes containing the rpoB deletion in Chlamydomonas, however, argues strongly that these genes play an important role in chloroplast gene expression, most likely in transcription.

167 known gene-specific translational activators or RNA stabilizers, do in fact exist. If they do, then one would expect mutants to be in hand already; however, one cannot exclude the possibility that one or a very few genes are controlled in this way, much as psbD/C, rrn16, and petG are the only reported examples of gene-specific transcriptional regulation in land plants (reviewed in Stern et al., 1997).

3. Evidence for a Nucleus-Encoded Chloroplast RNA Polymerase

2. Nucleus-Encoded Factors E. coli RNA polymerase holoenzyme contains a sigma factor in addition to the core subunits; however, genes encoding putative factors have not been found in cpDNA. This suggests that might be nucleus-encoded in plants. Recently, a nuclear SigA gene was reported for the red alga Cyanidium caldarium, providing additional evidence for this possibility (Liu and Troxler, 1996; Tanaka et al., proteins 1996). The partial purification of from land plant (Lerbs et al., 1988; Tiller and Link, 1993) and Chlamydomonas (Surzycki and Shellen­ barger, 1976; Troxler et al., 1994) chloroplasts has been reported. The next several years should see the biochemical data meshing with the molecular genetic. Another line of evidence for nucleus-encoded RNA polymerase components would be the isolation of nuclear mutants defective in chloroplast transcription. However, in land plants such mutants have not been reported and in Chlamydomonas, only a single nuclear mutant strain of this class is known (76-5EN). In 76­ 5EN, transcription of the rbcL gene is reduced, based on a run-on assay (Hong and Spreitzer, 1994). However, the reduction in the transcription rate may not fully account for the Rubisco deficiency of these cells. The dearth of transcription defects among the many non-photosynthetic Chlamydomonas strains that have been isolated has, then, two possible explanations. One is simply that the target genes do not exist, and the other is that the target genes are essential for cell viability. The latter limitation can be overcome by the isolation of temperature-sensitive mutants, of which there are numerous examples for the RNA polymerases of yeast and E. coli. While it is highly likely that one or more nuclear genes encode a subunit of chloroplast RNA polymerase in Chlamydomonas, an additional question is whether gene-specific transcription factors, analogous to well-

Several lines of evidence suggest that chloroplasts contain both nucleus- and chloroplast-encoded RNA polymerase activities. First, the existence of two biochemically-separable polymerase activities has been reported in Euglena gracilis and in land plants (Greenberg et al., 1984; Pfannschmidt and Link, 1994). These activities are a membrane-associated DNA-protein complex called transcriptionally active chromosome (TAC), thought to transcribe principally rRNA genes, and a soluble RNA polymerase that recognizes canonical E. coli-like promoters. Since only the E. coli type of RNA polymerase gene is found in cpDNA, this suggests that TAC is encoded in the nucleus. A second line of evidence is the transcription of plastid genes in organisms that lack either plastid ribosomes (Hess et al., 1993), or functional plastid-encoded RNA polymerase (Wolfe et al., 1992; Allison et al., 1996). Additionally, the observation that chloroplasts contain a T7-like RNA polymerase activity (Lerbs-Mache, 1993), and the existence of multiple promoter types (see Section I.C) are evidence for multiple RNA polymerases or at least different transcription factors. While RNA polymerases have been purified from the chloroplasts of E. gracilis and several plants, relatively little has been done in the case of Chlamydomonas. Fractionation of total cell RNA polymerases revealed a rifampicin-sensitive, activity that had a preference for cpDNA (Surzycki and Shellenbarger, 1976). The activity that incorporates nucleoside triphosphates into cpRNA of toluene-treated cells has similar characteristics (Guertin and Bellemare, 1979), and these features are consistent with an E. coli-like RNA polymerase. However, when Chlamydomonas RNA polymerases were separated based on their the ability to transcribe trnE1, encoding and activity was insensitive to both rifampicin, but was sensitive to heparin (Jahn, 1992).

168 Although rifampicin insensitivity distinguishes this activity from the E. coli-like polymerase, heparin sensitivity is typical of soluble chloroplast RNA polymerase activities from land plants when used for in vitro transcription experiments (Gruissem et al., 1983). The trnE1 transcription activity might be nucleus-encoded, especially since it has RNA polymerase III characteristics based on promoter deletion analysis of trnE1 (Section I.C).

David B. Stern and Robert G. Drager promoters are present, but inactive. For example, the spinach rrn16 gene has two upstream –35/–10 promoters which are active in vitro but not in vivo. Further analysis revealed that a third promoter of another type was located between them, and that initiation at this promoter blocked access of RNA polymerase to the E. coli-like promoters (Iratni et al., 1994).

2. Other Promoter Types C. Promoters 1. Prokaryotic-Like –10/–35 Promoters Canonical E. coli promoters consist of –35 and –10 elements with the consensus sequences TTGACA and TATAAT. Sequences similar or identical to these were recognized when chloroplast genes were first isolated from plants, and these were later verified to be bona fide promoter elements using in vitro transcription systems, especially from spinach (Gruissem and Zurawski, 1985a,b). In addition, it has been possible to analyze plastid gene promoters in E. coli. For example, mutations in the maize atpB promoter had the same effects either in E. coli or in a chloroplast in vitro transcription system (Bradley and Gatenby, 1985). Several Chlamydomonas chloroplast promoters have been identified by their ability to direct transcription of reporter genes in transformed chloroplasts, and alignment of these and additional sequences have revealed E. coli-like promoters upstream of some Chlamydomonas chloroplast genes, for example rrn16 and rbcL (Klein et al., 1992). Thus, the –35/–10 promoter type is widespread in chloroplast genomes, and in most cases has sequence requirements indistinguishable from those of E. coli promoters. At least two variants of the scenario described in the preceding paragraph are also known. In one case, the –35 element is apparently absent, leading to a ‘–10’ type of promoter. A consensus sequence TATAATAT has been proposed for Chlamydomonas, based on deletion analysis of the atpB promoter and inspection of sequences upstream of known mRNA 5´ termini. However, because Chlamydomonas chloroplast intergenic regions are extremely rich in A and T, and because many 5´ termini may be generated by RNA processing rather than by transcription initiation (Section II. A), these sequence alignments should be viewed with caution. A second variant of –35/–10 promoters occurs when these

In land plants, at least three types of chloroplast promoters are known apart from the prokaryotic types (–35/–10 or–10 only). These are so-called NEP promoters, driven by a nucleus-encoded RNA polymerase, light-regulated promoters (LRPs), and internal tRNA promoters. NEP promoters are those that principally drive components of the gene expression apparatus, for example rRNAs, ribosomal proteins and RNA polymerase subunits. The NEP promoters of plastid 16S rRNA genes are highly conserved, with a G+C content of approximately 50% (Vera and Sugiura, 1995; Allison et al., 1996). NEP promoters are also found upstream of some photosynthetic genes such as atpB and atpI; however, these genes also have E. coli-like promoters (Hajdukiewicz et al., 1997; Kapoor et al., 1997). Based on continued initiation at NEP promoters in the absence ofchloroplast-encoded RNA polymerase (Hess et al., 1993; Allison et al., 1996; Hajdukiewicz et al., 1997), they are assumed to be transcribed by a nucleus-encoded enzyme. The best-characterized LRP drives blue lightinduced transcription of a major psbD/C transcript in barley (Sexton et al., 1990) and other land plant species (Christopher et al., 1992). Elements of this promoter type have been dissected using in vitro transcription (Kim and Mullet, 1995; Satoh et al., 1997) and in vivo using transcriptional fusions to the uidA reporter gene in transformed tobacco chloro­ plasts (Allison and Maliga, 1995). These experiments revealed that the LRP contains both classical –35/– 10 elements as well as enhancing and light-responsive elements not found in other promoters. While Chlamydomonas has not been demonstrated to have this type of response to high light levels, there is strong evidence for circadian control of transcript levels in this organism. The amplitude of this variation can be more than ten-fold, and is exhibited by a number of genes, including rrn16, tufA and genes for photosynthetic proteins (Hwang et al., 1996). That

Chapter 10 Chloroplast Transcription transcription rate oscillation is circadian rather than light-controlled was shown by leaving lightsynchronized cells in continuous light or darkness, and finding that the circadian rhythms persisted for two days or more. Similar results were obtained in another study, with the additional finding that the psaB transcription rate is controlled by light rather than by endogenous rhythm (Salvador et al., 1993b). The promoter elements that mediate this trans­ criptional regulation remain to be defined. In spinach chloroplasts, two classes of tRNA gene promoters have been found. For example, trnM2 has a –35/–10 promoter with standard functional elements (Gruissem and Zurawski, 1985b). The trnS1 and trnR1 genes, however, do not require upstream elements for in vitro transcription (Gruissem et al., 1986), and thus resemble nuclear tRNA genes transcribed by RNA polymerase III. This latter class is also represented in Chlamydomonas chloroplasts, as evidenced by results obtained with an in vitro transcription system and the trnE1 gene (Jahn, 1992). Deletion analysis showed that neither 5´ nor 3´ flanking sequences were required for full activity, but that deletions into the coding region abolished in vitro transcription. Thus, trnE1 has an internal promoter. Whether this can be generalized to other Chlamydomonas chloroplast tRNA genes will require additional in vitro transcription analysis or deletion analysis in vivo. The occurrence of different classes of chloroplast RNA polymerases and of different promoter types in Chlamydomonas and land plants is summarized in Table 1.

3. Transcriptional Regulation by DNA Topology It is well-known that accessibility of promoters to RNA polymerase can influence transcription initiation rates. Unlike E. coli RNA polymerase, land plant chloroplast RNA polymerases have typically exhibited a preference for supercoiled over linear templates in vitro (Lam and Chua, 1987; Zaitlin et al., 1989), suggesting that DNA topology may play a role in establishing basal transcription rates of chloroplast genes. While similar in vitro transcription experiments have not been carried out for Chlamy­ domonas, treatment of cells with novobiocin, an inhibitor of DNA gyrase, has substantial effects on transcript accumulation patterns. It can be shown in vitro that Chlamydomonas chloroplasts have an ATPdependent supercoiling activity (Thompson and Mosig, 1985), and that the relative accumulation of

169

several transcripts is altered when this activity is inhibited by novobiocin (Thompson and Mosig, 1987), resulting in either increases or decreases for individual genes. These experiments suggested that torsional stress in DNA was being altered, which was subsequently examined by measuring the ability of an intercalating dye to bind to different regions ofthe Chlamydomonas chloroplast genome. It was found that DNA structure changed in response to light (Thompson and Mosig, 1990), consistent with a chromosome structure-mediated transcriptional response to light conditions. The light- and circadian rhythm-directed changes in chloroplast transcription rates that have been characterized (see previous section) may well result from, or require these alterations in chloroplast chromosome structure.

D. Transcription Termination 1. Inverted Repeats Are Inefficient Transcription Terminators The 3´-ends of most chloroplast protein-coding regions are flanked by inverted repeat sequences that can potentially form stem-loop structures. While these stem-loops superficially resemble bacterial terminators, they often lack the Urich tract immediately downstream that is required for efficient termination activity (Platt, 1986).

170 Measurements of termination using in vitro transcription systems from spinach showed that while terminators, for example the known E. coli trp attenuator and the bacteriophage T7 early terminator, could be recognized by RNA polymerase, chloroplast 3´ stem-loops were very inefficient terminators (Stern and Gruissem, 1987; Chen et al., 1990). A sole exception was trnS1, which had strong termination activity either within the tRNA coding region or downstream (Stem and Gruissem, 1987). The subsequent discovery that chloroplasts contain a T7-like RNA polymerase activity (Lerbs-Mache, 1993) raises the interesting question of whether the observed termination was due to a nuclear or chloroplast-encoded RNA polymerase activity. In Chlamydomonas chloroplasts, the termination efficiencies of 3´ UTRs have been measured by pulse-labeling RNA, and quantifying incorporation into sequences before and after possible of termination signals (Stern and Kindle, 1993; Rott et al., 1996). In these studies, no significant termination activity was seen (the atpB 3´ stem-loop imparted the highest level of transcription termination at ), even for known terminators. These results are ostensibly at odds with observations of polar effects of 3´ stem-loops on the transcription of downstream genes in transformed Chlamydomonas chloroplasts. For example, read-through of the rbcL 3´ stem-loop does not appear to occur when a reporter gene is flanked by three consecutive iterations ofthis sequence, because all accumulating transcripts end at the first stem-loop (Blowers et al., 1993). However, this result can also be explained by efficient processing at the first stem-loop, followed by degradation of downstream sequences. This is in fact what appears to occur at the 3´-end of the atpB gene (Stern and Kindle, 1993). Thus, what appears at first glance to be efficient termination may in fact represent efficient processing followed by or concurrent with polar RNA degradation. At an extreme, it can be envisioned that transcription termination simply does not occur in Chlamydomonas chloroplasts, at least not at a level sufficient to be a primary mechanism for generating 3´ termini. This would be akin to transcription of metazoan mitochondrial genomes, where the entire 16 kb genome is co-transcribed and subsequently processed to yield individual mRNAs and tRNAs (see Tracy and Stern, 1995 and references therein), although termination does occur by a special mechanism downstream of the rRNA genes (Daga et

David B. Stern and Robert G. Drager al., 1993). Another possibility is that transcription in chloroplasts is punctuated by tRNA genes capable of directing efficient termination (Stern and Gruissem, 1987), although this phenomenon has not yet been demonstrated in Chlamydomonas. Finally, termin­ ation may be stochastic, with the polymerase relying on the relative instability of RNA-DNA hybrids in the extremely A+T-rich intergenic regions in the chloroplast genome. Further studies in Chlamy­ domonas will probably have to await purification of the RNA polymerase or development of an in vitro transcription system.

2. Consequences of Transcriptional ReadThrough A lack of efficient transcription termination has several consequences for gene expression. First, rapid and precise RNA processing is required to generate functional transcripts. The known mechanisms of chloroplast mRNA processing are discussed in Section II, while chloroplast tRNA processing and rRNA processing have been described elsewhere (Gruissem et al., 1983; Keus et al., 1984; MarionPoll et al., 1988; Wang et al., 1988; Barkan, 1993). A second and related consequence is that the chloroplast must be able to distinguish, and degrade, the many untranslatable RNA segments that would accumulate initially as part of primary transcripts. Evidence for rapid degradation of non-coding RNA has been obtained by comparing transcript accumulation levels and run-on transcription rates in both Chlamy­ domonas (Stern and Kindle, 1993) and spinach (Deng et al., 1987) chloroplasts, and in maize mitochondria (Finnegan and Brown, 1990). The way in which these transcripts are recognized as non-coding is not known; however, it may be that the default pathway is degradation, and mature mRNAs are protected from degradation by specific RNA-binding proteins and/or secondary structures. A third consequence of transcriptional readthrough is the potential synthesis of antisense RNA, which could be detrimental to gene expression, especially in a genome with tightly clustered genes on both strands. Antisense RNA synthesis has been observed in run-on transcription experiments (Deng et al., 1987), but this RNA may be rapidly degraded as discussed above. In addition, because the chloroplast genome is polyploid, symmetric transcription does not necessarily pose topological problems. It may be possible to address the transcriptional activities of different genomes

Chapter 10 Chloroplast Transcription within a single plastid by using in situ methods, since cpDNA is organized as discrete bodies, called nucleoids(Fujie et al., 1994; Nakamura et al., 1994). Finally, in the absence of efficient termination, but with the existence of efficient RNA processing, the majority of chloroplast promoters are in effect obsolete. In the case of the Chlamydomonas petD gene, deletion of its promoter had no consequences for gene expression, since the upstream petA gene provided sufficient transcriptional readthrough (Sturm et al., 1994). Thus, the Chlamydomonas chloroplast may be evolving to contain fewer promoters, in keeping with a general trend of post­ transcriptional regulatory mechanisms.

II. Processing of Chloroplast mRNAs

A. General Considerations Messenger RNA processing in chloroplasts encom­ passes intron removal and steps that form the 5´ and 3´ transcript termini. Other forms of mRNA processing include polyadenylation, which in chloroplasts is related to RNA degradation (Kudla et al., 1996; Lisitsky et al., 1996), and RNA splicing and decay (Chapter 9, Nickelsen; Chapter 11, Herrin et al.). As discussed above, transcription termination is inefficient or absent in Chlamydomonas chloroplasts, requiring that 3´ termini be formed by processing. At the same time, researchers have been unable to detect primary transcripts in Chlamydomonas chloroplasts using vaccinia virus guanylyl transferase to ligate GTP to di- or triphosphate 5´ termini (Johnson and Schmidt, 1993; N. Sturm and D. B. Stem, unpub­ lished). In contrast, in vitro capping of land plant chloroplast transcripts is readily accomplished (Woodbury et al., 1989), and at least some Euglena gracilis chloroplast transcripts can be capped (Drager, 1993). This raises the possibility that most or all 5´ termini of Chlamydomonas chloroplast transcripts are also generated by RNA processing, rather than directly by transcription initiation. These considerations underscore the importance of RNA processing in the generation of translatable RNAs in Chlamydomonas chloroplasts. It is possible that the mechanisms of RNA processing are linked to those governing translation and RNA stability, which are subjects ofChapters 12 (Hauser et al.) and 9 (Nickelsen), respectively. These connections will

171 be revealed by continued biochemical and molecular genetic analysis of mRNA metabolism.

B. 5´-End Processing 1. The Extent of 5´-End Processing The determination that a mRNA 5´ terminus results from processing rather than initiation can be inferred from its inability to be capped in vitro, or from in vitro processing experiments using synthetic RNAs, In land plants, it is generally the case that the longest or primary transcript of a gene cluster can be capped, while a larger number of processed transcripts resulting from intercistronic cleavages accumulate to similar levels (e.g. Westhoff and Herrmann, 1988). In simpler cases, such as rbcL transcription, only a monocistronic mRNA accumulates, but two to three closely-spaced 5´ termini are found; one primary and the others processed (e.g. Erion, 1985; HanleyBowdoin et al., 1985; Mullet et al., 1985). In at least one case, this processing may have a function in regulating accumulation of the protein product (Reinbothe et al., 1993). In other cases, such as the spinach atpB transcripts, most multiple 5´ termini result from initiation and not processing (Chen et al., 1990). The situation in Chlamydomonas chloroplasts with respect to 5´-end formation is somewhat different, largely because many gene-specific probes hybridize with only a single mRNA, rather than the as many as 10–15 identified by the same probes in land plants. It has therefore been assumed that most Chlamy­ domonas genes include a promoter just upstream of the coding region which directs transcription of a monocistronic mRNA. However, several instances have now been documented where genes can be cotranscribed. Examples of co-transcribed Chlamy­ domonas reinhardtii chloroplast genes include psbD with psaA exon2 (Choquet et al., 1988), tscA-chlN (reviewed in Rochaix, 1996), psbB-psbT (Monod et al., 1992; Johnson and Schmidt, 1993), atpA-psbIcemA-atpH (Levy et al., 1997), atpE-rps7 (Robertson et al., 1990) and ycf9-psbM (D.C. Higgs, K.L. Kindle and D.B. Stern, unpublished observation). In addition, a single transcript can have multiple 5´ termini, as exemplified by petA (Matsumoto et al., 1991). The supposition that the 5´-ends of Chlamy­ domonas chloroplast transcripts are generated directly by transcript initiation is also supported by the identification of promoters immediately upstream of

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the mature 5´-ends of atpB and petD transcripts (see section I.C and Sakamoto et al., 1994). However, efforts to cap petD or other mRNAs in vitro have been unsuccessful, suggesting that Chlamydomonas chloroplast mRNAs may be processed at their 5´ends (although it cannot be ruled out that the 5´ di- or triphosphate is converted to a monophosphate without removal of nucleotides). In the only confirmed example of 5´ processing of a co-transcript emanating from an upstream promoter, it was found that the petD promoter could be deleted without substantially reducing the amount of monocistronic petD mRNA accumulating in vivo (Sturm et al., 1994). Whether the case for petD is typical for Chlamydomonas chloroplast mRNAs can only be determined by precisely deleting promoters for other mRNAs, and asking whether monocistronic mRNAs can still be produced by transcriptional readthrough ofupstream genes (the upstream gene is petA in the case of petD) followed by endonucleolytic processing. This type of experiment can also be performed in an ectopic context: for example, a promoterless petD 5´ UTR­ uidA fusion placed in the petA-petD intergenic region produced monocistronic uidA mRNA (Sakamoto et al., 1994).

2. Possible Mechanisms for 5´-End Processing Processed 5´-ends can in principle be generated by three post-transcriptional mechanisms which are illustrated in Fig. 1. In the first, an endonucleolytic cleavage generates directly the mature 5´ terminus; exonucleolytic processing in the second, trims the precursor to give the mature 5´-end; and in the third mechanism, endonucleolytic cleavage is exonucleolytic processing. In followed by the absence of in vitro RNA processing systems, it is difficult to distinguish between these possibilities. However, one approach is to use mutagenesis to define processing sites in vivo, and then determine whether a new upstream and presumably unprocessed 5´-end is produced, which could potentially be capped in vitro. In the case of petD, as described above, the promoter could be deleted without affecting 5´-end formation substantially (Sturm et al., 1994). In this sequences from –2 to –81 with deletion, termed respect to the mature 5´-end were removed. This suggests that the processing signal lies entirely within the 5´ UTR, a hypothesis that was confirmed by placing this promoterless 5´ UTR upstream of the uidA gene and showing that monocistronic uidA

mRNA could accumulate (Sakamoto et al., 1994). In genes, both the case of and the chimeric however, some unprocessed mRNA accumulated as co-transcripts with the upstream petA mRNA. This indicates that sequences upstream of–2 can contribute to the efficiency of mRNA processing.

3. The Relationship Between 5´-End Processing and mRNA Accumulation Post-transcriptional processes often affect RNA halflife and accumulation. For example, in maize nuclear mutants deficient in chloroplast RNA splicing, there is no increase in the accumulation of unspliced precursors, suggesting that the inability to remove introns may increase transcript susceptibility to nucleases (Jenkins et al., 1997). This does not appear

Chapter 10

Chloroplast Transcription

to be the case for Chlamydomonas nuclear mutants defective in psaA splicing, because unspliced precursors accumulate to increased levels (Choquet et al., 1988; Goldschmidt-Clermont et al., 1990). In other cases, nuclear or chloroplast mutations resulting in a translational defect have been shown to lead to increased degradation of the affected chloroplast transcripts, although in some cases there is no effect on transcript accumulation (reviewed in Rochaix, 1996). There are at least three land plant nuclear mutants that affect chloroplast mRNA processing. One is the crp1 mutant of maize, which is defective in the accumulation of monocistronic petB and petD mRNAs (Barkan et al., 1994), the 5´ termini of which are formed by intercistronic processing (Rock et al., 1987). The accumulation of 17 other transcripts from this complex gene cluster is unaffected, including the putative precursors to monocistronic petB and petD. This could be explained by a block in processing of the precursors coupled to some type of feedback control of precursor accumulation at the transcriptional level such that the precursor level remains constant, or by destabilization of the monocistronic products. Other possible processing mutants are Arabidopsis thaliana hcf5 and hcf109. The h c f 5 mutant accumulates a very low level of rbcL mRNA, and also has altered transcript patterns for the psbB gene cluster (Dinkins et al., 1997), whereas hcf109 fails to accumulate a subset of transcripts from the psbB, ndhC and ndhH gene clusters (Meurer et al., 1996). Again, in these cases it is difficult to say whether RNA processing or RNA stability is the primary defect, but the results nevertheless raise the possibility of a link between the two. In Chlamydomonas there are no confirmed nuclear mutants with defects in mRNA 5´ processing, although as for land plant mutants, nuclear mutants affecting mRNA half-lives could have primary defects in RNA processing. These RNA stability mutants are discussed in detail in Chapter 9 (Nickelsen). Two of them, nac2-26 (Kuchka et al., 1989) and F16 (Drager et al., 1998) were shown to destabilize psbD or petD mRNAs, respectively, via the 5´ UTR (Nickelsen et al., 1994). Thus, a defect in 5´ processing could be the primary lesion leading to increased RNA degradation, if processing were required to permit the binding of an RNA stabilizing protein, such as the 47 kD protein that binds to the psbD 5´ UTR in vitro (Nickelsen et al., 1994).

173 Other hints at the relationship between 5´ processing and RNA stability in Chlamydomonas chloroplasts come from deletions that remove processing signals. For example, in the chloroplast mutant FUD6 (Lemaire et al., 1986), the cytochrome complex fails to accumulate due to a 236-bp deletion that encompasses part of the petD 5´ UTR and also the upstream promoter region (Sturm et al., 1994). As a result, a petA-petD co-transcript accumulates instead of monocistronic petD mRNA. However, the level of this co-transcript is quite low, suggesting that it is unstable. This instability could be due to a lack of processing, its inability to be translated, and/or an altered RNA structure due to the deletion, that creates RNAse-hypersensitive sites. strain The petA-petD co-transcript of the related (section II.B.2.) also accumulates to a low level, and as noted above, there are several possible ways to account for the low abundance of this transcript. Overall, the discussion above emphasizes the interdependence ofpost-transcriptional processes in chloroplasts, and how a defect in one process can have pleiotropic effects. 5´ RNA processing is likely to interact with other mechanisms that stabilize transcripts and facilitate the initiation of protein synthesis. The isolation of appropriate mutants and the development of in vitro systems will help to define the interrelationships between these essential steps of chloroplast gene expression.

C. 3´ End Processing 1. Examples of 3´-End Processed Transcripts Like 5´ termini, mRNA 3´-ends can be formed by a direct mechanism, transcription termination, or indirectly through RNA processing. While some chloroplast RNA polymerases can recognize bacterial terminators (Stern and Gruissem, 1987; Chen and Orozco, 1988), there is little evidence for efficient termination of transcription at chloroplast 3´ UTR stem-loops (Stern and Gruissem, 1987; Stern and Kindle, 1993; Rott et al., 1996). However, these stem-loop structures define the ends of, and are required for accumulation of discrete transcripts (Stern et al., 1991; Lee et al., 1996). Thus, most or all chloroplast mRNA 3´-ends are probably formed by RNA processing. The strongest evidence for the existence of such mechanisms is the patterns of transcription from land plant, Euglena gracilis and Chlamydomonas complex operons or gene clusters,

174 where a primary transcript is processed to yield multiple smaller mRNAs (e.g. Rock et al., 1987; Westhoff and Herrmann, 1988; Stevenson and Hallick, 1994; Stollar and Hollingsworth, 1994; Hong et al., 1995;Levy et al., 1997). However, the existence of these transcripts does not prove a precursor-product relationship, i.e. that intercistronic termination does not occur in spite of the in vivo and in vitro results on transcription termination cited above. That the enzymatic machinery for processing exists in chloroplasts is proven adequately by a variety of in vitro results (Stern and Gruissem, 1987; Nickelsen and Link, 1993; Stern and Kindle, 1993; Hayes et al., 1996). Thus, the lack of termination and the presence of appropriate enzymatic activities processing is nearly support the notion that universal for chloroplast transcripts both in land plants and in Chlamydomonas. One confounding observation, however, has been that certain sequences appear to act as terminators in chimeric gene constructs introduced into Chlamydomonas (Blowers et al., 1993; Stern and Kindle, 1993; Sakamoto et al., 1994), whereas analogous constructs in transformed tobacco chloroplasts clearly allow readthrough of UTR, yielding polycistronic the chimeric gene transcripts (Staub and Maliga, 1994). RNA processing followed by degradation of downstream sequences, however, would give the same amount ofaccumulated RNA as transcription termination. This was inferred to be the case for the Chlamydomonas atpB mRNA (Stern and Kindle, 1993), and may explain the similar results cited above for the Chlamydomonas rbcL UTR (Blowers et al., 1993).

2. Mechanisms of 3´-End Processing As illustrated in Fig. 2, three mechanisms are possible for maturation, which in most cases results in immediately downstream of a stema mature loop or other stable secondary structure. In the first, is formed directly by a site-specific the mature endonucleolytic cleavage. In the second, the endonucleolytic cleavage occurs downstream of the and exonucleolytic resection is mature required. In the third mechanism, transcription termination occurs downstream of the mature either at a specific site or in a stochastic manner, exonucleolytic degradation continues and until the nuclease encounters a stable secondary structure, which thereby defines the transcript terminus.

David B. Stern and Robert G. Drager

In vitro evidence for each ofthe above mechanisms has been published for chloroplast transcripts, although for Chlamydomonas, only the maturation of the atpB has been studied; this maturation occurs by the two-step endonuclease-exonuclease mechanism (Stern and Kindle, 1993). Direct was reported endonucleolytic formation ofthe for spinach petD mRNA (Hayes et al., 1996); however, could be formed it was also reported that this by exonuclease alone (Stern and Gruissem, 1987, 1989). This inconsistency may merely reflect the fact that different protein fractions were used in the assays. Endonuclease activity was also implicated in the formation of the mustard trnK and rps16 mRNA (Nickelsen and Link, 1993). In the spinach petD study (Hayes et al., 1996), a partial cDNA sequence encoding a protein resembling E. coli polynucleotide phosphorylase was reported, as well as a protein which cross-reacted with an antibody raised against E. coli RNAse E. Poly­ exonuclease nucleotide phosphorylase is a which can process the 3´-end of petD mRNA correctly in vitro (Stern and Gruissem, 1989); RNAse E is an

Chapter 10 Chloroplast Transcription endonuclease involved in certain types of RNA maturation in E. coli (e.g. Hajnsdorf et al, 1994). Once the plant genes are cloned, reverse genetics may lead to an understanding of the roles of these enzymes in chloroplast mRNA metabolism. Other chloroplast ribonuclease activities have been reported (Chen and Stern, 1991; Yang and Stern, 1997); however, it is not clear whether the primary roles of these activities is in RNA processing or RNA degradation.

3. Nuclear Mutations Affecting Chloroplast mRNA Formation In the case of nuclear mutants affecting the accumulation of multiple RNAs from complex operons or gene clusters, such as maize crp1 or Arabidopsis hcf5 and hcf109 which were discussed above, one cannot rule out a primary defect in formation. For example, crp1 could be defective in petB formation and/or petD formation, since the data show only that the petD monocistronic transcript does not accumulate. In Chlamydomonas, two nuclear mutants clearly have defects in chloroplast processing and/or mRNA stability mediated by UTR elements. In the mutant ncc1, ATP synthase subunit accumulates to 50% of the wild-type level, and monocistronic atpA mRNA accumulates to 10% of the wild-type level (Drapier et al., 1992). However, other mRNAs emanating from the atpA promoter, for example the dicistronic atpA-psbI transcript, are unaffected. This suggests that ncc1 is defective in formation between atpA and psbI. The phenotype of ncc1 is wild-type, since the remaining ATP synthase is sufficient to support photoautotrophic growth. which carries a recessive The nuclear mutant mutation in the Crp3 (chloroplast RNA processing) formation within the gene, is also defective in atpA atpA gene cluster (Levy et al., 1997). In monocistronic mRNA accumulates to wild-type levels, whereas the dicistronic atpA-psbI transcript accumulates to 50% of the wild-type level, and the tricistronic atpA-psbI-ycf10 transcript fails to accumulate (the tetracistronic atpA-psbI-ycf10-atpH transcript is unaffected). These results indicate that the products of at least two nuclear genes participate maturation or stabilization of transcripts of in has at least the atpA gene cluster. Interestingly, two other phenotypes. First, it was identified by robust phototrophic growth on minimal medium

175 resulting from high levels of accumulation of a nuclease-susceptible atpB transcript that lacks the stem-loop structure (see section 4, below). Secondly, processing presumptive intermediates in petD accumulate in but not in its wild-type progenitor. Although these intermediates accumulate to only a low level, these results suggest that the Crp3 gene product functions in the maturation of multiple chloroplast mRNAs. Since screens for non-photosynthetic mutants of Chlamydomonas have yielded several that act on the level of RNA stability, one might naturally expect that a certain percentage would have defects in stabilization. However, as first shown for the psbD stability mutant nac2-26 (Nickelsen et al., 1994) and subsequently for the atpB stability mutant thm24 (Drapier et al., 1992), the petA stability mutant MØ11 (Gumpel et al., 1995; D. B. Stern and R. G. Drager, unpublished), and the petD stability mutant F16 (Drager et al., 1997), these nuclear gene products stabilize their cognate mRNAs by acting on the UTR. This raises the question of why more stabilitydeficient mutants have not been isolated. The two main possibilities are that such mutants would not have a detectable phenotype with the screen used, or that such mutants would be lethal. The fact that both ncc1 and crp3 (which we have shown to be unlinked loci by genetic crosses) are able to grow photo­ autotrophically under normal conditions supports the first possibility. In this case, it will be necessary to design screens for mutants defective in processing processing, for example by inserting elements ectopically into essential genes or selectable markers. If stability mutants are lethal, then temperature-sensitive mutant screens might get around this problem. In E. coli, however, mutations that inactivate enzymes such as RNAse II, RNAse III or polynucleotide phosphorylase are not lethal; only multiple mutations cause lethality (Donovan and Kushner, 1986; Babitzke et al., 1993). This again supports the notion that there is redundancy in the formation machinery, or that RNA secondary structure alone allows sufficient accumulation of transcripts to support photoautotrophic growth under the conditions most often used for mutant screening— that is, total inability to grow without added acetate (see Stern et al., 1991; Drager et al., 1996).

4. Does the

UTR Regulate mRNA Stability?

In both land plants and Chlamydomonas, chloroplast

176 mRNAs exhibit a wide range of half-lives, which can vary between developmental stages, within the cell cycle, or in a circadian manner (Deng and Gruissem, 1987; Mullet and Klein, 1987; Leu et al., 1990; Kim et al., 1993; Salvador et al., 1993a,b; Hwang et al., 1996). To what degree is the 3´ UTR a determinant of chloroplast mRNA half-life? As noted above, most chloroplast mRNAs have inverted repeats in their 3´ UTRs; these repeats pair to form stem-loop structures, at least in vitro (Stern et al., 1989), and such structures impede the progress of exoribonucleases (McLaren et al., 1991; Drager et al., 1996). Thus, RNAs lacking these structures are unstable in vitro in chloroplast protein extracts (Stem and Gruissem, 1987; Stern and Kindle, 1993). In Chlamydomonas cells altered by chloroplast transformation, an atpB gene lacking the IR yielded unstable and heterogeneous mRNA (Stern et al., 1991), while either removal of the psaB stem-loop or extension of the psaB coding region into the stemloop also destabilized the transcript (Lee et al., 1996). In contrast, measurement of RNA half-life by slot blots did not reveal an effect of stem-loop removal (Blowers et al., 1993). However, these experiments did not distinguish between intact and partially degraded or heterodisperse transcripts. Overall, it appears that the IR has an important role in defining the 3´-end of the transcript, and that in its absence, transcripts are less stable and more heterogeneous. On the other hand, mRNAs lacking 3´ IRs appear to be translatable, and no absolute requirement for a 3´ stem-loop to support photoautotrophic growth has been demonstrated. Although the atpB 3´ IR deletion referenced above did not lead to obligate heterotrophic growth, the cells accumulated only 20% of the wild-type level of the ATP synthase, and grew slowly on minimal medium, and were sensitive to high light or elevated (35°C) temperature. These phenotypes allowed selection or screening for phenotypic suppressors, of which two types were isolated. In the first type, a gene amplification event had occurred, resulting in a 20-fold increase in the number of atpB genes still carrying the IR deletion (Kindle et al., 1994). These amplification events were associated with complex rearrangements of the chloroplast genome, and were unstable when the suppressed cells were supertransformed with an atpB gene containing the IR, in which case the copy number of atpB returned to one per genome (Suzuki et al., 1997). One possible explanation for this would be that very high levels of

David B. Stern and Robert G. Drager atpB mRNA titrate a factor required for the expression ofother genes. The other type of atpB suppressor was a nuclear mutation that was impeded in the degradation of the normally unstable transcript (Levy et al., 1997). As discussed in section 3, this suppressor mutation was in the gene Crp3, which appears to mRNA degrada­ define a factor required for tion. This demonstrates that nuclear factors can, at least in certain cases, determine chloroplast RNA half-life by interacting with the 3´ UTR. The above discussion still leaves open the question of what regulates chloroplast mRNA half-lives that vary either intrinsically or according to cellular signals. In the case of rbcL, there is evidence for light control of mRNA stability via the 5´ UTR (Salvador et al., 1993a), and similar conclusions have been reached for psbA mRNA in tobacco (Staub and Maliga, 1994). Since in the case of psbA the same sequences also control light-regulated translation (Staub and Maliga, 1994), this raises the more general question of whether light-, developmentally-, or cell cycle-regulated changes in RNA stability are secondary consequences of translational control. The existence of a variety of Chlamydomonas nuclear mutants defective in chloroplast translation (Chapter 12, Hauser et al.) offers an opportunity to test this possibility. The intrinsic differences in chloroplast RNA half-lives, which vary from about 30 min to several days and include both protein-coding and structural RNAs, cannot be explained solely by translational differences. In addition, there is no clear correlation between transcript stability and translatability in Chlamydomonas (Chapter 9, Nickelsen). Rather, multiple determinants within the untranslated regions and coding regions may confer differential susceptibility to nucleases. Recently, a new pathway of land plant chloroplast mRNA degradation, which can involve the 3´ UTR, has been described. Two reports have shown that sequences within the 3´ UTR, as well as sequences within the coding region, can serve as sites for polyadenylate addition (Kudla et al., 1996; Lisitsky et al., 1996). Know sites of polyadenylate addition and aproposed degration mechanism (Lisitsky et al., 1997) are illustrated in Fig. 3. The resulting polyadenylated transcripts are particularly susceptible to exonuclease digestion. Similarpolyadenylation of transcripts also occurs in E. coli, where it also leads to transcript degradation (Cohen, 1995; HaugelNielsen et al., 1996). It is not yet clear whether polyadenylation is the rate-limiting step in land plant

Chapter 10 Chloroplast Transcription

chloroplast mRNA decay, nor is it known whether polyadenylation also occurs in Chlamydomonas chloroplasts. This should therefore prove a fertile area for the study of chloroplast mRNA 3´ UTR function over the next several years.

Acknowledgments Work on chloroplast RNA processing and stability in the Stern laboratory was supported by grants from the Department of Energy and the National Science Foundation. R. G. D. was supported by a fellowship from the National Institutes of Health.

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David B. Stern and Robert G. Drager Stern DB and Gruissem W (1989) Chloroplast mRNA 3´ end maturation is biochemically distinct from prokaryotic mRNA processing. Plant Mol Biol 13: 615–625 Stern DB and Kindle KL (1993) 3´ end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a twostep process. Mol Cell Biol 13: 2277–2285 Stern DB, Jones H and Gruissem W (1989) Function of plastid mRNA 3´ inverted repeats: RNA stabilization and gene-specific protein binding. J Biol Chem 264: 18742–18750 Stern DB, Radwanski ER and Kindle KL (1991) A 3´ stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo. Plant Cell 3: 285–297 Stern DB, Higgs DC and Yang J (1997) Transcriptional and translational activities of chloroplasts. Trends Plant Sci 2: 308–316 Stevenson JK and Hallick RB (1994) The psaA operon prc­ mRNA of the Euglena gracilis chloroplast is processed into photosystem I and II mRNAs that accumulate differentially depending on the conditions of cell growth. Plant J 5:247–260 Stollar NE and Hollingsworth MJ (1994) Expression of the large ATP synthase gene cluster from spinach chloroplasts. J Plant Physiol 144: 141–149 Sturm N, Kuras R, Büschlen S, Sakamoto W, Kindle KL, Stern DB and Wollman FA (1994)The petD gene is transcribed by f u n c t i o n a l l y redundant promoters in Chlamydomonas reinhardtii chloroplasts. Mol Cell Biol 14: 6171–6179 Surzycki SJ and Shellenbarger DL (1976) Purification and characterization of a putative sigma factor from Chlamy­ domonas reinhardii. Proc Natl Acad Sci USA 73: 3961–3965 Suzuki H, Ingersoll J, Stern DB and Kindle KL (1997) Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts. Plant J 11: 635–648 Tanaka K, Oikawa K, Ohta N, Kuroiwa H, Kuroiwa T and Takahashi H (1996) Nuclear encoding of a chloroplast RNA polymerase sigma subunit in a red alga. Science 272: 1932– 1935 Thompson RJ and Mosig G (1985) An ATP-dependent supercoiling topoisomerase of Chlamydomonas reinhardtii affects accumulation of specific chloroplast transcripts. Nucleic Acids Res 13: 873–891 Thompson RJ and Mosig G (1987) Stimulation of a Chlamy­ domonas chloroplast promoter by novobiocin in situ and in E. coli implies regulation by torsional stress in the chloroplast DNA. Cell 48: 281–287 Thompson RJ and Mosig G (1990) Light affects the structure of Chlamydomonas chloroplast chromosomes. Nucleic Acids Res 18:2625–2631 Tiller K and Link G (1993) Sigma-likc transcription factors from mustard Sinapis alba L. etioplasts arc similar in size to, but functionally distinct from, their chloroplast counterparts. Plant Mol Biol 21: 503–513 Tracy RL and Stern DB (1995) Mitochondrial transcription initiation: promoter structures and RNA polymerases. Curr Genet 28: 205–216 Troxler RF, Zhang F, Hu J and Bogorad L (1994) Evidence that sigma factors are components of chloroplast RNA polymerase. Plant Physiol 104: 753–759 Vera A and Sugiura M (1995) Chloroplast rRNA transcription from structurally different tandem promoters: An additional novel-type promoter. Curr Genet 27: 280–284

Chapter 10 Chloroplast Transcription Wang MJ, Davis NW and Gegenheimer P (1988) Novel mechanisms for maturation of chloroplast transfer RNA precursors. EMBO J 7: 1567–1574 Westhoff P and Herrmann RG (1988) Complex RNA maturation in chloroplasts: the psbB operon from spinach. Eur J Biochem 171:551–564 Wolfe KH, Morden CW and Palmer JD (1992) Function and evolution of a minimal plastid genome from a nonphotosynthetic parasitic plant. Proc Natl Acad Sci USA 89: 10648–10652

181 Woodbury NW, Dobres M and Thompson WF (1989) The identification and localization of 33 pea chloroplast transcription initiation sites. Curr Genet 16: 433–446 Yang J and Stern DB (1997) The spinach chloroplast endoribonuclease CSP41 cleaves the 3´ untranslated region of petD mRNA primarily within its terminal stem-loop structure. J Biol Chem 272: 12874–12880 Zaitlin D, Hu J and Bogorad L (1989) Binding and transcription of relaxed DNA templates by fractions of maize chloroplast extracts. Proc Natl Acad Sci USA 86: 876–880

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Chapter 11 RNA Splicing in the Chloroplast David L. Herrin, Tai-Chih Kuo Department of Botany and Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78713, U.S.A.

Michel Goldschmidt-Clermont Department of Molecular Biology and Department of Plant Biology, University of Geneva, Sciences II, 30 quai E. Ansermet, CH-1211 Genève 4, Switzerland

Summary I. Introduction II. Group I Introns A. Group I Introns: Splicing Mechanism and Structure B. Group I Introns in Chlamydomonas spp. Chloroplast Genes 1. Distribution and Classification 2. Identification of Self-Splicing Group I Introns 3. Biochemical Characterization of Two Self-Splicing Group I Introns C. Protein Factors Involved in Group I Splicing D. Regulation of Group I Splicing III. Group II Introns and Trans-Splicing A. Group II Introns: Splicing Mechanism and Structure B. Trans-Splicing of the psaA Introns 1. Structure of the psaA Gene 2. Trans-Splicing Mutants 3. The tscA RNA 4. Evolution of Trans-splicing C. Splicing of Heterologous Group II Introns IV. Perspective Acknowledgments References

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Summary The chloroplast genomes of Chlamydomonas spp. contain introns that belong to the two major classes found in organelles: group I and group II. Some of the members of both classes are ribozymes capable of self-splicing in vitro and, indeed, most of the group I introns studied in Chlamydomonas spp. are autocatalyic. The biochemical characterization of these introns suggests that, in vivo, their splicing must be dependent on protein factors. It is particularly interesting in this respect that light regulates the splicing of the four group I introns in the psbA gene of C. reinhardtii. The two group II introns of the psaA gene are split into two or more pieces which are transcribed separately from different chloroplast loci. The mature psaA RNA is assembled from the separate precursors in two steps of intermolecular trans-splicing. There are many nuclear mutants defective in psaA mRNA maturation, which identify a large number of protein factors that are required, directly or indirectly, for trans-splicing. J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 183–195. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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I. Introduction Some chloroplast genes contain intervening sequences, or introns as they are more often called, that interrupt the coding sequences (i.e. exons). Two main types of introns have been found in Chlamy­ domonas spp. chloroplast genes, group I and group II. These designations were originally given to the two structurally different families of introns that were found mainly, but not exclusively, in organelles, and was based primarily on conserved primary and secondary structure elements (Michel and Dujon, 1983). Subsequently, it was shown that these two intron groups also differ in their mechanism of splicing (see Cech, 1990; Michel and Ferat, 1995, for reviews). However, some members of both groups are self-splicing introns, i.e. introns that splice in vitro in the absence of proteins. Those remarkable findings indicated that the pre-RNA itself catalyzes the splicing reactions. Consequently, these molecules are often referred to as catalytic RNAs, or ‘ribozymes’. In vivo, however, proteins probably facilitate splicing of most group I and group II introns (Saldanha et al., 1993), and recent evidence indicates that, at least in the case of group I introns, some of the proteins act by promoting the correct folding of these large catalytic RNAs (Mohr et al., 1992; Shaw and Lewin, 1995; Weeks and Cech, 1995). Group I introns are more common than group II in Chlamydomonas spp. chloroplasts, a situation that differs from land plant chloroplasts, where group II introns are more common (Plant and Gray, 1988). The only group II introns described so far in Chlamydomonas spp. chloroplasts are the two introns in the psaA gene (Kück et al., 1987; Turmel et al., 1995a). Interestingly, both of these introns are spliced in trans: the psaA exons, with flanking intronic sequences, are found at widely separated sites in the chloroplast genome (Kück et al., 1987), and are apparently transcribed independently (Choquet et al., 1988; Herrin and Schmidt, 1988). In addition, an internal segment ofintron 1 is encoded at yet another site in the genome, the tscA locus, which is transcribed to produce a small RNA (Goldschmidt-Clermont et al., 1991). Thus, at least three RNAs must be brought together for the splicing of psaA intron 1. This mode of splicing shows a strong dependence on protein Abbreviations: LSU – large subunit ribosomal RNA; ORF – open reading frame

factors, since self-splicing has not been reported for any trans-spliced intron. It is also interesting that trans-splicing introns have only been found so far among group II, but not group I introns. Research with C. reinhardtii has played an important role in our current understanding of RNA splicing in green plant chloroplasts. The first selfsplicing RNAs from chloroplasts were identified using C. reinhardtii (Herrin et al., 1990, 1991), as were the first mutants blocked in splicing of chloroplast introns (Choquet et al., 1988; Herrin and Schmidt, 1988). In fact, mutants affected in chloroplast biogenesis played key roles in both those studies. In addition, chloroplast transformation in C. reinhardtii has greatly facilitated the study ofcis­ and trans-acting expression elements encoded in the chloroplast genome (Rochaix, 1996). Finally, Chlamydomonas is proving to be an excellent genus for studying the evolution of introns, due in part to the large number of related species, and the fact that most, if not all, contain these classes of introns (e.g. Turmel et al., 1993a, 1995a). The purpose of this chapter is to summarize our current state ofknowledge concerning RNA splicing in Chlamydomonas spp. chloroplasts.

II. Group I Introns

A. Group I Introns: Splicing Mechanism and Structure Figure 1 shows the splicing mechanism for group I introns (reviewed in Cech, 1990). The first step involves a free guanosine nucleotide (probably GTP), which makes a nucleophilic attack at the 5´ splicesite, breaking the pre-RNA chain, and becoming covalently attached to the 5´ end of the intron-exon molecule. In the second step, the 3´ OH of the 5´ exon attacks the 3´ splice-site, effectively displacing the intron and ligating the exons. The liberated intron often undergoes cyclization reactions via the conserved 3´ terminal G, which attacks one or more phosphodiester bonds near the 5´ end of the intron. Intron cyclization is commonly seen in vitro, but has also been seen in vivo (Daros and Flores, 1996). Because circular RNAs do not migrate strictly according to their size in electrophoresis gels, the principal fate ofthe free intron in vivo is not clear for most systems. We will briefly review the structure of group I

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185 5´ splice-site, which is also part of the P1 helix, is usually the sequence GU (U being the last nucleotide of the exon), with the U being involved in a wobble base-pairing interaction with a G in the intron. The 3´ terminal nucleotide of the intron is nearly always a G, and it binds to the guanosine-binding site in P7 during the second splicing step, exon ligation. A short sequence near the 5´ end of the intron, known as the internal guide sequence (IGS), base-pairs with the end of the 5´ exon, and during the second step, the beginning of the 3´ exon. Thus, the IGS plays a role in both steps by helping to align the splice-sites relative to the catalytic core. These conserved features make up only a fraction of the sequence for many group I introns, which range in size from ~250 to ~2000 bp. Most of the larger group I introns contain Open Reading Frames (ORFs), many of which have been shown to encode site-specific DNA endonucleases that promote intron mobility (reviewed in Dujon, 1989). Other less conserved domains in group I introns have also been found to play important roles in the ability of the respective introns to self-splice, apparently by contributing to the stable formation of the catalytic core (Jaeger et al., 1996). The presence or absence of these so-called peripheral domains have been used to classify Group I introns into four major classes (A,B,C, and D), and eleven subclasses in total (Michel andWesthof, 1990).

B. Group I Introns in Chlamydomonas spp. Chloroplast Genes introns; for a more detailed and recent review the reader is referred to Jaeger et al. (1996). Group I introns are identified according to their secondary structure, which includes a conserved set of basepaired regions (called P1, P2, etc.), but only a few highly conserved nucleotides. Figure 2 shows the secondary structure of the single intron in the 23S rRNA gene of C. reinhardtii (Rochaix et al., 1985), depicted in the ‘original’ style (Fig. 2A), and in the newer style (Fig. 2B); the latter is more representative of the intron’s three-dimensional structure in that the P4-P6, and the P8, P3 and P7 helices are stacked colinearly (Cech et al., 1994). The latter form also facilitates depiction of some of the tertiary interactions. Together, the paired regions P3-P8 constitute the catalytic core, and P7 contains the guanosine binding site. The ends of the intron, together with a few nucleotides into each adjacent exon, are important for splice-site recognition. The

1. Distribution and Classification Table 1 provides a list of published group I introns from Chlamydomonas spp. chloroplast genes; most of them fall into the A or B classes, but with only one B subclass, IB4, represented so far. A number of other Group I introns have been found in the rrnL gene of different Chlamydomonas species, although the sequences of these have not yet been published (Turmel et al., 1993a). The introns reported to date have been found mainly in rRNA genes (both rrnL and rrnS), and in genes coding for proteins involved in photosynthesis. It should be noted, however, that not all of the chloroplast genes in these species, including C. reinhardtii, have been sequenced. Thus, it is possible that additional Group I introns will be found in these and other species of Chlamydomonas. The irregular, but phylogenetically extensive,

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distribution of group I introns suggests that they have migrated during evolution. This migration could have occurred at the DNA level (see Dujon, 1989 for a discussion thereof), or at the RNA level via reverse splicing (Woodson and Cech, 1989), followed by reverse transcription of the RNA into cDNA, and then recombination with genomic DNA. One characteristic shared by the chloroplast genes that contain group I introns is that they encode abundant RNAs (see Table 1). The presence of abundant ‘substrate’ would certainly promote reverse splicing, although the successful completion of all steps is probably a rare event (Thompson and Herrin, 1994). Group I intron migration at the RNA level during evolution is also strongly supported by the preferential location of introns in regions of the rrnL gene that correspond to sites in the mature RNA that are exposed on the surface of the ribosome (Turmel et al., 1993a).

2. Identification of Self-Splicing Group I Introns Because not all group I (or group II) introns selfsplice in vitro, searching for self-splicing introns by subcloning each one (with some flanking exon sequences), synthesizing pre-RNA in vitro, and testing it for self-splicing activity could be a frustrating endeavor. However, an alternative approach is to take advantage of the group I splicing mechanism, which involves the covalent addition of a guanosine nucleotide to the intron (Fig. 1), to rapidly look for self-splicing introns in total cellular RNA. Of course, this approach requires the presence of at least some precursor RNA in the cells. Using RNA from the ac20 mutant of C. reinhardtii (which, as it turns out, contains increased levels ofunspliced RNAs from the rrnL and psbA genes), it was straightforward to identify self-splicing group I in the introns by incubating total RNA with

Chapter 11 Chloroplast RNA Splicing

presence of and then hybridizing the GTPlabeled RNA to total chloroplast DNA, or to cloned DNA fragments (Herrin et al., 1990, 1991). In vitro synthesis of pre-RNAs for each intron was used to confirm that they self-splice in vitro. The single intron in the rrnL gene, and all 4 introns in the psbA gene, proved to be catalytic RNAs (Herrin et al., 1990, 1991). Moreover, these 5 introns constitute all the known group I introns in C. reinhardtii. Using RNA synthesized in vitro, nine other Chlamydomonas chloroplast group I introns were tested for self-splicing activity, and eight of them were positive (Côté and Turmel, 1995; Table 1). Thus, of the group I introns from Chlamydomonas that have been tested, the vast majority have been found to be capable of self-splicing in vitro. It is also interesting to note that all of the self-splicing introns are in the A class, while the non-catalytic intron is from the B class (Table 1).

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3. Biochemical Characterization of Two SelfSplicing Group I Introns Since the RNA plays such an important role in group I splicing, acting both as catalyst, and providing specificity to the splicing reactions, it was of interest to carry out some biochemical analyses of selfsplicing reactions catalyzed by chloroplast group I introns. Table 2 shows the conditions that were necessary for efficient self-splicing of the CrLSU and CrpsbA2 introns; these data were gathered using synthetic RNAs produced by in vitro transcription of linearized plasmid with phage RNA polymerases. Both introns were highly specific for guanosine nucleotides, with only a weak reaction with ATP (at least 100-fold less than with GTP); there was no was strictly reaction detected with CTP or UTP. required for activity with either intron, and the optimal concentration range was 15–25 mM (in the presence

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of monovalent salt). The temperature optimum for self-splicing was quite high (45–47 °C), and splicing at 23 °C was very slow. Spermidine, which can promote RNA folding, stimulated the self-splicing of only the CrpsbA2 intron. Kinetic analyses of selfsplicing by both introns were also performed. The and values were similar to those of the Tetrahymena self-splicing intron (Bass and Cech, 1984), which has been the most studied group I intron (Cech, 1990); they were and for CrLSU, and and for CrpsbA2, respectively (Bao, 1993; T-C. Kuo and D.L. Herrin, unpublished). The rate values for self-splicing by these introns are also comparable to our estimated rates for splicing of these introns in vivo (Fig. 3), although, for the in vivo studies, the cells were grown and analyzed at 23 °C. Thus, from this perspective, these introns appear to splice more readily in vivo than in vitro. It is also apparent that these two introns are functionally related, which may not be surprising, since they are also in the same subclass, 1 A3 (Table 1). Finally, these studies showed that chloroplast group I introns self-splice using the same overall mechanism as the Tetrahymena intron.

C. Protein Factors Involved in Group I Splicing The splicing of most group I introns in vivo is probably facilitated by trans-acting factors that are proteins. This suggestion is supported by the following: (1) some group I introns are not capable of self-splicing in vitro, (2) of those that can, the conditions needed for efficient self-splicing are often non-physiological, and (3) fungal nuclear mutants have been isolated that are deficient in splicing mitochondrial group I introns in vivo, including introns that are capable of self-splicing in vitro. Proteins that promote splicing of mitochondrial group I introns in Saccharomyces cerevisiae and

Neurospora crassa were initially identified using genetic approaches; the proteins are encoded in the nuclear genome and within the introns themselves (Saldanha et al., 1993). The intron-encoded proteins are known only through genetics; they are usually found in-frame with the preceding exon, and they promote splicing of mainly the intron where they reside. However, two ofthe nucleus-encoded splicing factors have been biochemically characterized, these are products ofthe CYT18 and CBP2 genes. Cyt18 is a bifunctional protein that can promote splicing of many group I introns, including the non self-splicing NcLSU intron; its other function is in tyrosyl-tRNA charging (Guo and Lambowitz, 1992). In contrast, Cbp2 specifically promotes splicing of the Sc.cob.5 intron, which requires non-physiological (i.e. high) concentrations to self-splice in vitro (Gampel and Tzagoloff, 1987; Gampel et al., 1989). Both of these proteins seem to act by promoting folding of the pre-RNAs into their catalytically active forms (Mohr et al., 1992; Shaw and Lewin, 1995; Weeks and Cech, 1995). The conditions required for efficient self-splicing by the C. reinhardtii chloroplast group I introns, CrLSU and CrpsbA2 (see Table 2 and above), suggest that proteins probably facilitate splicing of these introns in vivo. However, splicing factors for chloroplast group I introns have not been clearly identified. Most of the intron-encoded ORFs described to date are free-standing rather than inframe like the mitochondrial maturases (although CrpsbA3 contains an ~18 kDa ORF that is in-frame with the upstream exon; N. Deshpande, unpublished). Moreover, several ofthese ORFs have been shown to encode DNA endonucleases that function in intron mobility (see Table 1), although that by itself does not preclude them from also having RNA maturase activity. However, it should be noted that deletion of most of the ORF from the CrLSU intron did not

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189 affect splicing of that intron in vivo (Thompson and Herrin, 1991). Previously, it was suggested that the ac20 mutant of C. reinhardtii, which is deficient in functional chloroplast ribosomes, might be a splicing mutant, since it over-accumulates unspliced CrLSU (i.e. pre­ 23S) RNA (Herrin et al., 1990). However, deletion of the CrLSU intron from ac20 did not restore the wildtype phenotype, which would have been expected if the primary defect in ac20 was a deficiency in CrLSU splicing (S. Holloway and D.L. Herrin, unpublished). Although this does not preclude a role for the ac20 gene product in group I splicing, it does indicate that the apparent splicing deficiency in this mutant is not the primary cause of its phenotype.

D. Regulation of Group I Splicing The fact that most group I introns probably require proteins for efficient splicing in vivo, suggests that splicing could be regulated. However, until recently, evidence for regulated splicing of group I introns was generally lacking. Under light-dark cycles, C. reinhardtii cell growth and division become synchronized, thereby providing a homogeneous population of cells (Harris, 1989). Moreover, under these conditions, there are extensive changes in gene expression (Herrin and Michaels, 1984). By focusing on the introns of the psbA gene of C. reinhardtii, a gene known to be strongly light-regulated (e.g. Herrin et al. 1986), Deshpande et al. (1997) obtained evidence that light promotes splicing of all four of the CrpsbA introns coordinately. These authors observed that unspliced psbA pre-RNAs accumulate in the dark, apparently due to a slow rate of splicing, and then disappear quickly upon illumination. This effect was observed despite a simultaneous, lightstimulated increase in transcription of the psbA gene (Deshpande et al., 1997). By blocking transcription with an inhibitor, the rate of splicing of each intron was estimated as shown in Fig. 3. The results indicate that light stimulates splicing ofpsbA introns six- to ten-fold, and that the introns splice with a half-life of ~12–15 min in the light. By using specific inhibitors, mutants, and chloroplast transformation, Deshpande et al. (1997) also showed that the light-regulated splicing of psbA introns requires photosynthetic electron transport, but not ATP synthesis. Thus, this study provided the first evidence that RNA splicing in chloroplasts can be regulated by light.

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III. Group II Introns and Trans-Splicing

A. Group II Introns: Splicing Mechanism and Structure Some members ofgroup II have the ability to undergo self-splicing in vitro under appropriate conditions of elevated temperature and high concentrations of salt and magnesium, which are clearly not physiological. This suggests that the reaction in vivo is facilitated by protein factors. Figure 4 shows the splicing mechanisms for group II introns (reviewed by Saldanha et al., 1993; Michel and Ferat, 1995; Jacquier, 1996). While the prevalent pathway involves two trans-esterification reactions, under certain conditions in vitro the first step can proceed via hydrolysis. In the first trans-esterification reaction, the 2´ OH of the A nucleotide at the branch site attacks the 5´ exon-intronjunction to produce a lariat intermediate (lariat intron- 3´ exon) and the separated 5´ exon. In the second step, the free 3´ OH of the 5´ exon attacks the 3´ intron-exonjunction, yielding the spliced exons and releasing the intron. Both transesterification reactions are reversible, so that a free intron can insert by reverse-splicing into the appropriate exon-exon junction. The excised intron is a versatile ribozyme and can also catalyze other reactions, such as reverse-splicing into DNA, or polymerization of RNA or DNA in a 3´– 5´direction (Möril et al., 1992; Mueller et al., 1993;Zimmerley et al., 1995 ;Hetzer et al., 1997). Group II introns share conserved secondary structure and tertiary interactions, but relatively few strictly conserved nucleotides in the primary sequence (Michel and Ferat, 1995; Jacquier, 1996; Costa etal., 1997). The secondary structure is usually represented as six partly helical domains surrounding a central wheel (see Section B.3 and Fig. 6). Domain V, which has a very conserved structure, and domain I are the most important for catalysis, while domain VI contains the bulging A at the branch point which is involved in the first trans-esterification reaction. An important tertiary interaction entails base-pairing of a sequence in domain I of the intron (exon binding site 1, EBS 1) with the last bases of the upstream exon (intron binding site 1, IBS1). The nucleotide just 5´ to EBS 1 can interact with the first nucleotide of the downstream exon and thus may act as a guide in the second trans-esterification. Another part of domain I (EBS2) can pair with nucleotides in the upstream exon (IBS2) adjacent to IBS 1. There are many further

tertiary interactions between different parts of the structure. Domain IV sometimes encodes a poly­ peptide which in some cases is required for splicing in vivo (maturase) and in other cases is involved in intron homing and transposition, with a domain related to reverse transcriptases (reviewed by Grivell, 1996; Curcio and Belfort, 1996).

B. Trans-Splicing of the psaA Introns 1. Structure of the psaA Gene In 1987, Kück and coworkers discovered that in C. reinhardtii, the psaA gene is composed of three exons encoded at widely separate loci of the chloroplast genome, and that sequences adjacent to

Chapter 11 Chloroplast RNA Splicing

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these exons share some of the conserved primary structure features of group II introns. This suggested that the psaA exons may be assembled via a process of splicing in trans. The first intron of the rps12 gene in land plants is similarly fragmented, and numerous split introns also occur in land plant mitochondria (reviewed by Bonen, 1993). The psaA gene in land plant plastids and the rps12 gene in C. reinhardtii are not fragmented (Liu et al., 1989). In other Chlamydomonas taxa, the psaA gene is also composed of three widely separated exons (Boudreau et al., 1994; Turmel et al., 1995). Even in the distantly related C. moewusii, the three exons are colinear with those of C. reinhardtii, indicating that the split structure of the psaA intron was present in a common ancestor of these Chlamydomonas taxa (Boudreau et al., 1994; Turmel et al., 1995). To our knowledge, efficient in vitro splicing has not been demonstrated for any of the fragmented group II introns, suggesting that splicing of these introns is strictly dependent on trans-acting factors.

2. Trans-Splicing Mutants The analysis of C. reinhardtii mutants defective in photosystem I activity revealed a large number of mutations in both the chloroplast and the nuclear genome that affect the maturation of the psaA mRNA (Choquet et al., 1988; Herrin and Schmidt, 1988; Roitgrund and Mets, 1990). The mutants accumulate unspliced precursors and partly spliced transcripts which are presumably intermediates in the psaA mRNA maturation pathway. Their analysis indicates that the three exons of psaA are transcribed independently, and that the mature psaA mRNA is normally assembled in two steps of splicing in trans (Fig. 5). Some of the precursors are polycistronic: exon 2 is transcribed together with the psbD gene upstream, and exon 1 is most likely transcribed together with trnI downstream (Choquet et al., 1988; Turmel et al., 1995). The mutants can be assigned to three classes, according to the precursors and intermediates that they contain. In one class ofmutants (class C), trans-splicing of exons 1 and 2 is blocked, but exons 2 and 3 are assembled normally. Conversely in a second class (class A), trans-splicing of exons 1 and 2 can proceed, but not of exons 2 and 3. In the third class (class B), neither trans-splicing reaction occurs. The mutants in each class belong to multiple nuclear loci, as determined by complementation tests: five in class A, two in class B and seven in class C

(Goldschmidt-Clermont et al., 1990). It is likely that there are many more than fourteen nuclear genes required for trans-splicing of psaA mRNA, because most loci in classes A and C are represented by only one allele. It is striking that the products of the numerous loci in classes A and C are specifically required, directly or indirectly, in the splicing ofjust one of the two split introns. The loci in class B may encode factors more generally involved in splicing of group II introns, since the two split introns of psaA are the only group II introns described so far in the C. reinhardtii chloroplast genome. Three of the nuclear genes required for trans-splicing have been cloned by gene-tagging or by complementation (M.

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Goldschmidt-Clermont, K. Perron, C. Rivier and J.D. Rochaix, unpublished; see Chapter 4, Kindle). The analysis of these and other genes should provide insight into the function of factors involved in group II intron splicing and trans-splicing. There are a few mutants of Saccharomyces cerevisiae which are affected in splicing ofmitochondrial group II introns (reviewed by Saldanha et al., 1993). Two nuclear mutants of maize (crs1 and crs2) are deficient in splicing of different subsets ofthe group II introns in the plastid (Jenkins et al., 1997). It will be interesting to determine whether the affected genes bear any relation to those affected in the splicing mutants of C. reinhardtii.

3. The tscA RNA There is also a chloroplast gene (tscA, trans-splicing in the chloroplast) which is required for trans-splicing of exons 1 and 2 (Roitgrund and Mets, 1990; Goldschmidt-Clermont et al., 1990). The product of tscA is a small RNA devoid of coding sequence which is probably involved in forming the conserved structure of group II introns (Goldschmidt-Clermont et al., 1991). In the proposed model (Fig. 6), the tscA RNA forms a helix with the precursor of exon 1 (domain I of the intron), contributes domains II and III, and forms a helix with the precursor of exon 2 (domain IV). The first intron of psaA is thus composed of (at least) three separate transcripts. A comparative sequence analysis of the first split intron in the related species C. gelatinosa and C. zebra supports this model (Turmel et al., 1995a). A puzzling feature is that conserved elements of domain I are absent, and in particular the EBS1 region. Whether the missing structures and tertiary interactions are replaced by protein factors, whether additional RNAs are involved, or both, remains to be determined.

4. Evolution of Trans-splicing From an evolutionary point of view, it is interesting that group II introns share structural and mechanistic features with nuclear introns, but their phylogenetic relation is still a matter of debate (reviewed by Jacquier, 1996). The split structure of the first intron of psaA, with part of the intron on a small separate transcript, can be taken as a simple example of how some of the structural or catalytic domains of nuclear introns may have been transferred to the snRNAs (Goldschmidt-Clermont et al., 1991; Sharp, 1991). Another issue is whether split introns are archaic

remnants of an ancestral situation, or alternatively, whether they have appeared more recently during evolution as the result of genome rearrangements, perhaps with the recruitment of novel factors to mediate intermolecular intron assembly (Laird, 1989). Group II introns are split in different genes in the lineages of land plants (rps12) compared to Chlamydomonas spp (psaA), but this difference does not allow a distinction between the two alternatives. The split introns may have appeared after the separation of the two lineages, or may have been present in both genes in a common ancestor of algae and land plants and subsequently differentially lost. It is possible that the mechanism of trans-splicing is ancestral, and that it has repeatedly allowed the appearance of novel split introns during evolution.

C. Splicing of Heterologous Group II Introns Group II introns from organelles of other organisms have been introduced into the C. reinhardtii chloroplast genome by transformation. The rI1 intron from the mitochondrial rDNA of Scenedesmus obliquus was found to excise accurately when it was inserted, together with the flanking EBS1 sequence, within the tscA RNA (Herdenberger et al., 1994). In contrast, when the chloroplast gene of spinach,

Chapter 11 Chloroplast RNA Splicing containing a group II intron, was introduced in the C. reinhardtii chloroplast genome in an expression vector, the chimeric atpF transcript accumulated to high levels but the heterologous intron was not spliced (Deshpande et al., 1995). According to details of their structure, group II introns can be subdivided in two classes, IIA and IIB (Michel et al., 1989). To understand the different fates ofthe two heterologous introns, it may be significant that the rI1 intron is a member of subgroup IIB, that the atpF intron belongs to subgroup IIA, and that the two split introns of C. reinhardtii, although untypical, are more closely related to subgroup IIB. This could indicate that splicing factors in the C. reinhardtii chloroplast are specific for introns from subgroup IIB. The analysis of maize mutants deficient in chloroplast splicing also suggests that different splicing factors are specific to one or other of the subgroups (Jenkins et al., 1997). However it should also be noted that the rll intron is capable of self-splicing in vitro (Kück et al, 1990), while the atpF intron is not: thus the requirements for splicing factors may be more stringent for the latter than for the former.

IV. Perspective An important goal for the future will be to study the factors that promote splicing of group I and group II introns in vivo, and this will be facilitated by the isolation of the corresponding genes. Mutations identify numerous nuclear loci which are required for trans-splicing of the psaA group II introns, some of which have recently been cloned (Section III.B.2). The putative proteins that promote splicing of the psbA group I introns should also be particularly interesting, since splicing of these introns is regulated by light via a redox pathway. It would also be important to determine whether splicing of any of the other introns in photosynthesis-related genes is light-regulated. Finally, the development of proteindependent assays for splicing in vitro, or in organello, would advance studies of the factors that mediate splicing and its regulation.

Acknowledgments We thank Drs. S. Holloway and H.-H. Kim for communicating results prior to publication, and N. Roggli for preparing some of the figures. DLH and TCK were supported by grants from the US Dept. of

193 Agriculture (96-35301-3420), and the Robert A. Welch Foundation (F-1164) during preparation of the manuscript. MGC was supported by a grant from the Swiss National Fund for Scientific Research (31 34014.92).

References Bao Y (1993) Studies of a self-splicing group I ribozyme in the chloroplast psbA gene of Chlamydomonas reinhardtii. PhD Dissertation, University of Texas at Austin Bao Y and Herrin DL (1993) Nucleotide sequence and secondary structure of the chloroplast group I intron Cr.psbA-2: Novel features of this self-splicing ribozyme. Nucleic Acids Res 21: 1667 Bass B and Cech TR (1984) Specific interaction between the selfsplicing RNA of Tetrahymena and its guanosine substrate: Implications for biological catalysis by RNA. Nature 308: 820–826 Bonen L(1993) Trans-splicing of pre-mRNA in plants, animals and protists. FASEB J 7:40–46 Cech TR (1990) Self-splicing of group I introns. Annu Rev Biochem 59: 543–568 Cech TR, Damberger SH and Gutell RR (1994) Representation of the secondary and tertiary structure of group I introns. Nature Struct Biol 1: 273–280 Choquet Y, Goldschmidt-Clermont M, Girard-Bascou J, Kück U, Bennoun P and Rochaix J-D (1988) Mutant phenotypes support a trans-splicing mechanism for the expression of the tripartite psaA gene in the C. reinhardtii chloroplast. Cell 52: 903–913 Costa M, Dème E, Jacquier A and Michel F (1997) Multiple tertiary interactions involving domain II of group II selfsplicing introns. J Mol Biol 267: 520–536 Côté M-J and Tunnel, M (1995) In vitro self-splicing reactions of chloroplast and mitochondrial group-1 introns in Chlamy­ domonas eugametos and Chlamydomonas moewusii. Curr Genet 27: 177–183. Côté V, Mercier J-P, Lemieux C and Turmel M (1993) The single group I intron in the chloroplast rrnL gene of Chlamydomonas humicola encodes a site-specific DNA endonuclease. Gene 129: 69–76 Curcio MJ and Belfort M (1996) Retrohoming: cDNA-mediated mobility ofgroup II introns requires a catalytic RNA. Cell 84: 9–12 Daròs J and Flores R (1996) A group I plant intron accumulates as circular RNA forms with extensive 5´ deletions in vivo. RNA 2: 928–936 Deshpande NN, Hollingsworth M and Herrin DL (1995) The atpF group-I I intron-containing gene from spinach chloroplasts is not spliced in transgenic Chlamydomonas chloroplasts. Curr Genet 28: 122–127 Deshpande NN, Bao Y and Herrin DL (1997) Evidence for light/ redox regulated splicing of the psbA introns in Chlamydomonas chloroplasts. RNA 3: 37–48 Dujon B (1989) Group I introns as mobile genetic elements: Facts and mechanistic speculations—a review. Gene 82: 91– 114 Durocher V, Gauthier A, Bellemare G and Lemieux C (1989) An

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optional group I intron between the chloroplast small subunit rRNA genes of Chlamydomonas moewusii and C. eugametos. Curr Genet 15: 277–82 Dürrenberger F and Rochaix J-D (1991) Chloroplast ribosomal intron of Chlamydomonas reinhardtii: In vitro self-splicing, DNA endonuclease activity and in vivo mobility. EMBO J 10: 3495–3501 Gampel A and Tzagoloff A (1987) In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA. Mol Cell Biol 7: 2545–2551 Gampel A, Nishikimi M and Tzagoloff A (1989) CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron. Mol Cell Biol 9: 5424–5433 Gauthier A, Turmel M and Lemieux C (1991) A Group I intron in the chloroplast large subunit rRNA gene of Chlamydomonas eugametos encodes a double-strand endonuclease that cleaves the homing site of this intron. Curr Genet 19: 43–47 Goldschmidt Clermont M, Girard Bascou J, Choquet Y and Rochaix JD (1990) Trans-splicing mutants of Chlamydomonas reinhardtii. Mol Gen Genet 223: 417–25 Goldschmidt-Clermont M, Choquet Y, Girard-Bascou J, Michel F, Schirmer-Rahire M and Rochaix J-D (1991) A small chloroplast RNA may be required for trans-splicing in Chlamydomonas reinhardtii. Cell 65: 135–143 Grivell LA (1996) Transposition: Mobile introns get into line. Curr Biol 6: 48–51 Guo Q and Lambowitz A (1992) A tyrosyl-tRNA synthetase binds specifically to the Group I intron catalytic core. Genes Dev 6: 1357–1372 Harris EH (1989) The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego Harris EH, Boynton JE and Gillham NW (1974) Chloroplast ribosome biogenesis in Chlamydomonas. Selection and characterization of mutants blocked in ribosome formation. J Cell Biol 63: 160–179 Herdenberger F, Holländer B and Kück U (1994) Correct in vivo RNA splicing of a mitochondrial intron in algal chloroplasts. Nucleic Acids Res 22: 2869–2875 Herrin D and Michaels A (1984) Gene Expression during the Cell Cycle of Chlamydomonas reinhardtii. In: Stein G and Stein J (eds) Recombinant DNA and Cell Proliferation, pp 87– 106. Academic Press, Orlando Herrin DL and Schmidt GW (1988) trans-Splicing of transcripts for the chloroplast psaA gene. In vivo requirement for nuclear gene products. J Biol Chem 263: 14601–14604 Herrin DL, Michaels AS and Paul A-P (1986) Regulation of genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase and the photosystem II polypeptides D-1 and D-2 during the cell cycle of Chlamydomomas reinhardtii. J Cell Biol 103: 1837–1845 Herrin DL, Chen Y-F and Schmidt GW (1990) RNA splicing in Chlamydomonas chloroplasts: Self-splicing of 23S preRNA. J Biol Chem 265: 21134–21140 Herrin DL, Bao Y, Thompson AJ and Chen Y-F (1991) Selfsplicing of the Chlamydomonas chloroplast psbA introns. Plant Cell 3: 1095–1107 Hetzer M, Schweyen RJ and Mueller MW (1997) DNA polymerization catalysed by a group II intron RNA in vivo. Nucleic Acids Res 25: 825–1829 Jaeger L, Michel F and Westhof E (1996) The structure of group

I ribozymes. In: Eckstein F and Lilley DMJ (eds) Nucleic Acids and Molecular Biology, Vol 10, pp 33–52. SpringerVerlag, Berlin Jacquier A (1996) Group II introns: Elaborate ribozymes. Biochimie 78: 474–487 Jenkins BD, Kulhanek DJ and Barkan AB (1997) Nuclear mutations that block group II RNA splicing in maize chloroplasts reveal several intron classes with distinct requirements for splicing factors. Plant Cell 9: 283–296 Kück U, Choquet Y, Schneider M, Dron M and Bennoun P (1987) Structural and transcriptional analysis of two homologous genes for the P700 chlorophyll a apoproteins in Chlamydomonas reinhardtii: Evidence for trans-splicing. EMBO J 6: 2185–2195 Kück U, Godehardt I and Schmidt U (1990) A self-splicing group II intron in the mitochondrial large subunit rRNA (LSUrRNA) gene of the eukaryotic alga Scenedesmus obliquus. Nucleic Acids Res 18: 2691–2697 Laird PW (1989) Trans-splicing in trypanosomes—archaism or adaptation? Trends Genet 5: 204–216 Liu X-Q, Gillham NW and Boynton JE (1989) Chloroplast ribosomal protein gene rps12 of Chlamydomonas reinhardtii. J Biol Chem 27: 16100–16108 Michel F and Dujon B (1983) Conservation ofsecondary structures in two intron families including mitochondrial-, chloroplastand nuclear-encoded members. EMBO J 2: 33–38 Michel F and Ferat J-L (1995) Structure and activities of group II introns. Annu Rev Biochem 64: 435–461 Michel F and Westhof E (1990) Modelling of the threedimensional architecture of Group I catalytic introns based on comparative sequence analysis. J Mol Biol 216: 585–610 Michel F, Umesono K and Ozeki H (1989) Comparative and functional anatomy of group II catalytic introns—a review. Gene 82: 5–30 Mohr G, Zhang A, Gianelos J, Belfort M and Lambowitz A (1992) The Neurospora cyt-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core. Cell 69: 483–494 Mueller MW, Hetzer M and Schweyen RJ (1993) Group II intron RNA catalysis of progressive nucleotide insertion: A model for RNA editing. Science 261: 1035–1038 Plant AL and Gray JC (1988) Introns in chloroplast proteincoding genes of land plants. Photosynth Res 16: 23–39 Rochaix J-D (1996) Post-transcriptional regulation of chloroplast gene expression in Chlamydomonas reinhardtii. Plant Mol Biol 32: 327–341 Rochaix J-D, Rahire M and Michel F (1985) The chloroplast ribosomal intron of Chlamydomonas reinhardtii codes for a polypeptide related to mitochondrial maturases. Nucleic Acids Res 13: 975–984 Roitgrund C and Mets LJ (1990) Localization of two novel chloroplast genome functions: Trans-splicing of RNA and protochlorophyllide reduction. Curr Genet 17: 147–153 Saldanha R, Mohr G, Belfort M and Lambowitz A (1993) Group I and Group II introns. FASEB J 7: 15–24 Shaw LC and Lewin AS (1995) Protein induced folding of a Group 1 intron in Cytochrome b pre-mRN A. J Biol Chem 270: 21552–21562 Thompson AJ and Herrin DL (1991) In vitro self-splicing reactions of the chloroplast Group I intron Cr.LSU from Chlamydomonas reinhardtii and in vivo manipulation via gene replacement.

Chapter 11 Chloroplast RNA Splicing Nucleic Acids Res 19: 6611–6618 Thompson AJ and Herrin DL (1994) A chloroplast group I intron undergoes the first step of reverse splicing into host 5.8S rRNA: Implications for intron-mediated RNA recombination, intron transposition and 5.8S rRNA structure. J Mol Biol 236: 455–468 Thompson AJ, Yuan X, Kudlicki W and Herrin DL (1992) Cleavage and recognition pattern of a double-strand-specific endonuclease (l-Crel) encoded by the chloroplast 23S rRNA intron of Chlamydomonas reinhardtii. Gene 119: 247–251 Tunnel M, Boulanger J and Lemieux C (1989) Two group I introns with long internal open reading frames in the chloroplast psbA gene of Chlamydomonas moewusii. Nucleic Acids Res 17: 3875–87 Turmel M, Boulanger J, Schnare MN, Gray MW and Lemieux C (1991) Six group I introns and three internal transcribed spacers in the chloroplast large subunit ribosomal RNA gene of the green alga Chlamydomonas eugametos. J Mol Biol 218: 293– 311 Turmel M, Gutell RR, Mercier JP, Otis C and Lemieux C (1993a) Analysis of the chloroplast large subunit ribosomal RNA gene from 17 Chlamydomonas taxa. Three internal transcribed spacers and 12 group I intron insertion sites. J Mol Biol 232: 446–467 Turmel M, Mercier J-P and Côté M-J (1993b) Group I introns interrupt the chloroplast psaB and psbC genes in Chlamy­ domonas. Nucleic Acids Res 21: 5242–5250

195 Turmel M, Choquet Y, Goldschmidt-Clermont, Rochaix J-D, Otis C and Lemieux C (1995a) The trans-spliced intron 1 in the psaA gene of the Chlamydomonas chloroplast: A comparative analysis. Curr Genet 27: 270–279 Turmel M, Côté V, Otis C, Mercier J-P, Gray MW, Lonergan KM, and Lemieux C (1995b) Evolutionary transfer of ORFcontaining group I introns between different subcellular compartments (chloroplast and mitochondria). Mol Biol Evol 12: 533–45 Turmel M, Mercier J-P, Côté V, Otis C and Lemieux C (1995c) The site-specific DNA endonuclease encoded by a group I intron in the Chlamydomonas pallidostigmatica chloroplast small subunit rRNA gene introduces a single-strand break at low concentrations of Nucleic Acids Res 23: 2519– 2525 Weeks KM and Cech TR (1995) Protein facilitation of group I intron splicing by assembly of the catalytic core and the 5´ splice site domain. Cell 82: 221–230 Woodson SA and Cech TR (1989) Reverse self-splicing of the Tetrahymena group I intron: Implication for the directionality of splicing and for intron transposition. Cell 57: 335–345. Zimmerly S, Guo H, Eskes R, Yang J, Perlman PS and Lambowitz AM (1995) A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell 83: 529–538

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Chapter 12

Regulation of Chloroplast Translation Charles R. Hauser, Nicholas W. Gillham and John E. Boynton DCMB Group, Departments of Botany and Zoology, Box 91000, Duke University, Durham, NC 27708-1000, U.S.A. Summary I. Introduction II. The Role of Physiological and Environmental Factors in Translational Control III. Current Biochemical and Genetic Approaches to Dissect Mechanisms of Translational Regulation IV. Cis-acting Sequences Involved in Translation Initiation V. Translational Regulation Involves Interactions between cis-Acting Sequences and trans-Acting Factors A. Regulation of Individual Photosynthetic Proteins B. Coordinate Regulation of Photosynthetic versus Ribosomal Proteins VI. Ribosomes, Membranes and Tethers VII. Translational Regulation of Complex Assembly A. Chloroplast Thylakoid Membranes B. Chloroplast Ribosomes VIII. How are the Regulatory Proteins Regulated? IX. Is there Hierarchical Control of Chloroplast mRNA Translation? Acknowledgments References

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Summary During the evolution of chloroplasts from cyanobacterial endosymbionts, many genes encoding components necessary for protein synthesis and photosynthesis have been transferred to the nucleus. Assembly of the machinery for both processes now relies on the concerted expression of genes in the nuclear and plastid genomes. Evidence accumulating in C. reinhardtii indicates that the expression and regulation of chloroplast genes probably differs in certain respects from the prokaryotic (E. coli) paradigm. Both mutational analysis of putative Shine-Dalgarno (SD) sequences, and creation of canonical SD sequences (–9 to –5) reveals that initiation is not mediated by SD-16S rRNA interactions for the majority of chloroplast-encoded mRNAs. Recent evidence in cyanobacteria, the most likely ancestors of the Chlamydomonas chloroplast, indicates that most genes lack SD sequences at the classical position. Interactions between cis-acting sequences in the 5´ untranslated regions (UTRs) and specific trans-acting nuclear gene products appear to mediate translation of chloroplast-encoded mRNAs for specific genes. Both UV crosslinking and gel mobility shift assays with several chloroplast leaders show that multiple proteins interact with these sequences. Some of these appear to be involved in gene-specific regulation, whereas others may be core proteins of a general ribonucleoprotein (RNP) complex. Inverted repeat sequences present in most chloroplast leaders predict higher order structures within the 5´ UTR of mRNAs that could serve as scaffolding for the formation of the RNP complex. One model predicts that the RNP complex directly enhances ribosome binding and translation initiation. A second model predicts that the RNP complex plays a role in making topological distinctions in the sites of synthesis of chloroplast mRNAs. A challenge in the coming years will be to determine which, if any, of these models are correct. J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 197–217. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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I. Introduction Posttranscriptional control plays a major role in regulating the expression of chloroplast and mitochondrial genes. This chapter specifically focuses on translational control mechanisms, whereas posttranscriptional control mechanisms including mRNA stabilization and degradation, processing, and splicing are reviewed elsewhere in this volume (Chapter 9, Nickelsen; Chapter 10, Stern and Drager; Chapter 11, Herrin et al.). Before considering trans­ lational control mechanisms in the chloroplasts of C. reinhardtii and land plants, we will briefly review comparable mechanisms operative in prokaryotes and eukaryotes. This is relevant because chloroplasts and mitochondria almost certainly arose from cyanobacteria and purple bacteria respectively by endosymbiosis (Gray, 1992) and many essential organelle genes were subsequently transferred to the eukaryotic nucleus (Gillham, 1994). Although the chloroplast translational apparatus is obviously of prokaryotic origin and resembles that of bacteria in many respects (Harris et al., 1994; Mache, 1990), it may have acquired certain eukaryotic characteristics over time. About two thirds of chloroplast ribosomal proteins and all the chloroplast tRNA synthetases in land plants and most algae are known to be encoded in the nucleus, synthesized in the cytosol and imported into the organelle (Gillham, 1994; Harris et al., 1994). Certain initiation and elongation factors appear to be chloroplast encoded (e.g. IF-1 in land plants, IF-2 in red algae and EF-Tu in C. reinhardtii and Euglena gracilis). However, analysis of several completely sequenced plastid genomes indicates that genes encoding the majority of the proteins involved in mediating translation on chloroplast ribosomes are absent from the chloroplast genomes of land plants and they are therefore assumed to be encoded by the nuclear genome of these organisms (Reith, 1995). We will begin by examining translational regulation in prokaryotes and then compare this to what is Abbreviations: 5´UTR – 5´ untranslated region; AAD – aminoglycoside adenine transferase; ASD-anti Shine-Dalgarno sequence; DBMIB – 2,5-dibromo-3-methyl-6-isopropyl-pbenzoquinone; DCMU – 3-(3,4 dichlorophenyl)-1,1 -dimethyl urea; DTNB–dithionitrobenzoicacid; PAR–photosynthetically active radiation; PQ – plastoquinone; p-protein – photosynthetic protein; RNP-ribonucleoprotein complex; r-protein–ribosomal protein; Rubisco – ribulose-1,5 bisphosphate carboxylase/ oxygenase; SD – Shine-Dalgarno sequence (GGAGG)

known in eukaryotes. In prokaryotes, control of translation initiation is dependent upon several factors (McCarthy and Brimacombe, 1994;Voorma, 1996). The pool of free 30S subunits available to begin translation is determined by the binding of initiation factors IF1 and IF3. These shift the equilibrium of the 70S ribosome monomers and their subunits towards dissociation into the 30S and 50S subunits. The 30S subunit, carrying IF 1, IF3, and probably IF2 as well, interacts with mRNA, and GTP through a series of intermediates giving rise to the 30S initiation complex. Recognition is governed largely by the extent of secondary structure in the mRNA and by two RNA-RNA interactions: 1) the codon-anticodon interaction between and the initiation codon (usually AUG), and 2) base pairing between a conserved purine rich ShineDalgarno (SD) sequence (GGAGG) 7 ± 2 nucleotides upstream of the mRNA initiator AUG codon and a complementary pyrimidine rich anti-SD (ASD) sequence near the 3´ end of the 16S rRNA in the small subunit. These two RNA-RNA interactions stabilize the 30S preinitiation complex in the proper reading frame. Other mRNA interactions may involve sites on the 30S subunit that might be regarded as providing an enlarged ‘catchment area’ capable of binding mRNA molecules that subsequently move into the major mRNA track (McCarthy and Brimacombe, 1994) Ribosomal protein S1 also plays a role in mRNA recognition in E. coli (Voorma, 1996). The amino terminus of this protein is associated with the 30S subunit by protein-protein interactions, but its long and flexible carboxyl terminal domain has a high affinity for pyrimidine sequences. Many E. coli mRNAs have pyrimidine rich sequences upstream of the SD sequence and the RNAs of phages f 2 and have U rich sequences upstream of the coat protein open reading frame which may represent S1 binding sequence sites. Site-directed mutagenesis of a upstream of rnd, an E. coli gene encoding RNase D, revealed that alteration of two to five U residues within this sequence had no effect on mRNA levels, but decreased RNase D protein and activity by as much as 95 percent. Furthermore, E. coli 30S subunits will recognize translational start sites of mRNAs lacking SD sequences such as alfalfa mosaic virus RNA4 and tobacco mosaic virus RNA only if S1 and IF3 are present. In fact, a recent scheme for initiation complex formation starts with S1-dependent formation of a 30S-mRNA binary complex followed

Chapter 12

Chloroplast Translation

by the SD-ASD interaction. Two major patterns of negative regulation have been identified in prokaryotes (Gold, 1988; McCarthy and Gualerzi, 1990;Voorma, 1996). The first involves repression of translation by trans-acting proteins that block formation of competent initiation complexes. Thus, the ribosomal (r-) protein operons are negatively auto-regulated by specific r-proteins whose target is usually the translational initiation region of the first gene in the operon. The second mechanism modulates mRNA secondary structure in order to control availability ofthe ribosome binding site for initiation of complex formation. In addition, nascent peptides have been found to play a role in translational regulation of specific genes via mechanisms as diverse as translational attenuation, ribosome hopping, and auto-regulation of mRNA half-life (Lovett, 1994). Translational regulation in eukaryotes has been reviewed by Standart and Jackson (1994) and in a recent volume edited by Hershey et al. (1996). In eukaryotic mRNAs the 5´ cap, the ‘context’ of the sequence flanking the initiator AUG, enhancer sequences (Kozak, 1983; Yamamoto et al., 1995; Schmitz et al., 1996), and the presence of upstream open reading frames (uORFs) in the 5´ UTR (Mathews et al., 1996) all affect the general efficiency of translation. Translational regulation of uORF­ containing mRNAs depends on many factors including the amino acid sequence encoded by the uORF, the length of intercistronic regions, and the sequence context of the termination codon of the uORF. In addition, the translation of arg-2 mRNA is negatively regulated in vitro by increasing concen­ trations of arginine (Wang and Sachs, 1997). Specific cis-elements and trans-acting factors are also important in regulation of translation. A wellstudied example of a eukaryotic translational repressor acting on the 5´ UTR is the iron-binding proteins (IRP1 and 2) which interact with a stemloop structure (IRE) in the 5' UTR of ferritin mRNA to turn off translation. In the absence of IRP, the IRE acts as a positive translational enhancer and causes preferential binding of initiation factors to ferritin mRNA (Standart and Jackson, 1994; Kim et al., 1995). Eukaryotic initiation factors (eIF) and the poly(A) tail at the 3´ end of eukaryotic mRNAs acting in synergy with the 5´ cap structure of the mRNA are also involved in regulating expression of eukaryotic mRNAs. Formation of the eukaryotic pre-initiation

199 complex with the 40S subunit is an elaborate process which requires not only the eIFs, but also novel properties of the poly(A) binding protein (Hentze, 1995, 1996; Proweller and Butler, 1996). The cap is bound by eIF-4E which plugs into the N-terminal portion of an adapter protein, eIF-4G whose central portion is associated with eIF-3, a multimeric complex bound to the 40S subunit. The multifunctional eIF­ 4G protein is also the site of interaction of the poly (A) binding protein which helps to recruit 40S subunits. Since the polyadenylation process itself depends on a whole array of proteins (Proudfoot, 1996), each of these is a potential target for translational regulation. Translational regulation in chloroplasts and mitochondria has been the subject of numerous reviews (Gillham et al., 1994; Mayfield et al., 1995; Fox, 1996a,b; Rochaix, 1996; Sugita and Sugiura, 1996; Cohen and Mayfield, 1997). Most of the data for mitochondria and chloroplasts derive from experi­ ments with yeast and C. reinhardtii respectively. In both organelles control is frequently exerted via trans-acting factors which bind to the 5´ untranslated regions (5´UTRs) of organellar mRNAs and affect message half-life or translatability. Fox (1996a,b) calls attention to the fact that translational activators binding to the 5´UTRs of specific mitochondrial mRNAs also interact with the small subunit of the mitochondrial ribosome, quite probably with specific ribosomal proteins. Although polyadenylation was considered to be a unique property of nucleus-encoded mRNA in eukaryotic cells, polyadenylation of chloroplastencoded mRNAs has recently been reported (Kudla et al., 1996, Lisitsky et al., 1996). In vivo, endonucleolytic cleavage of the petD mRNA is followed by polyadenylation ofthe cleavage products which in turn substantially increases their rate of degradation (Kudla et al., 1996). Similarly, mRNA can be targeted for rapid degradation in vitro by the addition of a poly(A)-rich sequence to an endo­ nucleolytic cleavage product (Lisitsky et al., 1996). Following transcription, chloroplast precursor mRNA may follow one of two paths: 1) undergo 3´ end formation to generate a stable product for translation, or 2) endonucleolytic cleavage and addition of poly(A)-rich sequences leading to rapid degradation. This branch point in mRNA metabolism is regulated in vitro by ATP concentration. However, Lisitsky et al. (1996) state that in vivo this branch point may be regulated by the chloroplast redox potential,

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photosynthetic electron flow or specific trans-acting proteins. Clearly, regulation of the enzymes responsible for chloroplast mRNA polyadenylation must now be considered among the factors determining which transcripts become available for translation. In addition, translation of chloroplast mRNAs may be regulated by other diverse mechan­ isms such as alternative processing of mRNA 5´ ends (Reinbothe et al., 1993, Shapira et al., 1997) or codon bias optimized for translational efficiency (Morton, 1996). These observations, taken together with the findings reviewed above relating to translational control mechanisms in eukaryotes and prokaryotes, should alert investigators studying translational control mechanisms in chloroplasts. The roles of specific chloroplast ribosomal proteins, initiation factors and other diverse factors (e.g. nascent peptides) will probably need to be taken into account in trying to reach a complete depiction of the events that accompany activation of chloroplast messages for translation.

II. The Role of Physiological and Environmental Factors in Translational Control Chloroplast development in response to light exposure in most land plants and green algae is controlled at the transcriptional, post-transcriptional and transla­ tional level. Light mediated reduction ofprotochloro­ phyllide accumulated in the dark to chlorophyllide triggers reorganization of the paracrystalline prolamellar body of the etioplast, synthesis of new chlorophyll pigment and photosynthetic proteins, formation of photosynthetic thylakoid membranes and their organization into grana stacks (von Wettstein et al., 1995). Clear evidence exists that transcription of many nuclear genes encoding photosynthetic proteins is activated by light (Gilmartin et al., 1990; Schindler and Cashmore, 1990). Concomitantly, light mediates chloroplast gene expression primarily at the translational level, although transcription and stability of chloroplast encoded mRNAs are also enhanced by light induced greening (Gruissem and Tonkyn, 1993; Gillham et al., 1994; Mayfield et al., 1995). In contrast to angiosperms, wild-type C. reinhardtii, like gymnosperms and liverworts (Gillham, 1994), has a light-independent protochloro­ phyllide reductase. Therefore, this alga reduces protochlorophyllide to chlorophyllide enzymatically

in the dark and maintains highly differentiated chloroplasts under heterotrophic growth conditions (Harris, 1989). Light has been shown to regulate translation of psbA mRNA encoding the D1 protein of Photo­ system II, which is degraded and re-synthesized more rapidly than any other chloroplast protein (Fig. 1). Synthesis of this protein is enhanced in wild-type C. reinhardtii grown phototrophically versus hetero­ trophically (Danon and Mayfield, 1991; Hauser et al., 1996). Pulse-labeling experiments indicate that D1 is not synthesized in heterotrophically grown cells of y1 and other mutants blocked in the enzymatic reduction of protochlorophyllide, but synthesis of D1 is activated by exposure of such mutants to light (Malnoë et al., 1988; Danon and Mayfield, 1991). However, a fast turn-over of D1 in these mutants cannot be ruled out. Binding of 47 and 60 kDa proteins to a 36 nt stem-loop structure upstream of the ribosome binding site in the 5´UTR of the psbA mRNA has been reported to correlate with light stimulated D1 synthesis in wild-type cells (Danon and Mayfield, 1991) and to be controlled by the redox potential in the chloroplast (Danon and Mayfield, 1994). However, a 47 kDa protein was also shown to crosslink to the leaders of the chloroplast rbcL and atpB mRNAs encoding photosynthetic proteins and the chloroplast rps7 and rps12 mRNAs encoding ribosomal proteins when extracts from heterotrophic and phototrophic cells were compared (Hauser et al., 1996; Rochaix, 1996). Whether the same or distinct 47 kDa proteins are involved in these binding reactions remains to be ascertained (Gillham et al., 1994; Hauser et al., 1996; Rochaix, 1996). Silk and Wu (1993) found that illumination of dark grown y1 cells is also accompanied by a five­ fold increase in the accumulation of tufA mRNA encoding the elongation factor EF-Tu due to an increased half-life of this mRNA in the light. While the significance of changes in tufA transcript levels is unknown, the authors speculate that EF-Tu might be essential for the accumulation ofchloroplast encoded proteins during greening of y1 cells. Transfer of low light adapted C. reinhardtii (CC­ PAR, 125) cells to high light (70 and respectively) has a dramatic transient effect on the differential translation of psbA and rbcL mRNAs (Shapira et al., 1997; Fig. 1). Synthesis of the D1 protein increases ten-fold during the first six hours whereas synthesis of ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit (Rubisco LSU)

Chapter 12 Chloroplast Translation

drops dramatically within 15 minutes and only gradually resumes at the end of the six hour period. Changes in D1 and LSU synthesis cannot be explained by shifts in the levels of accumulated psbA or rbcL mRNA or by the temporary decrease observed in the ratio of the short to long form of the rbcL transcripts. These several distinct effects of temporary light stress are correlated with a rapid, sustained increase a transient decline in in the reduction state of photosynthetic activity, a less rapid drop in total chlorophyll content and a delay in cell division. Whether these changes result in differential binding of translational activator and repressor proteins to the psbA and rbcL mRNA leaders in response to changes in the cell’s redox potential or to another regulatory mechanism involving the generation of free radicals, remains to be determined.

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Most studies of expression of nuclear and chloroplast genes affecting photosynthetic function in C. reinhardtii are carried out on cells grown mixotrophically on medium containing acetate as a reduced carbon source. The presence of reduced carbon sources represses expression of nuclear RbcS and Cab genes encoding the Rubisco small subunit and the chlorophyll a/b binding protein in C. rein­ hardtii (Gibbs et al., 1986; Kindle, 1987) and strongly inhibits the expression of nuclear genes encoding seven different photosynthetic proteins in maize tissue culture cells (Sheen, 1990). Much less has been published about the effect of carbon source on chloroplast gene expression. In C. reinhardtii cells grown in the light on acetate, the level of D1 protein increased and the level of the LSU protein was reduced, but no differences were detected in extracts

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from these cells with respect to the binding of six proteins that crosslink to the 5´UTRs of mRNAs for three genes encoding photosynthetic proteins and two genes encoding ribosomal proteins (Hauser et al., 1996). Internal concentration may also affect chloroplast gene expression at the translational level (Winder et al., 1992). Transfer of phototrophically grown cells of C. reinhardtii from elevated to limiting resulted in transient declines in synthesis of both the chloroplast-encoded large subunit and the nucleus-encoded small subunit of Rubisco with no changes in levels of the corresponding mRNAs. Nitrogen starvation in C. reinhardtii is known to effect a significant reduction in the levels of both chloroplast and cytoplasmic ribosomes as vegetative cells differentiate into sexual gametes (Harris, 1989). During a 10–12 h period following restoration of nitrogen, gametes begin to regenerate into vegetative cells, resynthesize rRNAs and ribosomal proteins and assemble the components into the normal complements of chloroplast and cytoplasmic ribosomes without cell division (Myers et al., 1984). Whether preferential translation of chloroplast mRNAs encoding chloroplast ribosomal proteins observed under conditions of reduced chloroplast protein synthesis (see below) allows C. reinhardtii cells to respond to fluctuations in nitrogen content in their environment remains to be determined.

III. Current Biochemical and Genetic Approaches to Dissect Mechanisms of Translational Regulation Development of techniques for stable transformation of chloroplast genes in C. reinhardtii (Boynton et al., 1988; Boynton and Gillham, 1993) and tobacco (Svab et al., 1990; Maliga, 1993; Svab & Maliga, 1993) has permitted the direct demonstration that the 5´UTR regions of chloroplast mRNAs are essential for their translation (Mayfield et al., 1994; Staub and Maliga, 1994; Zerges and Rochaix, 1994; Zerges et al., 1997). Recent efforts to identify cis-acting elements required for mRNA translation and stability have utilized the dominant eubacterial aadA gene (conferring spectinomycin resistance) in chimeric reporter constructs as a rapid means of accessing the functionality of putative cis regulatory elements in chloroplasts of C. reinhardtii (GoldschmidtClermont, 1991; Nickelsen et al., 1994; Zerges and

Rochaix, 1994; Fargo et al., 1997). Furthermore, analysis of AAD expression from reporter constructs in the progeny ofcrosses involving photosynthetic or ribosomal protein mutant strains and the wild type has allowed direct demonstration of the requirement for 5' UTR sequences in translalional regulation (Nickelsen et al., 1994; Zerges and Rochaix, 1994; C. R. Hauser, A. M. Johnson, N. W. Gillham and J. E. Boynton, unpublished). Chloroplast transformants containing 5´ UTR: aadA: 3´UTR reporter constructs should also be valuable for use in isolating cis-acting (5´ and/or 3´UTR) mutants in the chloroplast sequences and trans-acting nuclear mutations that suppress loss of function mutations in the cis-acting sequences. Gel mobility shifts (GMS), UV crosslinking and RNA affinity chromatography (Leibold and Munro, 1988; Meerovitch et al., 1989) have been utilized to identify the proteins mediating 5´UTR-driven selective translation in C. reinhardtii. Heparinenriched extracts from whole cells or chloroplast fractions isolated and analyzed in the presence of reductants have been used to identify 5´ UTR-binding proteins. Since the reduction state of certain 5´UTRbinding proteins has been shown to affect RNP complex formation in vitro (Danon and Mayfield, 1994b), caution should be exercised in extrapolating these in vitro results to in vivo regulation. GMS identifies a ribonucleoprotein complex (RNP) by the retarded migration of an RNA in a non-denaturing gel system, whereas UV crosslinking identifies the proteins in the complex which are in direct contact with labeled nucleotides in the RNA. Second dimension analysis by SDS PAGE of the proteins binding to an RNA identified as a gel mobility shifted band in a GMS assay, can identify additional proteins in the complex, regardless of whether the proteins participate via protein-RNA or protein-protein interactions. RNA affinity chromatography utilizing specific RNA ligands immobilized on matrices is a powerful approach to identify the trans-acting factors which bind to the chloroplast leader sequences (Rouault et al., 1989; Danon and Mayfield, 1991, Prokipcak et al., 1994). However, this method has so far seen only limited application in the identification of trans-acting proteins that bind to chloroplast 5' UTRs (Danon and Mayfield, 1991). The widely used yeast 2-and 3-hybrid systems designed to identify protein:protein or protein:RNA interactions required for gene expression (Fields and Sternglanz, 1994; Putz et al, 1996; SenGupta et al., 1996) should

Chapter 12

Chloroplast Translation

also be useful for the identification of trans-acting proteins that are part of the chloroplast 5´UTR complexes, particularly for those proteins that do not bind directly to RNA. With improved nuclear transformation efficiencies in C. reinhardtii and the development of dominant nuclear selectable markers (Stevens et al., 1996; Cerutti et al., 1997a,b), genomic complementation (Purton and Rochaix, 1994; Zhang et al. 1994; Vashishtha et al., 1996; Funke et al., 1997) and tagging of nuclear genes (Tam and Lefebvre, 1993; Gumpel and Purton, 1994) are viable routes for the identification of genes encoding trans-acting proteins which regulate the localization and/or translation of mRNAs.

IV. Cis-acting Sequences Involved in Translation Initiation Although chloroplast ribosomes share many structural and functional properties with prokaryotic ribosomes, questions have arisen as to whether translation initiation involves different mechanisms in the two systems. Conserved anti-SD sequences occur near the 3´ end of the highly conserved 16S rRNA molecule from chloroplast ribosomes of higher plants, green algae and cyanobacteria (Harris et al., 1994; Kaneko et al., 1996). However, complementary SD sequences are missing from the leaders of certain chloroplast and cyanobacterial genes, and when present, are hyper-variable in position and nucleotide composition (Gillham et al., 1994; Harris et al., 1994; Kaneko et al., 1996; Fargo et al., 1997). Furthermore, tracts and other prokaryotic UTR sequences reported to enhance translation are absent from the 5´UTRs of chloroplast mRNAs examined to date (Fargo et al., 1997). Experiments have been carried out in tobacco and C. reinhardtii to test whether SD-like sequences present in the 5´UTRs of chloroplast mRNAs function in translation as they do in E. coli. In tobacco, addition of a canonical SD sequence (GGAGG, –11 to –7) to the strong constitutive rRNA operon promoter (Prrn) fused to the uidA gene encoding the (GUS) reporter enzyme, leads to the efficient translation of nidA mRNA in chloroplast transformants (Staub and Maliga, 1994). In C. reinhardtii transformants, deletion of the SD-like sequence (GGAG) located 27 nt upstream of the ATG in the psbA mRNA reduced both half-life

203 and translation of this message greatly (Mayfield et al., 1994). Two dimensional modeling studies of the mutant leader using the M-fold algorithm (Zuker, 1994) suggest that the four nucleotide deletion is sufficient to alter the predicted secondary structure of the leader (Fargo et al., 1997). Strains containing a mutation (CUCC) within the putative 36 nt stemloop of the leader which sequesters the SD sequence (GGAG) by base pairing and extends the stem also blocked psbA expression and severely reduced mRNA stability (Mayfield et al., 1994). Mutant leaders containing a 32 nt deletion immediately upstream of the SD sequence translate psbA mRNA at high rates (Mayfield et al., 1994). This deletion, which eliminates 65% of the putative stem-loop sequence encompassing presumed binding sites for the 47 and 60 kDa proteins (Danon and Mayfield, 1991), is modeled to have an unstructured SD sequence with a short stem loop at the immediate 5´ end of the message. This stem loop may serve as a negative translational attenuator (Mayfield et al., 1994). Whether the observed reduction in psbA expression in these 5' UTR mutants resulted directly from a reduction in mRNA translation, or from a reduction in mRNA half-life, cannot be ascertained from the published data (Rochaix, 1996). Interpretation is further complicated by evidence that the half-life of chloroplast mRNAs can be enhanced or diminished by their association with ribosomes (Sakamoto et al., 1994; Yohn et al., 1996; Chapter 9, Nickelsen). Using an in vitro translation system derived from tobacco chloroplasts and a series of mutant psbA 5´UTRs, Hirose and Sugiura (1996) identified four elements within the psbA 5´UTR which are required for translation in vitro. Three of these 5´UTR elements appear to be putative ribosome binding sites (RBSs), RBS1 (AAG [–9]), RBS2 (UGAUGAU [–22]) and RBS3 (GGAG [–33]) based on complementarity to the 3´ terminus ofthe tobacco chloroplast 16S rRNA. Replacement mutagenesis of RBS1 or RBS2 reduced translation to 33% and 38% of wild type respectively, while mutagenesis of the canonical SD sequence, RBS3, had little effect (12% decrease) on translation. Disruption of both RBS1 and RBS2 resulted in a drastic decrease (92%) in translation whereas the mRNAs from two other double mutants (RBS1&3 and RBS2&3) were translated at levels similar to the single mutants RBS1 or RBS2. Deletion of a fourth element (UAAAUAAA [–17]) abolished translation and the authors suggest this sequence may be a target for potential trans-acting translation factor(s).

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Deletion of ca. 70% of the chloroplast petD 5´ UTR from a translational fusion with the uidA reporter gene reduced mRNA half-life four- to six-fold and glucuronidase activity 8 fold in chloroplast transformants of C. reinhardtii while the transcription rate of the uidA gene was unaltered (Sakamoto et al., 1993). In contrast, replacement mutagenesis of a trinucleotide SD-like sequence located 10 nt upstream of the ATG codon had no effect on the accumulation of the petD gene product subunit IV (Sakamoto et al., 1994a). Similarly, a change in the psbD SD sequence from GGAG to AAAG did not affect phototrophic growth or photosynthetic efficiency (unpublished data cited in Rochaix, 1996). The putative SD-like sequences in A-U rich 5´UTRs of two r-protein mRNAs (rps4 [SD at–149] and rps 7 [SD at –116]) and two photosynthetic protein mRNAs (atpB [SD at –85] and atpE [SD at –22]) were altered by replacement mutagenesis to destroy complementarity to the ASD in the 16S rRNA (Fargo et al., 1997). Transformants homoplasmic for a chloroplast expression cassette carrying the four upstream regulatory regions with either the wildtype or mutated SD-like sequences fused to the bacterial aadA reporter gene showed identical levels of spectinomycin resistance in vivo and identical levels of aminoglycoside adenine transferase (AAD) activity in vitro. Similar results were obtained when the uidA sequence encoding was substituted for the aadA coding sequence in the rps4 and atpB leader constructs. Based on the observation that certain chloroplast genes can be transcribed and their mRNAs translated in E. coli (Liu et al., 1989a), expression of these 5´UTR reporter constructs carried on pUC18 plasmids was also examined in E. coli. Whether wild-type or mutant SD-like sequences were present, all four chloroplast promoter/leader regions coupled to aadA resulted in comparable levels of spectino­ mycin resistance and AAD activity in the bacterial transformants (Fargo et al., 1997). Similar results were obtained in the case of E. coli transformants containing the uidA gene under the control of rps4 and atpB regulatory regions with the wild-type and mutant SD-like sequences. Creation of a canonical SD sequence (GGAGG) by replacement mutagenesis at positions –9 to –5 in the leaders of these constructs resulted in a slight elevation in reporter gene expression in E. coli and no detectable increase in C. reinhardtii. The use of two independent and

functionally unrelated reporter genes with the different chloroplast leaders greatly diminishes the possibility that internal coding sequences could be promoting formation of the initiation complexes in the absence of the SD-like sequences. In contrast, expression of both the aadA and uidA reporter genes in E. coli was also shown to require the presence of an active SD sequence when these genes were fused to the 5´UTR of mutant and wild-type leaders of the phage fI gene VII. Native gene VII mRNA contains a defective initiation site and is inactive in assays of independent initiation (Ivey-Hoyle and Steege, 1992). The chloroplast mRNA leaders are highly A-U rich (70–84%) and show no significant primary sequence identity to one another. However, analysis of these sequences using energy minimization algorithms (Zuker, 1994) predicts highly folded structures. Folding of the naked RNA depends on kinetic barriers to achieving the thermodynamically most stable structure. Thus the leader may assume a multitude of local energy minima conformations on a path to the most thermodynamically stable structure (Konings and Gutell, 1995). The structure(s) acquired by RNA in vivo are dependent also on their interaction(s) with trans-acting proteins, which once bound, may regulate accessibility to sequences required for translation. While certain 5´ UTR binding proteins have been reported to protect leader regions from RNase digestion (Danon and Mayfield, 1991; Hauser et al., 1996) there are no published accounts examining the effect(s) of protein binding to C. reinhardtii leader sequences on the higher order structure of the RNA. That the leaders of at least some chloroplast mRNAs undergo conformational changes prior to translation is implicit in a model proposed by Rochaix (1996) for PS II mRNAs. An RNP complex formed between the 5´ ends of the UTRs and trans-acting proteins is proposed to target the mRNA to a membrane site, at which point the structured 5´ end of the leader is clipped off, leaving a shorter and unstructured mRNA poised for binding to the 30S subunit and translation initiation. As in the case of certain E. coli genes (Zhang and Deutscher, 1992), changes in the initiation codon can affect translation of particular chloroplast mRNAs (Chen et al., 1993,1995). Mutating the AUG initiation codon of the chloroplast petD mRNA encoding complex, to AUU subunit IV of the cytochrome or AUC results in a temperature sensitive nonphoto­ synthetic phenotype with a 50% reduction in mRNA

Chapter 12

Chloroplast Translation

accumulation and a 80–90% reduction in both synthesis and accumulation of subunit IV under permissive photosynthetic conditions (Chen et al., 1993). A dominant nuclear mutation (sim30-1d ) appears to increase the translation initiation rate of the petD mRNA containing the mutant AUU codon (Chen et al., 1997). Changing the initiation codon of the chloroplast petA gene from AUG to AUU, ACG, ACC, ACU or UUC had variable effects on synthesis of cytochrome f and photosynthetic competence. All mutants except the one carrying the UUC mutation, accumulated detectable levels of cytochrome f, but only the AUU codon mutant grew well at 24 °C. Introduction of in-frame UAA stop codon mutations immediately at, or immediately downstream of, the initiation codon prevented accumulation of cyto­ chrome f, suggesting that neither the second codon nor codons downstream ofthe second codon serve as the initiation codon in vivo. In addition, strains containing a stop codon inserted immediately upstream of the AUG codon accumulated wild-type levels of cytochrome f, suggesting that initiation upstream of the AUG codon does not contribute significantly to cytochrome f accumulation. The authors suggest from these data that in each initiation codon mutant strain that accumulates cytochrome f, the mutant codon is used as the initiation codon. Therefore, the petA AUG codon is not required to specify the site oftranslation initiation in chloroplasts, but the strength of the initiation codon-anticodon interaction may determine the rate of translation initiation.

V.Translational Regulation Involves Interactions between cis-Acting Sequences and trans-Acting Factors

A. Regulation of Individual Photosynthetic Proteins Interactions between cis-acting sequences in the 5´UTRs of chloroplast mRNAs and specific trans­ acting nuclear gene products appear to be essential for the expression of the several chloroplast genes so far examined (Fig. 1). This gene-for-gene relationship has been deduced from characterization of nuclear mutants that either block translation of particular chloroplast mRNAs (Table 1) or perturb their halflife (Gillham et al., 1994; Mayfield et al., 1995; Rochaix, 1996). While there are many mutations

205 affecting the expression of genes encoding photo­ synthetic proteins, nuclear mutants in which the synthesis of a specific ribosomal protein is affected have not been identified. Nuclear gene products are required for the stabilization ofthe atpA and atpB transcripts encoding the and subunits of the chloroplast ATP synthase, respectively, as well as the translation of the atpA transcript (Stern et al., 1991, Drapier et al., 1992). The nuclear mutant F54 shows a four-fold increase in atpA mRNA accumulation over wild type, yet it fails to synthesize the subunit. These data suggest that the expression of the atpA gene is blocked at the level of translation in mutant F54, and that the translational block is accompanied by an increased half-life of the transcript. In contrast to the impaired subunit, the F54 mutant also synthesis of the displays a stimulation in the synthesis the subunit, while maintaining wild-type levels of atpB transcript. Together these data point to the interlinked expression of the chloroplast-encoded ATP synthase subunits. The molecular basis for the increased translation of the subunit in F54 is postulated to result from competition between atpA and atpB 5´UTRs for regulatory proteins which are present in limiting conditions in the wild type and which have a higher affinity for the atpA transcript. Under these conditions, a larger proportion of the regulatory proteins would be available for the translation of the atpB transcripts in mutant F54, where atpA transcripts would not associate with polysomes (Drapier et al., 1992). Deletion analysis carried out by Mayfield et al. (1994) has suggested that the putative stem-loop structure in the psbA 5´UTR located upstream of the SD-like sequence may function to modulate expression of the D1 protein. Mutants lacking the loop sequences or altering pairing within the stem accumulate only ~20% of the normal levels of D1 protein, so the authors speculate that this region continues to function as a translational attenuatorbut that its activity can no longer be overcome by binding of trans-acting activator proteins. The nuclear mutant F35 that blocks D1 synthesis (Girard-Bascou et al., 1992) has been studied in detail by Yohn et al. (1996). They report that F35 reduces association of psbA mRNA with chloroplast ribosomes and decreases the half-life of this transcript. Analysis of psbA mRNA distribution on polysomes suggests that the F35 mutant is impaired in translation initiation. The reduction in thepsbA mRNA loading onto polysomes in the F35 mutant correlates with a decrease in the

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formation of a psbA RNP complex compared to wild type as monitored by gel mobility shift analysis. However, in contrast to the results of experiments in which psbA expression was studied in illuminated versus dark adapted cells where formation of a RNP complex correlates with levels of translation (Danon and Mayfield, 1991), the total block of psbA translation in F35 cells is not paralleled by a complete loss of the psbA RNP complex. The authors explain the pleiotropic effects of the F35 mutant in terms of the modest reduction in formation of psbA mRNA initiation complexes causing a larger decrease in translation initiation of mRNA encoding the D1 protein. Preliminary results suggest that a 55 kDa protein may be absent from the complex in extracts from the F35 mutant. Similarly, deletion analysis of the 362 nt 5´UTR of the chloroplast petD gene denned two domains (+153 to 302 and +312 to 330) required for synthesis of subunit IV (Sakamoto et al., 1994). Isolation of nuclear suppressors that restore translation of the petD mRNA (Sakamoto et al., 1994) again points to the involvement of nucleus-encoded trans-acting

proteins in translational regulation of chloroplast mRNAs. A light-dependent psbA RNP complex has been identified and characterized (Danon and Mayfield 1991). The protein complex interacts with the aforementioned 36 base stem-loop RNA structure within the 5´untranslated region (UTR) of the psbA mRNA immediately 5´ to the putative ribosome binding site (GGAG). Binding of the protein complex to the RNA was detected using RNA gel mobility shifts, while its constituents were identified by UV crosslinking and RNA affinity chromatography. A minimum complex consists of proteins of 47,75,60, 55 and 38 kDa and possibly two additional proteins of 30 and 33 kDa (Danon and Mayfield, 1991,1994a). The 47 kDa protein was further shown to resolve into four species by two dimensional gel electrophoresis. Direct contact of the protein complex with the RNA appears to be mediated by the 47 kDa protein(s) whereas the 60 kDa protein may interact with the RNA through contacts with the 47 kDa protein. A correlation was found between RNP complex formation and the level of translation of the psbA

Chapter 12

Chloroplast Translation

mRNA under mixotrophic (light) and heterotrophic (dark) growth conditions as measured by RNA gel mobility shifts (Danon and Mayfield, 1991). Illuminated cells, which rapidly synthesize D1 protein, contained high levels of psbA RNP, whereas dark-grown cells, which accumulate low levels of Dl , showed only minimal levels of RNP formation. Dark-grown cells of the y1 mutant, which lack photosynthetic membranes and do not translate psbA mRNA, were completely deficient in RNP complex formation. However, only minor differences in the amounts of the 47 and 60 kDa proteins present in extracts from light- and dark-grown wild-type cells were detected as monitored by UV crosslinking. From these data, the RNP complex is hypothesized to regulatepsbA mRNA translation either by targeting the message to the thylakoid membranes for translation (Rochaix, 1996) or by directly enhancing ribosome binding and translation initiation (Danon and Mayfield, 1991, Mayfield et al., 1994). The D2 protein encoded by the chloroplast psbD gene in Chlamydomonas is also an integral thylakoid membrane constituent of the PS II reaction center. It is synthesized on thylakoid membrane associated ribosomes and is cotranslationally inserted into the membrane (see Rochaix, 1996). Synthesis of the D2 protein is dependent upon at least three nuclear genes: NAC1 and AC115 whose products promote D2 translation (Kuchka et al., 1988), and NAC2. The NAC2 product appears to be essential for protecting psbD mRNA from degradation (Kuchka et al., 1989) and also affects the ability of a 47 kDa protein (presumably also nucleus-encoded) to bind to the 74 nt psbD 5´UTR sequence (Nickelsen et al., 1994). Mutations at the nuclear NAC1 and AC115 loci that specifically reduce synthesis of the D2 protein by affecting translation of psbD mRNA (Kuchka et al., 1988) can be suppressed by a dominant nuclear mutation in strain sup4b (Wu and Kuchka, 1995). While the molecular basis for this suppression is not known, the hypothesis has been put forth that the sup4b mutation may bypass the requirement for NAC1 and AC115 gene products in psbD gene expression (Wu and Kuchka, 1995). Two recessive nuclear mutants (F34, F64) marking the TBC1 and TBC2 loci, respectively and a chloroplast mutant (FUD34) acting through the 5´UTR specifically block translation of psbC mRNA encoding PS II subunit P6 (Rochaix et al., 1989; Zerges and Rochaix, 1994, Zerges et al., 1997). Chloroplast transformants carrying a chimeric

207 construct in which the psbC 5´UTR is fused to the aadA reporter gene require the presence of both the TBC1 and TBC2 wild-type gene products for expression (Zerges and Rochaix, 1994). This confirms the specificity of cis-acting sequences in the psbC 5' UTR for the two nucleus-encoded, trans-acting proteins. Subsequent deletion analysis in which an inverted repeat sequence specifying a putative stemloop in the middle of the psbC 5´UTR was removed showed that deletion of this binding site for the TBC1 gene product reduced expression of the aadA reporter considerably (Zerges et al., 1997). The interaction of the wild-type TBC1 gene product with the stem-loop region in thepsbC 5´UTR is supported by the finding of a cis-acting chloroplast suppressor of the F34 mutation (psbC-F34suI) which contains a base change in the psbC 5´UTR (Rochaix et al., 1989). This suppressor mutation presumably diminishes the stability of the stem, partially restoring translation of the psbC mRNA. The nonphotosynthetic chloroplast mutant, FUD34, also alters the stability of the stem of a putative stem loop structure in the 5´UTR of the psbC mRNA (Rochaix et al., 1989). Insertions of two adjacent T residues and deletion of a C residue five nucleotides away are thought to increase the thermodynamic stability of the stem structure. Thus, the sequence alterations in F34suI and FUD34 affect two complementary sequences within the stem structure and have opposite effects on the trans­ latability of psbC mRNA which correlate with their ability to destabilize and over-stabilize the stem loop in the 5´UTR of psbC, respectively. A spontaneous partial phenotypic revertant of FUD34, tbc3-rb1, has also been isolated. This dominant nuclear mutation (tbc3-rb1) can restore expression ofthe aadA reporter gene from the 5´UTR bearing the psbC -FUD34 mutation or the deletion of the entire stem-loop (Zerges et al., 1997). The ability of tbc3-rb1 to suppress the deletion of the stem loop demonstrates that 5´UTR sequences outside of this region are sufficient for restoration of translation. That tbc3-rb1 also suppresses tbc1-F34 shows that the mutation alleviates the requirement for the functional interaction between the TBC1 gene product and the putative stem-loop region. From these data, Zerges and Rochaix (1997) propose two alternative models. A TBC1 -dependent protein interacts with the putative stem-loop to inhibit a TBC3-dependent translational repressor from acting on a site outside this region. In the absence of either

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the stem-loop or wild-type TBC1 function, the TBC3­ dependent repressor would block translation of the psbC mRNA. The tbc3-rb1 mutation acting as a dominant negative mutation would restore psbC translation. Alternatively, the TBC1 and TBC3 gene products activate psbC mRNA translation by binding to the stem loop. In the absence of TBC1 or the stemloop, wild-type TBC3 would be insufficient to promote translation. The dominant tbc3-rb1 mutation might alter the TBC3 gene product in such a way that it is sufficient to promote psbC mRNA translation in the absence of TBC1 or the stem-loop. Clearly, the stem-loop region of the psbC mRNA and the TBC1 and TBC3 gene products interact functionally to control translation of the psbC mRNA. This conclusion is tempered by the observation that in three of four strains carrying the tbc3-rb1 mutation analyzed, synthesis but not accumulation of Rubisco large subunit was apparently reduced relative to the wild type (Zerges et al., 1997). This may indicate that, in contrast to all other characterized mutations affecting chloroplast or mitochondrial translation, TBC3 function may not be gene specific, but required for the translation of other mRNAs as well. Molecular analysis of trans-acting proteins capable of interacting with chloroplast encoded 5´UTRs revealed that extracts of wild-type, F34 and F64 cells contained proteins of 95, 65, 40 and 33 kDa which crosslink to the 5´UTRs of psbC, psbA and psbD mRNAs, although the 33 kDa protein was not detected in extracts from purified chloroplasts (Zerges and Rochaix, 1994). As these proteins are present in all extracts, there is no reason to believe that they are related to the products of the wild-type TBC1 and TBC2 genes. However, a protein of 46 kDa (RBP46) specific to the extract from the F64 mutant was crosslinked to the psbC 5´UTR, suggesting that this protein may be responsible for the effect of the F64 mutation on psbC translation (Zerges and Rochaix, 1994).] RBP46 may well have other functions as this protein also crosslinks to the leaders of psbA and psbD (Zerges and Rochaix, 1994). Binding of RBP46 was inhibited by incubation of extract with poly(A) or poly(U) showing that the protein either has an affinity for AU-rich sequences, or has little sequence specificity. A mutation in the nuclear TAB1 gene, defined by strain tab1-F15, specifically interferes with the synthesis of the two reaction center subunits of Photosystem I (PS I), PsaA and PsaB encoded by the chloroplast psaA and psaB genes respectively

(Stampacchia et al., 1997). The tab1-F15 mutation was shown to affect either the preinitiation or initiation of psaB mRNA translation, but not that of psaA by examining the expression of aadA reporter constructs driven by the promoters and 5´UTRs of psaB and psaA. A photoautotrophic revertant of the tab1-F15 mutant, suF15, was isolated and shown to contain a G to A transition adjacent to a putative SD sequence 17 nucleotides upstream of the AUG translation initiation site of psaB. The loss of PsaA synthesis in the tab1-F15 mutant is a consequence of the absence of PsaB synthesis and not the presence of the tab1­ F15 mutation as demonstrated by the fact that the suF15 mutation in the psaB 5´UTR is capable of restoring the synthesis of both proteins. The authors speculate that TAB1 function is required for opening the stem structure to present an unstructured SD sequence analogous to the mom/com paradigm in bacteriophage Mu (Wulczyn et al., 1989).

B. Coordinate Regulation of Photosynthetic versus Ribosomal Proteins A syndrome ofphotosynthetic defects has been found in all chloroplast ribosome deficient mutants so far characterized (Harris, 1989; Harris et al., 1994). Experiments with the nuclear double mutant ac20 cr1 which is severely deficient in chloroplast ribosomes showed that synthesis and accumulation of chloroplast encoded photosynthetic proteins was greatly reduced while chloroplast-encoded ribosomal proteins (r-proteins) continue to be synthesized and accumulate (Liu et al., 1989b). A spectinomycin resistant mutant (spr-u-1-27-3) which carries a mutation in the chloroplast 16S rRNA, exhibits the same symptoms of reduced synthesis of photo­ synthetic proteins when grown in the presence of spectinomycin, but synthesizes and accumulates normal levels of r-proteins (Liu et al., 1989b). These results suggest that when the capacity for chloroplast protein synthesis is reduced, photosynthetic and rprotein mRNAs are distinguished in a class specific manner and a hierarchy of translation is established which favors expression of r-protein mRNAs (Gillham et al., 1994). Analysis of heparin-actigel purified whole-cell extracts fromwild-type cells grown phototrophically, mixotrophically or heterotrophically and extracts of mutants deficient in chloroplast protein synthesis demonstrated that proteins of 81, 62, 56, 47, 38, 36 and 15 kDa UV crosslink to the leaders of several

Chapter 12 Chloroplast Translation chloroplast, but not nucleus-encoded 5´UTRs (Hauser et al., 1996; Fig. 1). In extracts from mutant cells with reduced chloroplast protein synthesis (ac20 cr1 and spr-u-1-27-3) binding of the 36 kDa protein present in wild-type cells is undetectable (Hauser et al., 1996). This suggests that the 36 kDa protein may be required for the translation of photosynthetic, but not r-protein mRNAs. It is also possible that the 36 kDa protein is absent because it is synthesized on chloroplast ribosomes. The observation that six of the RNA binding proteins bind to five distinct chloroplast leaders under a variety of environmental conditions in conjunction with data cited above (Danon and Mayfield, 1991, 1994b; Zerges and Rochaix, 1994) suggest that at least some of these proteins are general leader binding proteins. Of particular interest is the 47 kDa protein family, whose members migrate as doublets when they are crosslinked to various leader sequences by UV irradiation (Hauser et al., 1996). The RBP46 protein identified in extracts of the F64 strain, in which psbC translation is inhibited (Zerges and Rochaix, 1994), and the 47 kDa protein that is involved in the lightactivated translation of psbA mRNA (Danon and Mayfield, 1991, 1994b) may either be genetically unrelated, or members of a related family of proteins. Together, these results suggest that members of the of 47 kDa RNA binding protein family may be either class-, message- or environment-specific, while others may bind ubiquitously to all chloroplast leaders. Proteins that UV crosslink directly to the 5´UTRs of chloroplast mRNAs may represent only a subset of the proteins constituting these RNP complexes. Other proteins which interact in the complex via protein-protein interactions will not be detected using this assay. Thus the aforementioned six to eight trans-acting proteins binding to the 5´UTRs of chloroplast mRNAs may be an underestimate of the number of the participants in the translational regulatory complex. Therefore, failure to observe differences in the pattern of proteins that U V crosslink to the 5´UTRs of chloroplast mRNAs in extracts from cells exposed to different environmental conditions may indicate that the key regulatory proteins do not bind directly to the mRNA, but rather to other proteins in the translation complex. Alternatively, since all of these extracts were prepared and analyzed under reducing conditions, differences in their state of reduction in vivo that might be related to their binding in response to environmental stimuli could be obscured. The 60 kDa protein

209 reported to be part of the complex on the psbA leader in C. reinhardtii (Danon and Mayfield, 1991) appears to be a regulatory protein of this type. Proteinprotein crosslinking experiments, use of the yeast two-hybrid system and/or isolation ofan intact 5´UTR binding complex will be necessary to determine whether additional translational regulatory proteins exist that are not in direct contact with the mRNA.

VI. Ribosomes, Membranes and Tethers A model has emerged, principally from studies of COX3 message translation in the yeast mitochondrion, which assumes that an inner-membrane-bound translational activator complex recognizes both the mRNA 5´UTR and the mitochondrial ribosome (Fox, 1996a,b). The possible application of this model to translational control in chloroplasts has already been recognized (Gillham et al., 1994; Rochaix, 1996). The model derives its appeal for chloroplasts from the fact that the COX3 protein, like many other proteins encoded in chloroplasts and mitochondria, is very hydrophobic. Coupling of the translation complex to the inner mitochondrial membrane would ensure that the nascent, hydrophobic polypeptide is synthesized close to its insertion site in the inner membrane. Translation of COX3 mRNA requires three proteins encoded by the nuclear genes PET54, PET122 and PET494 (Fox, 1996a,b). Several lines of evidence suggest that these three proteins form a complex. The PET54 product (Pet54p) is found in equal amounts as a soluble polypeptide and a peripheral membrane protein whereas the other two polypeptides are probably integral membrane proteins. Since the Pet54p plays a role in splicing of a mitochondrial intron in COX1 pre-mRNA in some, but not all, yeast strains, Fox speculates that the soluble form of the protein is responsible for intron splicing while the membrane-bound form is part of the COX3 mRNA translation complex. Other evidence suggests that Pet54p may participate in COX3 mRNA recruitment to the inner membrane while the Pet122p makes contact with the small subunit of the mitochondrial ribosome. Loss of function caused by carboxy­ terminal truncation of the Pet 122p through the use of deletion or nonsense alleles can be suppressed by mutations in the genes encoding three different proteins of the small subunit of the mitochondrial ribosome. All three proteins are required for

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mitochondrial protein synthesis in yeast, but none displays sequence similarity with ribosomal proteins from other organisms. Fox (1996a,b) believes that his model is consistent with data available for translation of other mitochondrial genes in yeast although they are less complete, and he further speculates that these proteins may play a role in making topological distinctions in the sites of

synthesis of each mitochondrion-encoded protein. Such specific inferences concerning the role of translational activator proteins in membrane binding and the recruitment ofmRNA and ribosomes are not yet possible to make in the case of the chloroplast of Chlamydomonas (Fig. 2). However, there is much evidence that many chloroplast encoded proteins are synthesized on thylakoid membrane-bound poly­

Chapter 12 Chloroplast Translation somes (see Gillham, 1994 and Harris et al., 1994 for reviews). During greening of etiolated barley, membrane-bound chloroplast ribosomes engaged in translation of the D1 protein pause at specific sites during the translation process (Kim et al., 1991). Six different pause sites have been identified which correspond roughly to the insertion of the five of D1 into the thylakoid hydrophobic membrane. There seems to be a correlation between pausing and the presence of the hydrophobic amino acids of these helices in the ribosome tunnel. Since the ribosome tunnel is itself somewhat hydrophobic, pausing may involve the interaction of the newly synthesized hydrophobic amino acids and those present in the ribosome tunnel. In addition, D1 translation intermediates at the third and fourth pauses may bind phaeophytin while those at the fifth pause may bind chlorophyll. Interestingly, nonphoto­ synthetic D1 mutants of C. reinhardtii with amino to Gln, Glu, His, and acid substitutions from Asp synthesize two forms of D1 one of which is slightly larger (33–34 kDa) and the other smaller (24–25 kDa) than the mature protein (Lardans et al., 1997). While the 33–34 kDa protein does not correspond to the precursor and may result from structural modification ofthe mature 32 kDa protein, the 24–25 kDa form is likely to be a translation intermediate. This polypeptide may result from abortion of translation in the C-terminal portion of IV and V during the loop connecting ribosome pausing at the next to last or last pause site. In summary, much evidence exists to support the idea that many chloroplast mRNAs program translation on thylakoid-bound polysomes and that some of the protein products are very hydrophobic. This would be consistent with the scheme envisioned by Fox (1996a,b) for yeast mitochondrial mRNAs in that these chloroplast mRNAs might bind close to the site on the membrane where their protein product is to be inserted. However, there is still no evidence that the nucleus-encoded proteins that bind to the 5´UTRs of chloroplast mRNAs either function to localize these mRNAs to the thylakoid membranes or that they make contact with the chloroplast ribosome. Recently, a nucleus-encoded homolog of E. coli ribosomal protein S1 which is targeted to the chloroplast has been identified (Franzetti et al., 1992; Harris et al., 1994). This protein could play a role in mRNA recognition as it does in E. coli (see above Section I).

211 VIl.Translational Regulation of Complex Assembly

A. Chloroplast Thylakoid Membranes The numerous nuclear mutants of Chlamydomonas deficient in photosynthesis or chloroplast protein synthesis also afford tools for the dissection of the mechanisms regulating assembly of these multimeric protein complexes (Fig. 1, Table 1). Nuclear mutants identified to date that affect the synthesis of chloroplast encoded photosynthetic proteins appear to function in a gene specific manner (Gillham et al., 1994; Rochaix 1995, 1996). A possible exception is the nuclear tbc3 mutant recently described by Zerges et al., (1997) that appears to affect expression of both psbC and rbcL mRNAs. Translational regulation of the synthesis of chloroplast-encoded proteins appears to mediate two distinct phenomena: (1) the coordinated assembly of photosynthetic and translational complexes, and (2) a response to specific environmental signals, such as light (reviews: Gillham et al., 1994; Rochaix, 1996). The precise role(s) these nucleus-encoded proteins play in regulating chloroplast mRNA translation is still being resolved, however intriguing models with parallels to the yeast mitochondrial translation system have been invoked (Gillham et al, 1994; Rochaix, 1996). The Photosystem II core complex is composed of proteins D1, D2, P5 and P6 encoded by psbA,psbD, psbB and psbC mRNAs. Previous work has revealed the concerted expression of the psbA,psbD and psbB genes (reviewed by Rochaix, 1996). During the assembly of the PS II complex the D1, D2 and P5 proteins form an intermediate complex prior to association with P6. Primer extension studies suggest the possibility that the mRNAs encoding these three hydrophobic proteins are synthesized as precursors and cleaved within their 5´UTRs to generate mature mRNAs. Analogous to the yeast mitochondrial system, discussed above, a protein complex by binding to the 5´ end of the chloroplast leader might serve to target and/or dock the mRNA to the thyla­ koid membrane (Fig. 2). Cleavage of the leader is hypothesized to induce a conformational change allowing ribosome binding and initiation of translation (Rochaix, 1996). The psbC product P6 is integrated in a subsequent step to form the PS II core complex. Assembly of the C. reinhardtii cytochrome complex appears to be regulated by two distinct

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mechanisms: 1) proteolytic degradation of unassem­ bled subunits and, 2) a process in which the pro­ duction of cytochrome f depends on its immediate interaction with cytochrome and subunit IV (Kuras and Wollman, 1994). Using insertional mutagenesis, mutants affected in the half-life of cytochrome f and have recently been isolated (Gumpel et al., 1995). Whether the tagged genes are required for constitutive expression of the subunits, or if they coordinate the synthesis of the subunits and thus assembly of the complex is unknown. Rubisco holoenzyme plays a pivotal role in photosynthesis because it catalyzes the carboxylation and oxygenation of RuBP (reviewed by Spreitzer, 1993; Chapter 27, Spreitzer). While most Rubisco­ deficient mutants result from alterations in the chloroplast encoded rbcL gene encoding LSU, which perturb its enzymatic functions, nuclear mutants not linked to rbcS encoding SSU, have been isolated which affect Rubisco at transcriptional, posttrans­ criptional and cotranslational steps (see Hong and Spreitzer, 1994). In addition, data from transgenic tobacco plants expressing antisense rbcS mRNA suggest that SSU protein abundance specifically contributes to the regulation of LSU protein accumulation at the level ofrbcL translation initiation (Rodermel et al., 1996). Sequences near the 5´ end of the rbcL leader were found to destabilize chimeric mRNAs in dark grown cells following illumination (Salvador et al., 1993). This effect is eliminated by the addition of sequences from the adjacent coding region of the rbcL gene, and these are probably responsible for stabilizing endogenous rbcL transcripts (Salvador et al., 1993).

B. Chloroplast Ribosomes The chloroplast translational apparatus is composed of five rRNA species and approximately 64 r-proteins (Harris et al., 1994). While all of the rRNAs are chloroplast encoded, only a third of the r-proteins are specified by the chloroplast genome with the remainder presumably being nuclear gene products (Gillham, 1994, Harris et al., 1994). To assemble such a molecular machine efficiently, the cell must provide equimolar amounts of each of the com­ ponents. While the mechanisms by which this is achieved in E. coli and the cytoplasm of eukaryotic cells have been extensively studied (see reviews by Nomura et al., 1984; Amaldi et al., 1989; Woolford and Warner, 1991), they remain to be determined in

chloroplasts. However, the identification of cis-acting sequences and trans-acting factors responsible for modulating the differential translation of chloroplastencoded photosynthetic and ribosomal protein subunits has begun (Section V).

VIII. How are the Regulatory Proteins Regulated? Except in the case of the psbA mRNA of C. reinhardtii, little is currently known about regulation of the nucleus-encoded trans-acting factors that govern expression of chloroplast mRNAs (Fig. 1). As discussed earlier, the 5´UTR of the psbA mRNA contains a 36 nt inverted repeat sequence thermo­ dynamically capable of forming a stem-loop which is crosslinked by UV to a complex of four closelyrelated 47-kDa proteins (Danon and Mayfield, 1991). A 60-kDa protein found to be associated with the isolated complex is not crosslinked to the 5´UTR by UV (Danon and Mayfield, 1994a). Although illuminated wild-type cells rapidly synthesize the D1 protein while dark-grown cells do not, there is less than a two-fold difference in the abundance of the 47-kDa proteins in light-and dark-grown cells. A serine/threonine phosphotransferase associated with the mRNA binding complex (Danon and Mayfield, 1994a) has been identified that utilizes the of ADP to phosphorylate the 60-kDa protein in vitro. Although phosphorylation of this protein inhibits the mRNA binding activity of the complex, the high levels ofADP required in vitro are only attained in vivo in the chloroplasts of plants grown in the dark. Phosphorylation of the 60 kDa protein in vivo appears to be sufficient to overcome any effect of reduction of the 47 kDa protein in these in vitro assays. Thus, the activator complex would bind well to the psbA 5´UTR in light- but not darkgrown cells. Synthesis of the 47-kDa protein appears to require the presence of chlorophyll and/or a fully developed chloroplast, since the 47-kDa protein is not detected in dark-grown cells of the y1 mutant which, unlike wild type, does not synthesize chlorophyll, chloroplast membranes or D1 (Danon and Mayfield, 1991). Light-modulation of psbA mRNA expression via redox potential has been proposed as a translational regulatory mechanism (Danon and Mayfield, 1994b). Once again the evidence is based mostly on in vitro experiments and shows that oxidation of the

Chapter 12 Chloroplast Translation regulatory protein complex with dithionitrobenzoic acid (DTNB) blocks binding ofthe complex topsbA­ mRNA. This effect is reversed by incubation of the complex with reducing agents like dithiothreitol (DTT). Furthermore, an even greater restoration of RNA-binding activity of the complex was observed when reduced thioredoxin was added to the DTNBoxidized complex suggesting that thioredoxin was the agent responsible for reducing the protein complex in vivo. In chloroplasts, reduction of thioredoxin by ferredoxin is stimulated by photosynthetic electron transfer in response to light (Buchanan et al., 1994; Chapter 26, Jacquot et al.). Reduced ferredoxin activates three of the four major regulatory enzymes of the carbon cycle. This led Danon and Mayfield (1994b) to postulate that reduced ferredoxin can also activate the protein complex that binds to the 5´UTR of psbA mRNA and stimulates translation of D1. The model was tested in vivo using a Photosystem I-deficient mutant (ac-u-g-2-3) which can no longer mediate reduction of thioredoxin by ferredoxin (Danon and Mayfield, 1994b). The mutant, as predicted, contained much less of the psbA mRNA protein binding complex and much less D1 than wild type although psbA mRNA accumulation was similar in the mutant and wild type. Synthesis of three other chloroplast-encoded Photosystem II proteins (D2, CP43 and CP47) was also reduced in ac-u-g-2-3. Rochaix (1996), states that these results are at variance with earlier pulse-labeling studies with other Photosystem I-deficient mutants of C. reinhardtii which did not reveal a diminution in the synthesis of the core Photosystem II polypeptides including D1. Obviously this discrepancy will have to be resolved before the redox model for D1 regulation can be accepted as explaining light-regulation of psbA mRNA translation in vivo. Redox regulation, possibly via the thioredoxin-ferredoxin pathway, has also been implicated recently in splicing of group I introns from the psbA pre-mRNA in C. reinhardtii (Desh­ pande et al., 1997) so the mechanisms by which redox regulation of chloroplast gene expression is exerted are potentially far-reaching. In addition, experiments with Dunaliella tertio­ lecta, a relative of Chlamydomonas, reveal that the redox state of plastoquinone may modulate tran­ scription rates of the nuclear Cab genes encoding the chlorophyll a/b proteins via a phosphorylation cascade (Escoubas et al., 1995). The reduction state of the plastoquinone (PQ) pool can be manipulated using the electron transport inhibitors DCMU [3-

213 (3,4 dichlorophenyl)-1,1 -dimethyl urea) and DBMIB [2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone] to block the reduction and oxidation of the PQ pool respectively. DCMU induced a doubling of Cab mRNA abundance mimicking the effect of shifting cells from high to low light intensity while DBMIB induced a 75% decrease in Cab mRNA, mimicking the effect of a shift to higher irradiance. Gel shift assays identified a protein-DNA complex in extracts from cells grown at high, but not low light. Phosphatase inhibitors blocked derepression of Cab gene expression in low light. These results have been interpreted in terms of a model in which reduced PQ is sensed by a chloroplast kinase which phos­ phorylates a chloroplast phosphoprotein. This phosphoprotein then directly or indirectly activates a cytosolic kinase which phosphorylates a repressor which can bind in the Cab gene promoter region in its phosphorylated state and block transcription. Whether similar models will prove applicable to nucleus-encoded regulators of chloroplast mRNA translation remains to be seen.

IX. Is there Hierarchical Control of Chloroplast mRNA Translation? Specific nuclear gene products are clearly essential for expression of individual chloroplast genes in C. reinhardtii at the transcriptional, posttrans­ criptional or translational levels as discussed in this and earlier reviews (Gillham et al., 1994; Mayfield et al., 1995;Rochaix, 1995,1996; Cohen and Mayfield, 1997). The notion that translation initiation is modulated by a complex of proteins bound to the 5´UTRs of chloroplast mRNAs is supported by both genetic and biochemical evidence. Such translational activation complexes are likely to contain both genespecific proteins discussed earlier and ubiquitous proteins required for the translation of all chloroplast mRNAs (Hauser et al., 1996). Binding of specific proteins to the translational complexes may also effect concordant regulation of classes of chloroplast genes with related functions or in response to particular environmental stimuli (Gillham et al., 1994). Binding of gene-specific, class-specific or environment-specific proteins to the complex could exert either a negative or positive effect on translation initiation. This might occur directly by altering the ability of the mRNA to associate with the small subunit of the chloroplast ribosome to form an

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initiation complex. Alternatively, it could occur indirectly, by preventing binding of certain of the ubiquitous proteins essential for initiation to occur. In addition, these proteins may play a role in targeting the messages to specific sites within the organelle to facilitate coordinated translation of members of multisubunit complexes (Fox 1996b, Rochaix, 1996, Gillham et al., 1994). Clear evidence exists for classspecific translational regulation of ribosomal protein genes versus photosynthetic protein genes under conditions of reduced chloroplast protein synthesis in the C. reinhardtii chloroplast (Gillham et al., 1994; Liu et al., 1989b). The precise mechanism(s) regulating the entry of a mRNA into a translationally competent state in a gene, class or environment specific manner remains to be determined.

Acknowledgments This research was supported by NIH Grant GM­ 19427 to JEB and NWG. We thank D. Stern and J.-D. Rochaix for providing unpublished results.

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215 Kim HY, Klausner RD and Rouault TA (1995) Translational represser activity is equivalent and is quantitatively predicted by in vitro RNA binding for two iron-responsive elementbinding proteins, IRP1 and IRP2.J Biol. Chem 10:4983–4986 Kindle K (1987) Expression of a gene for a light-harvesting chlorophyll a/b-binding protein in Chlamydomonas reinhardtii: Effect of light and acetate. Plant Mol Biol 9: 547–563 Kozak M (1983) Comparison of initiation of protein synthesis in procaryotes, eucaryotes and organelles. Microbiol Rev 47: 1– 45 Kuchka M, Mayfield SP and Rochaix J-D (1988) Nuclear mutations specifically affect the synthesis and/or degradation of the chloroplast-encoded D2 polypeptide of Photosystem II in Chlamydomonas reinhardtii. EMBO J 7: 319–324 Kuchka M, Goldschmidt-Clermont M, van Dillewijn J and Rochaix J-D (1989) Mutation at the Chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PS II. Cell 58: 869–876 Kudla J, Hayes R and Gruissem W (1996) Polyadenylation accelerates degradation of chloroplast mRNA. EMBO J 15: 7137–7146 Kuras R, Wollman F-A (1994) The assembly of cytochrome b6/ f complexes: An approach using genetic transformation of the green alga Chlamydomonas reinhardtii. EMBO J 13: 1019– 1027 Lardans A, Gillham NW and Boynton JE (1997) Site-directed mutations at residue 251 of the Photosystem II Dl protein of Chlamydomonas that result in a nonphotosynthetic phenotype and impair D1 synthesis and accumulation. J Biol Chem 272: 210–216 Leibold EA and Munro HN (1988) Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5´ untranslated region of ferritin heavy- and light-subunit mRNAs. Proc Natl AcadSci USA 85: 2171–2175 Lisitsky I, Klaff P and Schuster G (1996) Addition ofdestabilizing poly(A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA. Proc Natl Acad Sci USA 93: 13398–13403 Liu X-Q, Gillham NW and Boynton JE (1989a) Chloroplast ribosomal protein gene rps12 of Chlamydomonas reinhardtii. J Biol Chem 264: 16100-16108 Liu X-Q, Hosler JP, Boynton JE and Gillham NW (1989b) mRNAs for two ribosomal proteins are preferentially translated in the chloroplast of Chlamydomonas reinhardtii under conditions of reduced protein synthesis. Plant Mol Biol 12: 385–394 Lovett P (1994) Nascent peptide regulation of translation. J Bact 176: 6415–6417 McCarthy JE and Brimacombc R (1994) Prokaryotic translation: The interactive pathway leading to initiation. Trends Genet 10: 402–407 McCarthy JE and Gualerzi C (1990) Translational control of prokaryotic gene expression. Trends Genet 6: 78–85 Mache R (1990) Chloroplast ribosomal proteins and their genes. Plant Sci 72: 1–2 Maliga P (1993) Towards plastid transformation in flowering plants. Trends Biotech 11: 101–107 Malnoë P, Mayfield SP and Rochaix J-D (1988) Comparative analysis of the biogenesis of Photosystem 11 in the wildtype and y-1 mutant of Chlamydomonas reinhardtii. J Cell Biol

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106:609–616 Mathews MB, Sonenberg N and Hershey JWB (1996) Origins and targets of translational control. In: Hershey JWB, Mathews MB and Sonenberg N (eds), Translational Control, pp 1–29. Cold Spring Harbor Laboratory Press, Plainview Mayfield SP, Cohen A, Danon A and Yohn CB (1994) Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5´ untranslated region. J Cell Biol 127: 1537–1545 Mayfield S, Yohn CB, Cohen A and Danon A (1995) Regulation of chloroplast gene expression. Annu Rev Plant Physiol Plant Mol Biol 46: 147–166 Meerovitch K, Pelletier J and Sonenberg N (1989) A cellular protein that binds to the 5´-noncoding region of poliovirus RNA: Implications for internal translation initiation. Genes Dev3: 1026–1034 Morton BR (1996) Selection on the codon bias of Chlamydomonas reinhardtii chloroplast genes and the plant psbA gene. J Mol Evol 43: 28–31 Myers AM, Harris EH, Gillham NW and Boynton JE (1984) Mutations in a nuclear gene of Chlamydomonas cause the loss of two chloroplast ribosomal proteins, one synthesized in the chloroplast and the other in the cytoplasm. Curr Genet 8: 369– 378 Nickelsen J, van Dillewijn J, Rahire M and Rochaix J-D (1994) Determinants for stability of the chloroplast psbD RNA are located within its short leader region in Chlamydomonas reinhardtii. EM BO J 13: 3182–3191 Nomura M, Gourse R and Baughman G (1984) Regulation of the synthesis of ribosomes and ribosomal components. Ann Rev Biochem 53: 75–117 Prokipcak RD, Herrick DJ and Ross J (1994) Purification and properties of a protein that binds to the C-terminal coding region of human c-myc mRNA. J Biol Chem 269: 9261–9269 Proudfoot N (1996) Ending the message is not so simple. Cell 87: 779–781 Proweller A and Butler JS (1996) Ribosomal association of poly(A)-binding protein in poly(A)-deficient Saccharomyces cerevisiae. J Biol Chem 271: 10859–10865 Purton S and Rochaix J-D (1994) Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library. Plant Mol Biol 24: 533–537 Putz U, Skehel P and Kuhl D (1996) A tri-hybrid system for the analysis and detection of RNA-protein interactions. Nucl Acids Res 24: 4838–4840 Reinbothe S, Reinbothe C, Heintzen C, Seidenbecher C and Parthier B (1993) A methyl jasmonate-induced shift in the length of the 5´ untranslated region impairs translation of the plastid rbcL transcript in barley. EMBO J 12: 1505–1512 Reith M (1995) Molecular biology of rhodophyte and chromophyte plastids. Annu Rev Plant Physiol. Plant Mol. Biol. 46: 549–575 Rochaix J-D (1995) Chlamydomonas reinhardtii as the photosynthetic yeast. Annu Rev Genet 29: 209–230 Rochaix J-D (1996) Post-transcriptional regulation ofchloroplast gene expression in Chlamydomonas reinhardtii. Plant Mol Biol 32: 327–341 Rochaix J-D, Kuchka M, Mayfield S, Schirmer-Rahire M, GirardBascou J and Bennoun P (1989) Nuclear and chloroplast mutations affect the synthesis or stability of the choroplast psbC gene product in Chlamydomonas reinhardtii. EMBO J 8:

1013–1021 Rodermel S, Haley J, Jiang, C-Z, Tsai, C-H and Bogorad L (1996) A mechanism for intergenomic integration: Abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA. Proc Natl Acad Sci USA 93: 3881–3885 Rouault TA, Hentze MW, Haile, DJ and Harford JB (1989) The iron-responsive element binding protein: A method for the affinity purification of a regulatory RNA-binding protein. Proc Natl Acad Sci USA 86: 5768–5772 Sakamoto W, Kindle KL and Stern DB (1993) In vivo analysis of Chlamydomonas chloroplast petD gene expression using stable transformation of beta-glucuronidase translational fusions. Proc Natl Acad Sci USA 90: 497–501 Sakamoto W, Chen X, Kindle KL and Stern DB (1994) Function of the Chlamydomonas reinhardtii petD 5´ untranslated region in regulating the accumulation of subunit IVof the cytochrome complex. Plant J 6: 503–512 Salvador ML, Klein U and Bogorad L (1993) 5´ sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts. Proc Natl Acad Sci USA 90: 1556–1560 Schindler U and Cashmore AR (1990) Photoregulated gene expression may involve ubiquitous DNA binding proteins. EMBO J 9: 3415–3427 Schmitz J, Prüfer D, Rohde W and Tacke E (1996) Non-canonical translation mechanisms in plants: Efficient in vitro and in planta initiation at AUU codons of the tobacco mosaic virus enhancer sequence. Nucl Acids Res 24: 257–263 SenGupta DJ, Zhang B, Kraemer B, Pochart P, Fields S and Wickens M (1996) A three-hybrid system to detect RNAprotein interactions in vivo. Proc Natl Acad Sci USA 93: 8496–8501 Shapira M, Lers A, Heifetz PB, Irihimovitz V, Osmond CB, Gillham NW and Boynton JE (1997) Differential regulation of chloroplast gene expression in Chlamydomonas reinhardtii during photoacclimation: Light stress transiently suppresses synthesis of the Rubisco LSU protein while enhancing synthesis of the PS II D1 protein. Plant Mol Biol 33: 1001–1011. Sheen J (1990) Metabolic repression of transcription in higher plants. Plant Cell 2: 1027–1038 Silk GW and Wu M (1993) Posttranscriptional accumulation of chloroplast tufA (elongation factor gene) m R N A during chloroplast development in Chlamydomonas reinhardtii. Plant Mol Biol 23: 87–96 Spreitzer RJ (1993) Genetic dissection of Rubisco structure and function. Annu Rev Plant Physiol Plant Mol Biol 44: 411–434 StampacchiaO, Girard-Bascou J,ZanascoJL, Zerges W, Bennoun P and Rochaix JD (1997) A nuclear-encoded function essential for the translation of the chloroplast psaB mRNA in Chlamydomonas. Plant Cell 9:773–782 Standait N and Jackson RJ (1994) Regulation of translation by specific protein/mRNA interactions. Biochimie 76: 867–879 Staub JM and Maliga P (1994) Translation of psbA mRNA is regulated by light via the 5´-untranslated region in tobacco plastids. Plant J 6: 547–553 Stern D, Radwanski ER, Kindle KL (1991) A 3´ stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo. The Plant Cell 3: 285–297 Stevens DR, Rochaix JD and Purton S (1996) The bacterial phleomycin resistance gene ble as a dominant selectable marker

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217 Cold Spring Harbor Press, Cold Spring Harbor Wu HY and Kuchka MR (1995) A nuclear suppressor overcomes defects in the synthesis of the chloroplast psbD gene product caused by mutations in two distinct nuclear genes of Chlamydomonas. Curr Genet 27: 263–269 Wulczyn FG, Bolker M and Kahman R (1989) Translation of the bacteriophage MU mom gene is positively regulated by the phage com gene product. Cell 57:1201–1210 Yamamoto Y, Tsuji H and Ogokata J (1995) 5´-Leader of a Photosystem I gene in Nicotiana sylvestris, psaDb contains a translational enhancer. J Biol Chem 270: 12466–12470 Yohn CB, Cohen A, Danon A and Mayfield SP (1996) Altered mRNA binding activity and decreased translational initiation in a nuclear mutant lacking translation of the chloroplast psbA mRNA. Mol Cell Biol 16: 3560–3566 Zerges W and Rochaix JD (1994) The 5´ leader of a chloroplast mRNA mediates the translational requirements for two nucleusencoded functions in Chlamydomonas reinhardtii. Mol Cell Biol 14: 5268–5277 Zerges W, Girard-Bascou J and Rochaix J-D (1997) Translation of the chloroplast psbC mRNA is controlled by interactions between its 5´ leader and the nuclear loci TBC1 and TBC3 in Chlamydomonas. Mol Cell Biol 17: 3440–3448 Zhang J and Detitscher MP (1992) A uridine-rich sequence required for translation of prokaryotic mRNA. Proc Natl Acad Sci USA 89: 2605–2609 Zhang H, Herman PL and Weeks DP (1994) Gene insolation through genomic complementation using an indexed library of Chlamydomonas reinhardtii DNA. Plant Mol Biol 24: 663– 672 Zuker M (1994) Prediction of RNA secondary structure by energy minimization. Methods Mol Biol 25: 267–294

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Chapter 13

Chloroplast Protein Translocation Mireille C. Perret, Karen K. Bernd and Bruce D. Kohorn Developmental Cell and Molecular Biology, Box 91000, LSRC, Duke University, Durham, NC 27708, U.S.A.

Summary I. Introduction II. Chloroplast Import A. Signal Sequences B. The Import Apparatus III. Sorting of Proteins Within the Chloroplast IV. Thylakoid Translocation V. Mutations Affecting Translocation A. Chloroplast Suppressors B. Nuclear Suppressors VI. Perspectives Acknowledgments References

219 220 220 220 221 222 223 226 226 227 228 229 229

Summary The signal hypothesis, first put forth by G. Blobel and B. Dobberstein over 20 years ago, suggested that all proteins had as part of their primary amino acid sequence an amino-terminal region that defined the destination of proteins carrying that signal. This hypothesis has stood the test of numerous experiments and appears to be pertinent to most cellular compartments and proteins within eukaryotes and prokaryotes, including the chloroplast. Studies with Chlamydomonas reinhardtii, the subject of this chapter, have tested these concepts in live cells and have provided both new findings and the confirmation of in vitro results derived from vascular plants. Proteins synthesized outside the plastid carry chloroplast specific signals, often at their amino terminus. This signal can be removed upon entry of the protein into the chloroplast, but those destined for internal compartments within the plastid also contain additional signals. This sub-organelle targeting information can be removed once the protein is correctly localized. Thus chloroplast sorting is determined by multifunctional signal sequences. Experiments with isolated organelles have defined a number ofdifferent energetic parameters that are required for the translocation of proteins with distinct types of signal sequences, and models suggest that multiple mechanisms exist within the chloroplast. C. reinhardtii can be grown either heterotrophically or photoautotrophically thereby allowing for the selection and propagation of mutations that affect the biogenesis of chloroplasts, and thus some that affect protein translocation directly. The genetic analysis of thylakoid protein translocation in C. reinhardtii has revealed at least six loci whose products are involved in the process, and has provided genetic means to dissect those paths in vivo that have been described in isolated organelle studies. While there are likely to be a variety of requirements for the targeting and translocation of proteins to and within the chloroplast, these mechanisms may well share components between apparatuses, and between diverse groups of organisms. J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 219–231. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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I. Introduction Yellow green shapes (Greek: chloros plastos) containing sac-like forms (Greek: thytakos) were detected in the early days of microscopy and were named by Schimper in the 1880’s (Reed, 1942). Since this initial observation, we have come to realize the importance of this organelle in a variety of roles, including amino acid biosynthesis, lipid production and photosynthesis (Gillham, 1994). While chloro­ plasts and other plastids have been studied in a variety of photosynthetic species, C. reinhardtii has provided a unique contribution. The ability of C. reinhardtii to survive on acetate supplemented media without photosynthesis has allowed the creation and propagation of mutants impaired in chloroplast function. Moreover, the rapidity and ease of radiolabeling proteins in live C. reinhardtii has led to important conclusions about processes in vivo. The import of proteins into chloroplasts and the translocation ofproteins into and across the thylakoid membrane has been recently reviewed extensively (Cline and Henry, 1996; Kouranov and Schnell, 1996), and it would be redundant to provide another detailed account. Rather, we will highlight important findings that describe the mechanisms and, where appropriate, indicate how studies with C. reinhardtii have contributed. In most cases, work with C. reinhardtii has provided a basis for in vivo mechanisms, while studies with vascular plants have been restricted to either in vitro studies or gene identification. While many have touted one or another organism as ‘model,’ and C. reinhardtii is no exception, it is perhaps more useful to realize that we will discover something interesting wherever we look and that it is easier to do certain experiments with particular organisms. The internal structure of C. reinhardtii chloroplasts appears ultrastructurally and compo­ sitionally extremely similar to that of higher plants, and thus what we find true for events within the C. reinhardtii chloroplast is also likely true for other algae and angiosperms. Differences, such as reduced amounts of granal stacks in the algal chloroplasts Abbreviations: Cyt f – cytochrome f; DHFR – dihydrofolate reductase; LHCP– light harvesting chlorophyll a/b protein; OEC – oxygen evolving complex; OEC33, OEC23, OEC17 – 33 kDa, 23 kDA and 17 kDa subunits of the oxygen evolving complex; PC – plastocyanin; Rubisco SSU –small subunit of ribulose-1,5bisphosphate carboxylase oxygenase; SRP54 – 54 kDa subunit of the signal recognition particle; Tip – thylakoid insertion protein

(Harris, 1989) and the presence ofalternative electron (Wood, 1978; acceptors such as cytochrome Merchant and Bogorad, 1987), may reflect adaptive strategies. There are distinct differences between the organization of a chloroplast within a C. reinhardtii cell and within a vascular plant cell. The most obvious is that C. reinhardtii hasjust one chloroplast that fills most of the cell and is usually oriented in the same way within the cell. Vascular plant cells are normally characterized as having multiple chloroplasts that can stream with the cytoplasm or reside in numerous cellular locations. Thus, a priori, one might expect to find that there are unique features in C. reinhardtii versus vascular plants in mechanisms that target proteins from the cytoplasm to the chloroplast surface.

II. Chloroplast Import

A. Signal Sequences The signal hypothesis, first put forth by Blobel and coworkers (Blobel and Sabatini, 1971; Blobel and Dobberstein, 1975) over 20 years ago, suggested that all proteins had, as part of their primary amino acid sequence, an amino-terminal region that defined the destination of proteins carrying that signal. This concept has dominated and driven the field of protein targeting, and most experiments are consistent with the hypothesis. Not long after putting forth their signal hypothesis, which was based on studies with the endoplasmic reticulum, Blobel and Dobberstein (Blobel and Dobberstein, 1977) detected a ‘putative precursor’ of C. reinhardtii Rubisco small subunit (SSU) that had been translated in vitro, was precipitated by anti-SSU antibody, and could be processed to the size of SSU. They proposed that the extra sequence included chloroplast targeting information. Indeed, chloroplast proteins whose translation is initiated in the cytosol usually contain amino-terminal signals that are necessary and sufficient for targeting to the chloroplast envelope. These signals are often removed by one or more stromal peptidases upon entry into the chloroplast (Su and Boschetti, 1993, 1994; Rufenacht and Boschetti, 1995). While there are a number of predicted signal sequences in C. reinhardtii (von Heijne et al., 1991), in only a few cases have these sequences been shown to be functional and removed upon import (Yu et al., 1988; Schnarrenberger et al., 1994; Funke et al., 1997). The definition of a signal

Chapter 13 Chloroplast Protein Translocation

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sequence has often assumed that only the portion that is removed constitutes the signal. However, it has been observed that regions of the mature protein of two vascular plant proteins can influence the import reaction in vitro, suggesting that some signals may extend beyond the processing site into the sequence of the mature protein (Kohorn et al., 1986; Wan et al., 1995). Comparisons of amino-terminal signals from a variety of vascular plant proteins reveal little or no sequence conservation, although the region is rich in hydroxylated amino acids and alanine (von Heijne et al., 1989). The amino-terminal regions of nucleusencoded C. reinhardtii chloroplast proteins also appear to have no conserved primary structure, are rich in hydroxylated amino acids, and particularly rich in alanine. In solution, the signal has no apparent secondary structure, although some have suggested that signals can form helices in the presence of lipids (Horniak et al., 1993; see Chapter 26, Jacquot et al.). A structural study of the chloroplast signal sequence of the C. reinhardtii gamma subunit of the chloroplast ATP synthase showed that this region has a random coil conformation in a nonpolar environment (Theg and Geske, 1992). C. reinhardtii chloroplast signals resemble mitochondrial targeting signals in that they contain amphipathic, positively charged regions (Franzen et al., 1990; Franzen and Falk, 1992). Indeed, the amino-terminal region of the Rubisco SSU from C. reinhardtii can direct a fusion protein of CoxIV to the mitochondrion of coxIV deficient yeast (Hurt et al., 1986) and an SSU-DHFR fusion can be imported into isolated yeast mito­ chondria (Hurt et al., 1986). These experiments demonstrate that the presequence can function as a mitochondrial targeting signal if the cell requires such targeting for survival or if the signal is only presented with isolated mitochondria. The C. reinhardtii nucleus-encoded chloroplast protein PsaF can be imported in vitro into isolated mitochondria from spinach even when its presequence is removed (Hugosson et al., 1995) suggesting that a cryptic signal may be revealed under in vitro conditions. However, it is not yet clear whether in live C. reinhardtii the sequence functions as both a mitochondrial and a chloroplast signal. In C. rein­ hardtii neither SSU nor PsaF are found in the mitochondrion, suggesting that if mistargeting occurs, then the proteins are rapidly degraded. Alternatively, the chloroplast signal may be dominant to the cryptic mitochondrial one, or, more likely, additional

regulatory mechanisms exist in the cytosol that differentiate between chloroplast and mitochondrial proteins in vivo as is the case for angiosperms (Whelan and Glaser, 1997). In summary, C. reinhardtii chloroplasts can be distinguished from those in vascular plants by their mitochondrial-like chloroplast targeting signals and by their dominating presence within a single cell. Whether these two unique characteristics are related remains to be tested. There are many reports of molecular dissection of chloroplast targeting signals. The studies provide conflicting results depending on whether the signals are assayed in vitro or by transformation into a plant. However, all studies do conclude that the region is necessary and sufficient for chloroplast import. For example, deletion studies (Hageman et al., 1990) have shown that the plastocyanin (PC) transit sequence is necessary for import into the chloroplast, and fusion protein studies have shown that the PC transit sequence is sufficient to transfer dihydrofolate reductase (DHFR) across the chloroplast envelope. Also, the pea SSU transit sequence can direct the bacterial protein neomycin phosphotransferase into pea chloroplasts (Van den Broeck et al., 1985). A systematic deletion analysis ofthe C. reinhardtii PC import signal (K. Kindle, personal commun­ ication) reveals that the region is required for import, but that the in vitro reactions are more sensitive to signal sequence perturbations than are live cells. Overall, it is clear that small deletions or substitutions can be accommodated in vivo and this may indicate a plasticity in the targeting and import apparatus. The absence of a phenotype provided by signal sequence mutations is not conducive to a genetic suppressor screen to identify proteins that interact with the signal sequence. Moreover, mutations in this import process may be lethal (Smith and Kohorn, 1994; Schnell, 1995), and together, these restrictions may explain the lack of reports utilizing C. reinhardtii or any vascular plant for a genetic analysis of chloroplast import. C. reinhardtii may provide the best genetic system for this analysis as at least problems from pleiotropic effects on photosynthesis could be avoided by growth on acetate-containing medium.

B. The Import Apparatus All ofthe studies that have characterized chloroplast envelope proteins involved in import have been performed with isolated vascular plant chloroplasts,

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and therefore will not be reviewed extensively. Briefly (as reviewed by Kouranov and Schnell, 1996), the chloroplast envelope contains two lipid bilayers and translocation occurs as a result of the coordinated action of protein translocation machinery in each bilayer at specialized regions called contact zones where the envelope membranes meet. Outer envelope proteins include the translocation-intermediate associating IAP34, IAP75, and IAP86 integral membrane protein complex and two Hsp70-like proteins, COM70 on the cytoplasmic side of the outer envelope and Hsp70-IAP on the inner side of the outer envelope. The Hsp70 proteins and the GTPbinding domain-containing IAP34 and IAP86 are considered to be components of a two-step translocation mechanism where initial precursor binding occurs with ATP and GTP hydrolysis and is then followed by translocation that requires higher amounts of ATP hydrolysis. IAP75 and IAP86 have been cross-linked to the transit sequences in vitro, as have the inner envelope proteins IAP21 (integral) and IAP25 (localization unknown). Inner envelope proteins that associate with import intermediates in vitro have also been found (IAP100, IAP36). It has also been suggested that the targeting of precursor proteins to the outer envelope machinery is aided by the unique lipid composition of this bilayer (van’t Hof et al., 1993). Interaction between the signal sequence and receptor components may be predicated by lipid-precursor protein contact that induces a more translocation-competent secondary structure in the signal sequence. Many of the conclusions concerning chloroplast targeting have been based upon the assumption that import is post-translational. This assumption is derived from the early observation that completed protein chains can be imported into chloroplasts (Chua and Schmidt, 1978; Mishkind et al., 1985) and many have used this post-translational assay to further our understanding of the process. It is quite difficult to determine whether this is indeed what occurs in vivo for all precursors. Estimations that have been made suggest that the in vivo rate of import exceeds that seen in vitro by orders of magnitude (Hoober et al., 1994). What has been lacking in the literature is a thorough analysis of the possible co-translational insertion of at least some proteins into the chloroplast. This has been shown to be a distinct possibility with yeast mitochondria, and ribosomes can be detected on the surface of mitochondria (Fujiki and Verner, 1991, 1993; Verner, 1993). C. reinhardtii would be

well suited for such studies as pulse labeling and ultrastructural observations could be coupled with in vitro analysis.

III. Sorting of Proteins Within the Chloroplast There are six identifiable compartments within the chloroplast: the outer and inner membranes of the envelope, the space between these bilayers, the stroma, the thylakoid membrane, and the thylakoid lumen. In 1986, Smeekens et al. proposed that some chloroplast signals were multifunctional, a concept that has also been supported in mitochondrial import studies (Smeekens et al., 1986; Schatz and Dobberstein, 1996). According to this hypothesis, the signal is bipartite; the amino-terminal region mediates import across the chloroplast envelope and the remaining portion ofthe signal specifies sub-organellar targeting (Fig. 1). Their experiments and subsequent analysis in other labs addressed the targeting of thylakoid lumen proteins and provided evidence suggesting that upon entry into the chloroplast, the chloroplast import region was removed by a stromal peptidase, and the remaining amino-terminal region then directed the protein to the thylakoid membrane where it mediated translocation into the lumen. Many in vitro experiments with vascular plant chloroplasts demonstrate that newly imported proteins destined for the thylakoid vesicle indeed pass through the stroma (Cline and Henry, 1996). The fact that insertion into and passage through the thylakoid membrane can be performed posttranslationally in vitro with isolated thylakoid membranes is indeed consistent with the passage of precursors through the stroma in vivo. In most cases, the thylakoid transfer signal is required for the in vitro translocation into the lumen, but the envelope signal is not. Despite their rigor and clarity, none of these experiments address directly the possible transfer of some proteins directly from the envelope to the thylakoid. While many have confirmed this sorting model for chloroplast proteins destined for the thylakoid membrane or lumen by using in vitro experiments, there is only one publication supporting this view for events that occur in vivo, and this was carried out with C. reinhardtii. Howe and Merchant (1993) were able to detect the intermediate form of lumen-destined plastocyanin (PC), and the 33 kDa cytochrome oxygen-evolving complex protein (OEC33) in which

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the envelope targeting signal had been removed, but the thylakoid signal remained. Some of this intermediate was detected in a soluble fraction which supported the idea that it was stromal. Pulse chase experiments showing the concomitant disappearance of the intermediate and increase in the amount of mature protein supported the identity of the larger form as a true intermediate. These data stand alone as the only in vivo evidence of stromal intermediates in sub-chloroplast targeting. However, some of the intermediate was insoluble, and since its location remains unknown, the issue requires more attention.

triphosphates (NTPs), and the precursor of light harvesting chlorophyll a/b binding protein (LHCP) (Kirwin requires a stromal protein and GTP and a et al., 1989; Mould and Robinson, 1991; Cline et al., 1992; Hulford et al., 1994; Nielsen et al., 1994; Yuan and Cline, 1994; Kouranov and Schnell, 1996). The stromal protein required for pre-LHCP import is likely to be CP54, a homologue of the SRP 54-kD protein, as its depletion from stroma prevents insertion (Li et al., 1995), although it has not been reported that CP54 can be added back to restore depleted extracts. Studies of the integral membrane protein found that nucleoside triphosphates and stromal extracts are not required for its integration into the membrane, and that a only slightly enhances its integration (Michl et al., 1994). The energetic requirements for the translocation of precursors of thylakoid lumen proteins appear to be dependent upon the thylakoid transfer signal as exchange of signals also exchanges the energetic requirement. A chimeric construct consisting of the pea OEC23 thylakoid transit sequence fused to the mature region of PC does not require stromal extract or NTPs for thylakoid translocation, and the translocation of both this chimera and an OEC17 transit/mature PC fusion protein is inhibited by across the nigericin, which dissipates the

IV. Thylakoid Translocation A number of detailed studies with isolated pea chloroplasts show that differentproteins have varying energetic requirements for successful translocation into and across the thylakoid membrane. All of these precursors require thylakoid membranes and thylakoid proteins, but in addition, pre-PC and pre­ OEC33 (the 33-kD oxygen-evolving complex protein) require SecA and ATP, the precursors of the oxygen-evolving complex 23- and 17-kD proteins (pre-OEC23 and pre-OEC 17) and the photosystem I but no nucleoside N-subunit protein require a

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thylakoid membrane (Robinson et al., 1994). Another study (Henry et al., 1994) showed that the translocation of an OEC23/PC chimera is not sensitive to sodium azide, an inhibitor of the SecA ATPase, while the translocation of an OEC33/OEC17 chimera is inhibited by azide. From these experiments, it appears that the energetic requirements of a chimeric protein for translocation are determined by the thylakoid transfer signal. In order to determine the region within the thylakoid signal sequence that specifies the required energetics, chimeras were made within this region (Henry et al., 1997). A protein construct consisting of the charged region of the OEC23 lumen targeting domain followed by the hydrophobic core of the PC lumen targeting domain and the mature PC sequence was able to cross the thylakoid membrane efficiently, andthis translocation and competitively was dependent upon ATP and inhibited by an OEC23 translocation intermediate. Thus, this ‘dual-targeting’ signal sequence resulted in a combination of energetic requirements. However, the flexibility and function of chimeric thylakoid signal sequences is limited: a chimera consisting of the charged region of the PC signal fused to the hydrophobic core of OEC23 and the mature sequence of PC was not translocated, and the ‘dual-targeting’ signal used for PC allowed only dependent transport when OEC23 and OEC17 were used as passenger proteins. In contrast, pre-LHCP does not have a simple cleaved thylakoid membrane-targeting signal sequence (Auchincloss et al., 1992, Huang et al. 1992). Some information resides in the mature protein because correct insertion requires multiple trans­ membrane helices. Collectively, the energetics data have been interpreted to indicate the presence of multiple pathways of translocation across the thylakoid membrane. However, the results are also consistent with there being a variety of requirements necessary for different precursors to interact with the components of just one pathway. The truth may lie somewhere between these two extremes and in the identification of the transport apparatus. Homologues of both the bacterial SecA and SecY proteins that mediate translocation across the bacterial inner membrane (Schatz and Dobberstein, 1996) have been identified in vascular plant and algal chloroplasts and cyanobacteria (Nakai et al., 1992, 1994; Laidler et al., 1995). A SecA requirement for translocation in vitro has been demonstrated for PC, cytochrome f(Cyt f) and OEC33 (Nakai et al., 1994;

Yuan et al., 1994; Nohara et al., 1996). As SecY of bacterial membranes and its mammalian homologue, Sec61p of the ER, are protein translocases, it is assumed that chloroplast SecY has a similar function. One series of studies so far has established that indeed SecA has a role in the thylakoid translocation apparatus in vivo in vascular plants. Several nuclear mutants ofmaize are impaired in the translocation of a number of thylakoid proteins in a way that is entirely consistent with the models supported by the in vitro experiments with pea chloroplasts. The tha1 mutant has a transposon-disruption of the chloroplast SecA, and it has reduced levels of properly localized PC, OEC33, PSI-F, and Cyt f while the translocation of OEC23 and OEC17 is unaffected. These results suggest that SecA can function in the translocation of some proteins across the thylakoid membrane in vivo, but that it is not strictly required for the translocation of these proteins (Voelker et al., 1997). The hcf106 mutant has the opposite translocation phenotype of tha1. It has reduced levels of properly localized OEC23 and OEC17, while the levels of PC, PSI-F, and Cyt f are reported to be unaffected (Voelker and Barkan, 1995). However, in some cases Cyt f is reduced in the hcf106 mutant (R. Martienssen, personal communication), suggesting that Cyt f pathway. translocation is influenced by the Recent crosslinking experiments suggest that CP54 can associate with LHCP, Cyt f and the Rieske FeS protein (High et al., 1997), and these were proteins thought to be on separate pathways defined by energetics. Thus, the multiple pathways may indeed involve overlapping sets of components. Consistent with the view that different precursors may interact with similar translocation apparatuses are the results obtained from in vivo studies of the translocation of C. reinhardtii Cyt f into the thyla– koid lumen (Smith and Kohorn, 1994). Mutations within the 31 amino acid signal sequence of chloroplast encoded pre-apo Cyt f (Fig. 2) reduce or eliminate its translocation into the thylakoid membrane, causing the cells to become nonphotosynthetic. Point mutations (A15E and V16D) and deletions (Fig. 2) in the predicted hydrophobic core of the signal sequence had the most pronounced effects and other mutations delineated the boundaries of this essential region (Smith and Kohorn, 1994; Baillet and Kohorn, 1996) The signal sequence mutation A15 E inhibits not only the insertion ofpreCyt f but also decreases the accumulation of integral thylakoid membrane proteins LHCP and D1

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(Fig. 3A). The accumulation of PC and OEC 33, which are lumenal, is not affected (Fig. 3 A). OEC 17 and 23, lumenal proteins having different energetic requirements for in vitro translocation, are increased in the A15E strain (Fig. 3A). As expected, pre-Cyt f A15E also reduces the translocation of wild type pre-

Cyt f (Smith and Kohorn, 1994). Since the Cyt f A15E mutant is found as a precursor in the stroma and thylakoid membrane surface, it may be jamming the translocation apparatus. Thus, precursors using the same path would also be jammed while those on alternative routes would not be affected (we call this

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the Los Angeles effect). This is diagrammed in Fig. 3B. The decrease in LHCP levels suggests that Cyt f A15E blocks a common step, while the increase in OEC 23 is consistent with the view that it enters the pathway after the pre-Cyt f A15E induced block. This implies that components of the translocation apparatus may be limiting. The steady state level of PC is not affected by Cyt f A15E, and thus PC may have different requirements than do Cyt f and LHCP. Some studies show that Cyt f and PC are assisted by SecA (Voelker and Barkan, 1995; Voelker et al., 1997), but if SecA is not limiting, then Cyt f A15E may not be expected to affect PC. These data collectively suggest that integral thylakoid proteins and lumenal proteins use distinct translocation apparatuses, but these pathways may have common components (Smith and Kohorn, 1994). The changes in steady state levels of other thylakoid proteins may indicate that those precursors utilize the same apparatus, but small changes in other protein levels may go undetected, and thus, this analysis is not comprehensive. The experiments on the translocation of pre-Cyt f in C. reinhardtii are consistent with the early microscopic observations of ribosomes on the thylakoid surface which suggests that translocation of some thylakoid proteins is co-translational (Falk, 1969; Margulies and Michaels, 1974). Processing of Cyt f occurs in the thylakoid lumen and only those proteins being translocated will be processed. Therefore, those that are stromal and not processed are necessarily blocked from entry into the lumen. Precursor Cyt f is never detected in wild type cells, and could only be detected in mutants defective in translocation (Smith and Kohorn, 1994). These data are consistent with co-translational translocation of Cyt f.

ability to grow photosynthetically (Smith and Kohorn, 1994; see above, Section IV). Mutations that suppressed the Cyt f signal sequence alterations were selected for their ability to restore photosynthetic growth (Fig. 4). Suppressor mutations were identified as these were thought to represent gain of function alleles, and the generation of null mutations in the translocation process might be lethal. This scheme was based on a genetic analysis of protein translocation in E. coli (Emr et al., 1981). It was expected that some ofthe suppressors would reveal proteins that were involved in the translocation of Cyt f and perhaps interact with the signal sequence. The suppressor mutations map to six nuclear loci and therefore represent extragenic suppressors of the signal sequence mutations in Cyt f (Smith and Kohorn, 1994; Bernd and Kohorn, 1998). These loci defined by the suppressor mutations have been termed TIP for thylakoid insertion proteins. Two suppressors that lie within the Cyt f signal were also isolated (Smith and Kohorn, 1994; Baillet and Kohorn, 1996).

A. Chloroplast Suppressors V. Mutations Affecting Translocation To gain an understanding of thylakoid translocation in vivo, and to produce a selection scheme aimed at identifying proteins that are directly involved in the process, C. reinhardtii was used for a systematic characterization of the Cyt f thylakoid signal se­ quence and genetic analysis of the translocation mechanism of this protein. Strains carrying point and deletion mutations of the Cyt f signal sequence were unable to translocate pre-Cyt f into the thyla­ koid membrane, and this resulted in an impaired

The two chloroplast suppressor mutations result in the removal ofthe positively charged amino acid that borders the amino-terminus of the signal sequence hydrophobic core, and replaces this arginine with either a cysteine or a leucine (Fig. 5). The original mutations at A15E and V16D are unchanged. The occurrence of this type of suppressor mutation suggests that the hydrophobic core can be shifted in position within the signal sequence. One of the two isolates of the V16D suppressor strain was used for site-directed mutagenesis to create a triple mutant, in which the hydrophobic core of the signal sequence

Chapter 13 Chloroplast Protein Translocation

was further disrupted by a charged residue, A12E, which, when alone, has no phenotype. The R10L substitution is no longer capable of restoring photosynthetic growth in this triple mutant in a context of insufficient stretches of hydrophobic core regions (Fig. 5; Baillet and Kohorn, 1996). Thus, the signal that mediates translocation into the thylakoid membrane is characterized by a hydrophobic region whose exact amino acid content is not critical, but must be a minimum length of residues. Furthermore, the hydrophobic region of the signal sequence does not have to be flanked on its amino terminus by a charged residue.

B. Nuclear Suppressors Genetic analysis reveals six unlinked loci that were selected for their suppression of the Cyt f A15E mutation. The tip suppressors allow translocation of both wild-type thylakoid proteins and Cyt f A15E, indicating that they are mutations that make the translocation machinery more permissive. Thus it is not surprising that the tip mutants show no detectable change in photosynthetic growth in a wild-type Cyt f background, relative to the A15E Cyt f strain (Bernd and Kohorn, 1998). Wild-type Cyt f is processed from a 3 5-kD stromal precursor to the mature 32-kD thylakoid membrane form by a lumenal peptidase (reviewed in Gray, 1992). Pulse-chase experiments that detect Cyt f processing revealed that the kinetics of Cyt f maturation in the tip strains ranges from rates indistinguishable from wild type to rates where the precursor of Cyt f accumulates and is detected only after relatively long chase periods (Bernd and Kohorn, 1998). As processing is a measure of translocation,

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these data confirm that the tip suppressors restore the translocation of mutant Cyt f signal sequences but show that the strength of suppression varies between tip alleles. The six tip alleles were selected for their ability to suppress the A15E mutation, but crosses with C. reinhardtii strains carrying other Cyt f signal sequence mutations show that the tip mutants can also suppress these, albeit to differing degrees (Table 1). Thus the suppressor mutations are not allele specific. The original expectation of this selection scheme was that a given suppressor would compensate for a specific signal mutation. However, as the tip suppressors can also suppress the phenotype of strains carrying a deletion of the hydrophobic core of the signal, the data suggests that the suppressors render the thylakoid more permissive to translocation of proteins without a signal or a less than ideal signal. This same observation is made for similar selection schemes in bacteria (Emr et al., 1981). Significantly, improperly localized proteins are not found in the thylakoid membrane nor in bacterial membranes in these permissive mutants. Double tip mutant analysis indicates that a tip4/ tip5 strain is not photosynthetic under stringent conditions of selection, while all other combinations of tip mutations have no affect larger than the single alleles. Thus the proteins encoded by TIP4 and TIP5 may interact. The tip5 mutation, and to a lesser degree tip2, can suppress V16D and and the V16D and mutations block the signal sequence from binding the thylakoids (Smith and Kohorn, 1994). Thus we propose that Tip4, TipS and Tip2 act early in the translocation pathway and this is shown in Fig. 6. Mutations in Tip 1, 3 and 6 might not

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suppress V16D as this altered signal sequence may never reach the later steps mediated by these proteins. The A15E mutation blocks the Cyt f precursor at the membrane and mutations in Tip1, 3, and 6 can therefore suppress this mutation.The tip suppressor alleles are being cloned by transformation of the Cyt f A15E strain with a library, and characterization of these loci should clarify some of these issues. The TIP loci may well describe SecA and SecY homologues, but the identity of the other four is unknown. The thylakoid membrane contains a number of lipids unique to the chloroplast, but their role in protein translocation has yet to be studied (Kouranov and Schnell, 1996). These lipids help to determine the shape of the flattened thylakoid structure (Janero and Barrnett, 1981), and perhaps an alteration in the structure of the lipids by a translocation protein apparatus could prepare the bilayer for the translocating protein. This model is still consistent with the various energetic requirements that are dependent upon the type of signal rather than the mature sequence (Robinson et al., 1994), and with the signal sequence hypothesis that states that only the signal is of importance. Chlamydomonas can also be used to address the question of whether the in vitro energetic experiments truly reflect multiple independent translocation mechanisms as suggested (Robinson and Klosgen, 1994; Cline and Henry, 1996) as Cyt f, PC and OEC23 appear to have different energetic require­ ments for translocation. Null mutations of C. rein­ hardtii PC, OEC33 and OEC23 (ac208, Fud44 and Fud39, respectively) have been isolated (Gorman and Levine, 1966; Mayfield et al., 1987a; de Vitry et al., 1989) and the genes cloned (Mayfield et al., 1987b; Mayfield et al., 1989; Quinn et al., 1993), making the construction and analysis of signal sequence mutants that inhibit their translocation

possible. If there is interaction or sharing between translocation components used by Cyt f and PC or OEC23, then at least some of the tip mutants should suppress inhibitory mutations within those signals. Different tip mutants might be expected to suppress different signal types if there are several pathways that overlap at different points (as proposed in Fig. 3). If the tip suppressor alleles only suppress Cyt f mutations, then new tip mutants specific to PC or OEC33 or OEC23 can be isolated to describe participants in those import processes.

VI. Perspectives It is assumed that the ultimate goal of many researchers is to create a thylakoid translocation system that has been reconstituted from isolated components identified through in vitro or genetic approaches. This would enable a detailed analysis of mechanism. However, these experiments will still leave the question of what occurs in vivo. Hoober has proposed that proteins transversing the envelope do

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not pass through stromal intermediates, and perhaps those intermediates that are detected are rare escapes (Hoober et al., 1994). Those PC, and OEC 33 soluble intermediates detected in C. reinhardtii (Howe and Merchant, 1993) may be species that escape from the general path, or are in vesicles that fractionate with soluble components; the insoluble intermediates remain to be characterized. This alternate view is not generally accepted but is consistent with the large amounts of vesicular traffic and contacts between thylakoid and envelopes observed in chloroplasts active in thylakoid biogenesis (Hoober et al., 1994; Chapter 19, Hoober et al.). Direct transfer oflipophilic proteins such as the 22-kD thylakoid heat-shock protein and LHCP, which do not contain simple thylakoid signal sequences (Grimm et al., 1989; Auchincloss et al., 1992), from the envelope to the thylakoid membrane would obviate the need in vivo for stromal proteins such as CP54. Alternatively, CP54 may mediate envelope to thylakoid transfer. While most in vitro experiments are clear and concise and support the concept of multifunctional signal sequences that produce stromal intermediates, these alternate ideas remain to be tested. C. reinhardtii may well be best suited to determine the translocation pathways involved in intact cells.

with mutant signal sequences. Genetics, in press Blobel G and Dobberstein B (1975) Transfer of proteins across membranes. Presence of proteolytically processed and unprocessed nascent immunoglobin light chains on the membrane-bound ribosomes of murine myeloma. J Cell Biol 67: 835–851 Blobel G and Dobberstein B (1977) In vitro synthesis and processing of a putative precursor for the small subunit of ribulose-l,5-bisphosphate carboxylase of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 74: 1082–1085 Blobel G and Sabatini D (1971) Ribosome-membrane interaction in eukaryotic cells. In: Manson LA (ed) Biomembranes, pp 193–195. Plenum Publishing Corporation, New York Chua N-H and Schmidt G (1978) Post-translational transport into intact chloroplasts of a precursor to the small sub-unit of ribulose-l,5-bisphosphate carboxylase. Proc Natl Acad Sci USA 75: 6110–6111 Cline K and Henry R (1996) Import and routing of nucleusencoded chloroplast proteins. Ann Rev of Cell and Devel Biol 12: 1–26 Cline K, Ettinger W and Theg S (1992) Protein-specific energy requirements for protein transport across or into thylakoid membranes. J Biol Chem 267: 2688–2696 de Vitry C, Olive J, Drapier D, Recouvreur M and Wollman F-A (1989) Posttranslational events leading to the assembly of photosystem II protein complex: A study using photosynthesis mutants from C. reinhardtii. J Cell Biol 109: 991–1006 Emr S, Hanley-Way S and Silhavy T (1981) Suppressor mutations that restore export of a protein with a defective signal sequence. Cell 23: 79–88 Falk H (1969) Rough thylakoids: polysomes attached to chloroplast membranes. J Cell Biol 42: 582–587 Franzen L-G and Falk G (1992) Nucleotide sequence of cDNA clones encoding the beta subunit of mitochondrial ATP synthase from the green alga C. reinhardtii: The precursor protein encoded by the cDNA contains both an N-terminal presequence and a C-terminal extension. Plant Mol Biol 19: 771–780 Franzen L, Rochaix J and von Heijne G (1990) Chloroplast transit peptides from the green alga C. reinhardtii share features with both mitochondrial and higher plant chloroplast presequences. FEBS Lett 260: 165–168 Fujiki M and Verner K (1991) Coupling of protein synthesis and mitochondrial import in a homologous yeast in vitro system. J Biol Chem 266: 6841–6847 Fujiki M and Verner K (1993) Coupling of cytosolic protein synthesis and mitochondrial protein import in yeast. Evidence for cotranslational import in vivo. J Biol Chem 268: 1914– 1920 Funke RP, Kovar JL and Weeks DP (1997) Intracellular carbonic anhydrase is essential to photosynthesis in C. reinhardtii at Demonstration via genomic atmospheric levels of mutant ca-1. Plant complementation of the high Physiol 114: 237–244 Gillham N (1994) Organelle genes and genomes. Oxford University Press New York Gorman D and Levine R (1966) Photosynthetic electron transport of C. reinhardtii VI. Electron transport in mutant strains lacking either cytochrome 553 or plastocyanin. Plant Physiol 41: 1648–1656 Gray JC (1992) Cytochrome f: Structure function and biosynthesis. Photosyn Res 34: 359–374

Acknowledgments We would like to thank Phillip Hartzog, Benoit Baillet and Lib Harris for their help in the work presented, which was supported by the Pew Charitable Trusts, the National Institutes of Health, and the United States Department of Agriculture. We have attempted to represent the field fairly, and apologize to any of those that were excluded, as it was by no means intentional.

References Auchincloss A, Alexander A and Kohorn BD (1992) Requirement for three membrane spanning alpha-helices in the post­ translational insertion ofa thylakoid membrane protein. J Biol Chem 267: 10439–10446 Baillet B and Kohorn BD (1996) Hydrophobic core but not amino-terminal charged residues are required for translocation of an integral thylakoid membrane protein in vivo. J Biol Chem 271: 18375–18378 Bernd KK and Kohorn BD (1998) Tip loci: Six Chlamydomonas nuclear suppressors that permit the translocation of proteins

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Grimm B, Ish-Shalom D, Even D, Glaczinski H, Ottersbach P, Ohad I and Kloppstech K (1989) The nuclear-encoded chloroplast 22kD heat-shock protein of C. reinhardtii. Evidence for translocation into the organelle without a processing step. Eur J Biochem 182: 539–46 Hageman J, Baecke C, Ebskamp M, Pilon R, Smeekens S and Weisbeek P (1990) Protein import into and sorting inside the chloroplast are independent processes. Plant Cell 2: 479–494 Henry R, Kapazoglou A, McCaffery M, and Cline K (1994) Differences between lumen targeting domains of chloroplast transit peptides determine pathway specificity for thylakoid transport. J Biol Chem 269: 10189–10192 Henry R, Carrigan M, McCaffery M, Ma X and Cline K (1997) Targeting determinants and proposed evolutionary basis for the Sec and delta pH protein transport systems in chloroplast thylakoid membranes. J Cell Biol 136: 823–832 Harris EH (1989) The C. reinhardtii Sourcebook: A Compre­ hensive Guide to Biology and Laboratory Use. Academic Press, San Diego High S, Henry R, Mould R, Valent Q, Meacock, S, Cline K, Gray J and Luirink J (1997) Chloroplast SRP54 interacts with a specific subset of thylakoid precursor proteins. J Biol Chem 272: 11622–11628 Hoober J, White R, Marks D and Gabriel J (1994) Biogenesis of thylakoid membranes with emphasis on the process in C. reinhardtii. Photosyn Res 39: 15–31 Horniak L, Pilon M, van’t Hof R and de Kruijff B (1993) The secondary structure of the ferredoxin transit sequence is modulated by its interaction with negatively charged lipids. FEBS Lett 334: 241–246 Howe G and Merchant S (1993) Maturation of thylakoid lumen proteins proceeds post-translationally through an intermediate in vivo. Proc Natl Acad Sci USA 90: 1862–1866 Huang L, Adam Z and Hoffman NE (1992) Deletion mutants of chlorophyll a/b binding proteins are efficiently imported into chloroplasts but do not integrate into thylakoid membranes. Plant Physiol 99: 247–255 Hugosson M, Nurani G, Glaser E and Franzen LG (1995) Peculiar properties of the PsaF Photosystem J protein from the green alga C. reinhardtii: Presequence independent import of the PsaF protein into both chloroplasts and mitochondria. Plant Mol Biol 28: 525–535 Hulford A, Hazell L, Mould R and Robinson C (1994) Two distinct mechanisms forthe translocation ofproteins across the thylakoid membrane, one requiring the presence of a stromal protein factor and nucleotide triphosphates. J Biol Chem 269: 3251–3256 Hurt E, Soltanifar N, Goldschmidt-Clermont M, Rochaix JD and Schatz G (1986) The cleavable pre-sequence of an imported chloroplast protein directs attached polypeptides into yeast mitochondria. EMBO J 5: 1343–1350 Jancro DR and Barrnett R (1981) Cellular and thylakoidmembrane phospholipids of C. reinhardtii 137+. J Lipid Res 22:1126–1130 Kirwin P, Meadows J, Shackleton J, Musgrove J, Elderfield P, Hay N and Robinson C (1989) ATP-dependent import of a lumenal protein by isolated thylakoid vesicles. EMBO J 8: 2251–2255 Kohorn BD, Harel E, Chitnis PR, Thornber JP and Tobin EM (1986) Functional and mutational analysis of the lightharvesting chlorophyll a/b protein of thylakoid membranes.

J Cell Biol 102: 972–981 Kouranov A and Schnell D (1996) Protein translocation at the envelope and thylakoid membranes of chloroplasts. J Biol Chem 271: 31009–31012 Laidler V, Chaddock AM, Knott TG, Walker D and Robinson C (1995) A SecY homolog in Arabidopsis thaliana, J Biol Chem 270: 17664–17667 Li X, Henry R, Yuan J, Cline K and Hoffman N (1995) A chloroplast homologue of the signal recognition particle subunit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes. Proc Natl Acad Sci USA 92: 3789–3793 Margulies M and Michaels A (1974) Ribosomes bound to chloroplast membranes in C. reinhardtii. J Cell Biol 60: 65–77 Mayfield S, Bennoun P and Rochaix JD (1987a) Expression of the nuclear encoded OEE1 protein is required for oxygen evolution and stability of Photosystem II particles in C. reinhardtii. EMBO J 6: 313–318 Mayfield S, Rahire M, Frank G, Zuber H and Rochaix JD (1987b) Expression of the nuclear gene encoding oxygenevolving enhancer protein 2 is required for high levels of photosynthetic oxygen evolution in C. reinhardtii. Proc Natl Acad Sci USA 84: 749–753 Mayfield S, Schirmer-Rahire M, Frank G, Zuber H and Rochaix J-D (1989) Analysis of the genes of the OEE1 and OEE3 proteins of the Photosystem II complex from C. reinhardtii. Plant Mol Biol 12: 683–693 Merchant S and Bogorad L (1987) The Cu(II)-repressible plastidic cytochrome c: Cloning and sequence of a complementary DNA for the pre-apoprotein. J Biol Chem 262: 9062–9067 Michl D, Robinson C, Schackleton J, Herrmann R and Klosgen R (1994) Targeting of proteins to the thylakoids by bipartite presequences: CFoll is imported by a novel, third pathway. EMBO J 13: 1310–1317 Mishkind M, Wessler S and Schmidt G (1985) Functional determinants in transit sequences: Import and partial maturation by vascular plant chloroplasts ofthe ribulose-1,5-bisphosphate carboxylase small subunit of C. reinhardtii. J Cell Biol 100: 226–234 Mould R and Robinson C (1991) A proton gradient is required for the transport of two lumenal oxygen-evolving proteins across the thylakoid membrane. J Biol Chem 266: 12189– 12193 Nakai M, Tanaka A, Omata T and Endo T (1992) Cloning and characterization of the secY gene from the cyanobacterium Synechococcus PCC7942. Biochem Biophys Acta 1171:113– 116 Nakai M, Goto A, Nohara T, Sugita D and Endo T (1994) Identification of the SecA protein homolog in pea chloroplasts and its possible involvement in thylakoidal protein import. J Biol Chem 269: 31338–31341 Nielsen V, Mant A, Knoetzel J, Moller B and Robinson C (1994) Import of barley photosystem I subunit N into the thylakoid lumen is mediated by a bipartite presequence lacking an intermediate processing site. J Biol Chem 269: 3762–3766 Nohara T, Asai T, Nakai M, Suguira M, and Endo T (1996) Cytochrome f encoded by the chloroplast genome is imported into thylakoids via the SecA-dependent pathway. Biochem Biophys Res Com 224: 474–478 Quinn J, Li H, Singer J, Morimoto B, Mets L, Kindle K and Merchant S (1993) The plastocyanin-deficient phenotype of

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C. reinhardtii ac-208 results from a frame-shift mutation in the nuclear gene encoding plastocyanin. J Biol Chem 268: 7832– 7841 Reed HS (1942) A Short History of the Plant Science. Chronica Botanica Company, Waltham, MA Robinson C and Klosgen R (1994) Targeting of proteins into and across the thylakoid membrane—a multitude of mechanisms. Plant Mol Biol 26: 15–24 Robinson C, Cai D, Hulford A, Brock I, Michl D, Hazell L, Schmidt I, Herrmann R and Klosgen R (1994) The presequence of a chimeric construct dictates which of two mechanisms are utilized for translocation across the thylakoid membrane: evidence for the existence of two distinct translocation systems. EMBO J 13: 279–285 Rufenacht A and Boschetti A (1995) Specificity of processing enzymes in chloroplasts of C. reinhardtii In: P. Mathis (ed) Photosynthesis: From Light to Biosphere, Vol III, pp 767–770. Kluwer Academic Publishers, Dordrecht Schatz G and Dobberstein B (1996) Common principles of protein translocation across membranes. Science 271: 1519– 1526 Schnarrenberger C, Pelzer-Reith B, Yatsuki H, Freund S, Jacobshagen S and Hori K (1994) Expression and sequence of the only detectable aldolase in C. reinhardtii. Arch Biochem Biophys 313: 173–78 Schnell D (1995) Shedding light on the chloroplast protein import machinery. Cell 83: 521–524 Smeekens S, Bauerle C, Hageman J, Keegstra K and Weisbeek P (1986) The role of the transit peptide in the routing of precursors toward different chloroplast compartments. Cell 46: 365–375 Smith T and Kohorn BD (1994) Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors. J Cell Biol 126: 365–374 Su Q and Boschetti A (1993) Partial purification and properties of enzymes involved in the processing of a chloroplast import protein from C. reinhardtii. Eur J Biochem 217: 1039–1047 Su Q and Boschetti A (1994) Substrate- and species- specific processing enzymes for chloroplast precursor proteins. Biochem J 300: 787–792 Theg SM and Geske FJ (1992) Biophysical characterization of a transit peptide directing chloroplast protein import. Bio­ chemistry 31: 5053–5060 Van den Broeck G, Timko MP, Kausch AP, Cashmore AR, Van Montagu M and Herrera-Estrella L (1985) Targeting of a

foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase. Nature 313: 358–363 van’t Hof R, van Klompenburg W, Pilon M, Kozubek A, de Korte-Kool G, Demel R, Weisbeek PJ and de Kruijff B (1993) The transit sequence mediates the specific interaction of the precursor of ferredoxin with chloroplast envelope membrane lipids. J Biol Chem 268: 4037–4042 Verner K (1993) Co-translational protein import into mito­ chondria: An alternate view. Trends Biochem Sci 18: 366–371 Voelker R and Barkan A (1995) Two nuclear mutations disrupt distinct pathways for targeting proteins to the chloroplast thylakoid. EMBO J 14: 3905–3914 Voelker R, Mendel-Hartvig J and Barkan A(1997)Transposondisruption ofa maize nuclear gene, tha 1 encoding a chloroplast SecA homologue: In vivo role of cp-SecA in thylakoid protein targeting. Genetics 145: 467–478 von Heijne G, Steppuhn J and Herrmann R (1989) Domain structure of mitochondrial and chloroplast targeting peptides. EurJ Biochem 180: 535–545 von Heijne G, Hirai T, Klosgen RB, Steppuhn J, Bruce B, Keegstra K, and Herrmann R (1991) CHLPEP—A database of chloroplast transit peptides. PMB Reporter 9: 104–126 Wan J, Blakely SD, Dennis DT and Ko K (1995) Import characteristics of a leucoplast pyruvate kinase are influenced by a 19-amino-acid domain within the protein. J Biol Chem 270: 16731–16739 Whelan J and Glaser E (1997) Protein import into plant mitochondria. Plant Mol Biol 33: 771–789 Wood PM (1978) Interchangeable copper and iron proteins in algal photosynthesis. Studies on plastocyanin and cytochrome c-552 in C. reinhardtii. Eur J Biochem 87: 9–19 Yu LM, Merchant S, Theg SM and Selman BR (1988) Isolation of a cDNA for the gamma subunit of the chloroplast ATP synthase of C. reinhardtii import and cleavage of the precursor protein. Proc Natl Acad Sci USA 85: 1369–1373 Yuan J and Cline K (1994) Plastocyanin and the 33-kD a subunit of the oxygen-evolving complex are transported into thylakoids with similar requirements as predicted from path way specificity. J Biol Chem 269: 18463–18467 Yuan J, Henry R, McCaffery M and Cline K (1994) SecA homolog in protein transport within chloroplasts: Evidence for endosymbiont-derived sorting. Science 266: 796–798

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Chapter 14

Supramolecular Organization of the Chloroplast and of the Thylakoid Membranes Jacqueline Olive

Institut Jacques Monod, CNRS/Université Denis Diderot,

2 Place Jussieu, 75251 Paris Cedex, France

Francis-André Wollman

Laboratoire de Photosynthèse, Institut de Biologie Physico-chimique,

13 rue Pierre et Marie Curie, 75005 Paris, France

Summary I. Introduction II. Cell and Chloroplast Morphology A. Cell B. Chloroplast 1. Envelope 2. The Eyespot 3. The Pyrenoid 4. Thylakoid Membranes III. Ultrastructural Organization of Thylakoid Membranes A. Lateral Distribution of Membrane Components 1. PS II Reaction Centers 2. PS I Reaction Center and its Peripheral Antenna 3. Light-Harvesting Complex of PS II 4. Cytochrome Complex 5. ATP Synthase 6. The Static Picture B. Transmembrane Organization IV. Dynamic Aspects of Thylakoid Membrane Organization A. The Stacking-Destacking Process B. State Transition: Phosphorylation-Dependent Changes C. Fusion of Thylakoid Membranes During Sexual Reproduction V. Biogenesis A. Greening: Synthesis of Membrane Complexes B. Assembly of PS II VI. Conclusion Acknowledgment References

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J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 233–254. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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Jacqueline Olive and Francis-André Wollman

Summary From a comparative ultrastructural analysis of the wild-type and mutant strains of Chlamydomonas reinhardtii, it has been established that the complexes involved in photosynthesis are heterogenously distributed between stacked and unstacked membranes: PS II centers and their peripheral antenna are essentially localized in stacked regions of the membranes, whereas PS I centers, their peripheral antenna and ATP synthase are complex is distributed in both membrane domains. exclusively located in unstacked areas; the cytochrome Each of these protein complexes can be visualized by freeze-fracture as individual particles, but they can also be found in association with other proteins: in particular, the peripheral antennae are, in part, associated with their corresponding reaction centers to form large PFu or EFs particles. It has also been suggested that some Cyt complexes may be associated within the same particle with PS I or PS II reaction centers. Another important characteristic of intramembrane complexes is their lateral mobility. Destacking of the membranes by removal of divalent cations induces a random redistribution of the proteins in the membrane. State transitions also cause complexes, along a lateral displacement of the mobile part of the PS II peripheral antenna, and of some Cyt the thylakoid membranes. Fusion of thylakoid membranes from two distinct gametes, upon zygote formation, allows their photosynthetic protein complexes to mix. The identification, as authentic photosynthetic complexes, of a large proportion of the intramembrane thylakoid particles permitted biogenesis studies at the ultrastructural level. Here we discuss the assembly of the Photosystem II complexes.

I. Introduction For two decades, the supramolecular organization of the thylakoid membranes has been the subject of intensive investigations aimed at the identification of substructures corresponding to individual protein complexes and at the description of their spatial and functional integration in the photosynthetic apparatus (for reviews, see Staehelin, 1986; Gantt, 1994; Mustárdy, 1996; Staehelin and van der Staay, 1996). In the chloroplasts of green algae and vascular plants, intramembrane complexes located in specialized membrane discs, the thylakoids, carry out a series of interrelated photochemical and redox reactions by which light energy is trapped and converted into utilizable chemical energy. The complexes involved in the photosynthetic process are the two photosystems, Photosystem I (PS I) and complex, Photosystem II (PS II), a cytochrome operating between PS II and PS I in an electron to nicotinamide-adeninetransfer chain from dinucleotide phosphate and an adenosine triphosphate (ATP) synthase, utilizing the gradient Abbreviations: CPo – chlorophyll-protein complex of Photosystem I antenna; – chlorophyll-protein complex I ; OEE – oxygen evolving enhancer; EFs – stacked external face; EFu – unstacked external face; PS I – Photosystem I reaction center; PS II – Photosystem II reaction center; PFs – stacked protoplasmic face; PFu – unstacked protoplasmic face; RCI – reaction center I; RuBP – ribulose bisphosphate; RuBisCO – ribulose bisphosphate carboxylase/oxygenase

generated by the electron transfer chain to produce ATP. The four protein complexes are supramolecular assemblies of polypeptide chains, pigments and/or electron carriers. In addition, two small electron carriers, plastoquinones and plastocyanin, are located respectively within the hydrophobic core of the membrane and in a soluble form in the thylakoid lumen. The first thin sections of Chlamydomonas spp. were observed by electron microscopy by Sager and Palade (1954). They reported the presence ofa regular laminated organization in the chloroplast, corres­ ponding to thylakoid membranes. Further progress in the knowledge of the ultrastructure of membranes came with the development of the freeze-fracture technique. For this technique, the biological material is frozen at very low temperature (–196 °C) and Torr). fractured under high vacuum conditions ( The membranes of the cells split along an interior plane, exposing face views of the hydrophobic interior of the lipid bilayer. Particles, which are correlated to intramembranous complexes are visualized by shadowing with platinum and carbon layers (Branton, 1966). For Chlamydomonas spp. (Goodenough and Levine, 1969; Goodenough and Staehelin, 1971; Ojakian and Satir, 1974) as well as for vascular plants (Armond and Arntzen, 1977; Armond et al., 1977), distinct membrane domains were described. They were attributed to stacked and unstacked membranes respectively, and were characterized by their content in various classes of particles which

Chapter 14

Supramolecular Organization of Chloroplast and Thylakoid Membranes

differed in size and density. One of the main goals of the freeze-fracture study of thylakoid membranes has been the identification of the different categories of intramembrane particles. For that purpose, research with Chlamydomonas spp. has been of primary importance because of the numerous mutants that were characterized as specifically lacking one type of photosynthetic complex. Thus a correlation could be drawn between the loss of a category of intramembrane particles and the absence of a specific set of polypeptides, corresponding to the different subunits of an oligomeric protein. More recently, the biochemical characterization of the subunits of photosynthetic protein complexes and significant progress in their purification has opened the way to the preparation of specific antibodies. This led to the development of an immunocytochemical approach. For this technique, the cells are embedded at low temperature in a hydrophilic resin (Lowicryl K4M) which preserves the antigenicity of the molecules. Labeling with the antigens is performed on thin sections so that antibodies can access antigens. The bound antibodies are revealed by electron-dense colloidal gold particles, coupled either to protein A or to anti-rabbit IgG antibodies (Carlemalm et al., 1982). This technique provided direct detection ofthe protein complexes in the thylakoid membrane domains. Critical to that technique is the estimation of the proportion of background labeling. The availability of Chlamy­ domonas mutants totally devoid of an antigen has been a most valuable tool to assess the significance of the labeling with the corresponding antibody in the wild type strain.

II. Cell and Chloroplast Morphology

A. Cell There are many species in the Chlamydomonas genus, but mainly four species are used experimentally: C. reinhardtii, C. eugametos, C. moewusii and C. monoica (Harris, 1989). Moreover, the ultra­ structural organization of the thylakoid membranes has been extensively characterized only in C. rein­ hardtii. This is largely due to the availability of a great number of mutant strains of this species. C. reinhardtii was described for the first time by Dangeard (1888). Early descriptions of its architecture were derived from electron micrographs obtained by

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Sager and Palade (1954). It is an ovoid cell, in diameter (Figs. 1A and B). It is delimited by a plasma membrane surrounded by a glycoprotein cell wall. It has a polar structure with two anterior flagella and a single basal Chloroplast that may partially surround the nucleus. The two flagella, each about 10 long, arise from a basal body (Fig. 1C). The

236 chloroplast bounded by a double envelope membrane contains numerous lamellae, the thylakoids, and two typical substructures: the pyrenoid surrounded by starch-containing bodies (Fig. 1B) and the eyespot located just beneath the chloroplast envelope membrane (Fig. 1D). Like other eucaryotic cells, Chlamydomonas spp. cells contain a nucleus, mitochondria, an endoplasmic reticulum and a Golgi apparatus. The cell wall described in a thin section study (Roberts et al., 1972) has also been analyzed by the quick-freeze, deep-etch technique (Goode­ nough and Heuser, 1985). The major wall constituents are hydroxy-proline-rich glycoproteins, with arabinose, mannose, galactose and glucose as the predominant sugars in C. reinhardtii. The intact wall has been shown to consist of a discrete central triplet bisecting a meshwork formed of a distinct set of components. Other species of Chlamydomonas vary in cell shape, thickness of the cell wall or vary in the number of pyrenoids (for a review, see Ettl, 1976).

B. Chloroplast In C. reinhardtii, the chloroplast appears as a highly organized body, with a U-shape profile which occupies about 40% of the volume of the cell (Schötz et al. 1972). It is surrounded by an envelope and contains not only stacks of lamellae which are the site of photosynthesis but also, localized in the stroma, differentiated regions such as the eyespot associated with phototaxis and the pyrenoid associated with starch synthesis and storage, and with accumulation of ribulose-1 -5 bisphosphate carboxylase/oxygenase (Rubisco).

1. Envelope The algal chloroplasts, like those of vascular plants, are delimited by a basic envelope consisting of two parallel membranes, the inner and outer envelope membranes, each with a distinct set of polypeptides. The inner envelope membrane regulates the transport of metabolites into and out of the chloroplast, whereas the outer envelope is highly permeable to many low molecular weight substances. The chloroplast envelope is the site where the organelle interacts with other cellular components. The envelope not only regulates the transport of metabolites between the stroma and the cytosol but also mediates the import of nucleus-encoded chloroplast proteins. Most

Jacqueline Olive and Francis-André Wollman of these are synthesized as precursor proteins with N-terminal extensions, the transit peptides. These transit peptides are necessary and sufficient to direct the import of proteins into the chloroplast. The envelope also plays a major role in the biosynthesis of various lipids including galactolipids, the predominant lipids in chloroplast membranes (Douce and Joyard, 1990; see also Chapter 21, Trémolières). The inner membrane has been shown to have a higher protein to lipid ratio (Douce and Joyard, 1990). Consistent with this result, freezefractured cells show that the inner envelope contains five to ten times more intramembranous particles than the outer envelope membrane (Fig. 2, A and B). As suggested by Staehelin (1986), patches of large particles within the outer envelope membrane make contact with the inner envelope membrane.

2. The Eyespot In C. reinhardtii, the eyespot is found in the chloroplast, midway between the anterior and posterior regions of the cell (Sager and Palade, 1957; Goodenough et al., 1969;Gruber and Rosario, 1974; Melkonian and Robenek, 1980). The eyespot lies directly beneath the chloroplast envelope and consists of plates of large dense granules closely packed together (Figs. 1D and 3A). There is an alternating arrangement of plates and discs. Each plate is composed of a single layer of spherical, uniform bodies, about 100 to 140 nm in diameter (Sager and Palade, 1957). The number of rows of granules is constant for most of the species but can differ from one species to another. The eyespot of C. eugametos possesses only one layer of granules whereas that of C. reinhardtii consists of three or four layers (Lembi and Lang, 1965). The presence of microtubules has been described in the eyespot region but, in the green algae, the eyespot and flagella are separated by a fairly broad expanse cytoplasmic domain (Gruber and Rosario, 1974) and a direct association between the two organelles seems to be unlikely (Walne and Arnott, 1967). Freeze-fracture studies show that the plasmalemma and the outer envelope membranes overlying the eyespot contain a greater number of intramembrane particles than do other areas of both membranes (Nakamura et al., 1973; Bray et al., 1974; Melkonian and Robenek, 1980) (Fig. 3B). It has been shown that the eyespots in all flagellated algae act as a photoreceptor for phototaxis. It has

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been proposed that the eyespot could act as a shading device with the photoreceptor sites lying in the membrane adjacent to the eyespot globules (Arnott and Brown, 1967) or that it could reflect and intensify light of a specific spectral range (Foster and Smyth, 1980).

3. The Pyrenoid The pyrenoid is a differentiated region of the chloroplast stroma of many eucaryotic algae. It is a (Sager spherical body with a diameter of 1.5 to and Palade, 1957; Gibbs, 1962a,b; Griffiths, 1980). It appears in thin sections as uniformly dense (Fig. 1B). Pyrenoids have been isolated from C. reinhardtii and from several species of algae (Holdsworth, 1971; Kerby and Evans, 1978; Sato et al., 1984; Kuchitsu et al., 1988), and their main component was shown to be Rubisco . The functional significance of pyrenoids is controversial. Due to their close spatial relationship with starch granules, a role in starch synthesis has been proposed (Sager and Palade, 1957). Griffiths (1980) and Sato et al. (1984) assumed that the pyrenoid functions as a reservoir of Rubisco protein

whereas Kuchitsu et al. (1988) suggested that RuBP carboxylase is active in the pyrenoid and shows distinct kinetic characteristics. The pyrenoid might function as a specific metabolic compartment providing efficient coupling between carbon fixation and starch metabolism.

4. Thylakoid Membranes The basic structural components of the chloroplast are membranes. They are organized into very long, flat vesicles, called discs (Sager and Palade, 1957; Gibbs, 1962a; Ohad et al., 1967a; Goodenough and Levine, 1969). The discs, in turn, are appressed to one another in such a way as to form an elaborate array of stacked membranes. In thin sections, the membranes inside the wild-type chloroplast are seen either as single discs, or more frequently in stacks of two to ten discs (Fig. 4). A given disc may be

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associated in some places with one stack and in other places with another stack but the length of the intervening unstacked disc region is rarely extensive. In this respect it differs widely from the chloroplast membrane system of vascular plants, in which short, uniform segments of many stacked discs (grana) are interconnected by a network of long, single discs (Staehelin et al., 1977). All components of the photosynthetic electron transport chain are known to be associated with the thylakoid membranes. The freeze-fracture technique, which allows visualization of intramembrane complexes, had been utilized early on to describe the organization of thylakoid membranes. The first pictures of freeze-fractured membranes of C. rein­ hardtii were obtained by Goodenough and Staehelin (1971) and by Ojakian and Satir (1974). In the same period, similar studies were conducted with Euglena gracilis (Miller and Staehelin, 1973) and vascular plant chloroplasts (Sjolung and Smith, 1974; Staehelin, 1975). The two complementary hemi­ membranes, the exoplasmic (EF) and protoplasmic (PF) layers have a different organization in appressed and non-appressed regions, giving rise to four

Jacqueline Olive and Francis-André Wollman

different types of fracture faces: EFs and PFs originate from the stacked regions while EFu and PFu originate from the unstacked membrane domains (Fig. 5). The EF hemi-membrane is facing the exterior of the chloroplast, while the protoplasmic one is facing the lumen. Each fracture face was characterized by classes of particles differing in size and density (Fig. 6 and Table 1). On these grounds a search for the proteins constituting these particles was started, and this search lasted for over two decades. With the help of many Chlamydomonas mutant strains, most of the classes of intramembrane particles were correlated with a defined protein complex. From this supramolecular mapping study has emerged the description of extensive lateral heterogeneity in the distribution of the photosynthetic complexes along the thylakoid membranes. Given this knowledge, it became possible to study how controlled changes in the supramolecular organization of the membranes altered photosynthetic functions.

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III. Ultrastructural Organization of Thylakoid Membranes

A. Lateral Distribution of Membrane Components

1. PS II Reaction Centers The most spectacular correlation between intramembrane particles and protein complexes came with the identification of the PS II-associated particles. In mutant strains of Chlamydomonas devoid of PS II activity, a dramatic loss in EF particles was observed (Olive et al., 1979; Wollman et al., 1980; Olive et al., 1992). The EF particle density decreased in the wild type to 350 from 1450 particles particles in the tbc1-F34 mutant (Fig. 7 A and B) (Wollman et al., 1980). The tbc1-F34 strain lacks the PS II polypeptide P6 (CP43) because the psb C mRNA cannot be translated owing to a mutation in a nuclear regulatory gene (Rochaix et al., 1989) and, as in most mutants defective in PS II primary photoactivity, the whole set of subunits engaged in

the formation of the PS II protein complex is not accumulated in the membranes (Delepelaire, 1984; Bennoun et al., 1986). Moreover, in a partly suppressed strain, F34SU3, which showed restoration of half of the PS II activity, the EF particle density was found to be intermediate between those in the (Wollman et WT and tbc1-F34 mutant al., 1980). In the chloroplast mutant FuD34, which is also devoid of the P6 polypeptide owing to an alteration in the psbC gene (Rochaix et al., 1989), the EF particle density was calculated in both the stacked and unstacked membrane domains (Olive et al., 1992). A decrease in EF particle density was observed in

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Jacqueline Olive and Francis-André Wollman subunits of the PS II reaction center or against the extrinsic oxygen evolving enhancer (OEE) subunits showed that most of the labeling was concentrated in the stacked membrane domains (Vallon et al., 1985). This is illustrated for the labeling of the PS II core antenna subunit P5 (CP47) in Fig. 8A and Table 2. For the various components of the PS II reaction center, 10–20% of the labeling was found in unstacked regions. Similar distribution between stacked and unstacked membranes was also obtained for spinach (a barley (Vallon et al., 1985) whereas for mutant incapable of synthesizing D1), the traces of CP43 (P6) and CP47 (P5) found in this mutant were not concentrated into stacked regions (Simpson et of cytochrome al., 1989). However, the was found at a near normal level and accumulated in stacked regions. The results obtained for C. reinhardtii or spinach were in accordance with numerous fractionation studies, performed with vascular plant thylakoid membranes which indicated a pronounced depletion of PS II in stroma lamellae (Andersson and Anderson, 1980; Andersson and Melis, 1983; Vallon et al., 1987;Bassi et al., 1988).

both membrane domains, which argues for the presence of some PS II reaction centers, not only in the stacked membrane regions where most of the EF particles in the wild-type strain are found but also in the unstacked membrane regions: 533 and 243 p in EFs and EFu respectively versus 1500 and 600 p in wild type. Similar results were observed with another green alga, Chlorella sorokiniana (Lacambra et al., 1984). Taking into account the ratio of unstacked versus stacked membranes, the decreased content in EF particles in the PS II mutants led to an estimate of about 20% of the PS II centers present in the unstacked regions (Olive et al, 1992). From studies in which a double mutant more completely devoid of PS II subunits (Section V.B) was used, it was concluded that PS II centers appear to be responsible for about 95% of the EF particles. Several studies with PS II mutants from vascular plants concluded an even higher heterogeneity in the distribution of PS II, with only minor changes on the EFu faces when there was a considerable drop in the density of the EFs particles (Miller and Cushman, 1979; Simpson et al., 1989). The immunocytochemical approach also supported the heterogeneity in lateral distribution of PS II between the stacked and unstacked domains. Studies using various antibodies against the main integral

2. PS I Reaction Center and its Peripheral Antenna In the freeze-fractured membranes, the PS I reaction center and its peripheral antenna were expected to correspond to a subpopulation of the PF particles, and ATP the other particles corresponding to Cyt synthase. Since, in contrast with PS II mutant strains, neither mutation actually caused the disappearance of only the PS I complex or its peripheral antenna, both complexes were treated together. A comparative freeze-fracture study was conducted with the F14 and Ac40 mutants. The former strain is which contains the PS I primary donor devoid of P700 and the PS I core antenna, and the latter strain which corresponds to the two complexes lacks LHCI-705 and LHCI-680 forming the peripheral antenna of PS I in C. reinhardtii (Wollman and Bennoun, 1982; Ish-Shalom and Ohad, 1983). These two complexes from Chlamydomonas have a very similar polypeptide composition although they are different with respect to Chl a/b and Chl/carotenoid ratio (Bassi et al., 1992). This is in clear contrast to vascular plants where LHCI-730 and LHCI-680 were shown not to have common polypeptides (Bassi et al., 1987).

Chapter 14 Supramolecular Organization of Chloroplast and Thylakoid Membranes

Most of the PF particles above 11 nm in size, which are mostly found on PFu in the wild type, were or complex was lacking. missing when the As a result, PFu and PFs faces could no longer be distinguished on the basis of particle size. The overall particle density on the PF faces of the mutants was similar to that of the pooled PF faces of WT (6800 in the mutant versus 5830 in WT) particles (Olive et al., 1983). It was suggested that the large PFu particles in WT resulted from the association of the two protein complexes. Size measurements on freeze-fracture replicas of isolated PS I complexes

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from pea, containing, in addition to PSI polypeptides, 110 chlorophylls/P700, reconstituted into artificial phospholipid liposomes, led to particles 11 nm in diameter (Mullet et al., 1980). A similar location for PS I reaction centers on the PFu faces in unstacked membrane domains was reported in maize (Miller, 1980) and in barley (Simpson, 1982), based on comparative studies with mutants lacking the PS I complex. In part because of the difficulty in raising antibodies to C. reinhardtii reaction centers, immunocyto­ chemical localization of PS I has proved difficult.

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Using an immunocytochemical approach with an antibody to a spinach reaction center I (RCI) protein which cross-reacted with that of C. reinhardtii, Vallon et al. (1986) observed that most of the label was found over unstacked membranes (Fig. 8B). The weak cross-reaction observed in the wild-type thin sections was proved significant because a control strain, the C3 mutant, totally devoid of the antigen, showed no significant labeling. In contrast, the same workers observed a strong reaction with spinach thylakoid membranes. It confirmed the localization of PS I reaction centers in unstacked membranes. Similar conclusions were derived from phase partition studies with spinach thylakoid membranes (Anders­ son and Hähnel, 1982).

3. Light-Harvesting Complex of PS II As discussed in the section on light-harvesting complexes (Chapter 19, Hoober et al.), the green algae possess Chl a and Chl b, carotenes and xanthophylls linked to a few specific proteins. The light harvesting proteins maintain high concentrations of pigments in a suitable orientation for maximal energy transfer to the reaction centers. Their supramolecular organization is also aimed at protecting the photosynthetic membranes from photodamage. The pigment-protein complexes are embedded in the membrane as components of the supramolecular protein complexes of PS II and PS I. According to the classification derived from studies with vascular plant chloroplasts, C. reinhardtii possesses three types of peripheral antenna complexes, the ‘minor’ antenna complexes, CP24, CP26, CP29, (Bassi and Wollman, 1991), the LHCII

Jacqueline Olive and Francis-André Wollman and LHCI antenna complexes (Delepelaire and Chua, 1981). Antenna complexes from the first and third categories participate respectively to PS II and PS I light harvesting processes. The function of LHCII has been considerably reassessed in the past decade. Originally presented as a major PS II antenna complex, at a time where the existence of the minor antenna proteins was not yet recognized, it is nowadays considered, at least in C. reinhardtii, as a regulating antenna which may associate with PS II as well as PS I depending whether the algae are in state 1 or in state 2 (Delosme et al., 1995; Chapter 30, Ohad). It follows that one may be somewhat confused in reading the original papers on freeze-fractured thylakoid membranes, because of the systematic use of LHC (or LHCII) for some particles which are more likely to correspond to the minor peripheral antenna complexes. We have tried, in the presentation below, to replace the conclusions of these older studies with our more recent knowledge. Greening studies with chloroplasts from vascular plants showed that the variability in EF particle size was, in part, due to the association of PS II core complexes with a variable amount of Chl a/b proteins (Armondetal., 1977). In wild-type Chlamydomonas, the EFs particles have widely different sizes, from 8 to 20 nm, while the EFu particles show a narrower distribution, in the smaller size range, from 6 to 15 nm (Fig. 6). This suggests that PS II centers in nonappressed regions have a smaller antenna, as has been shown in vascular plants. In PS II mutants, the PFs particle density increased upon disappearance of most of the PS II-core containing EFs particles (Wollman et al., 1980), suggesting that antenna complexes can form PFs particles when they are no longer associated with PS II centers. In two other instances, complementary changes were observed between the PFs and EFs faces, with no concomitant changes in the actual content in Chl a/b proteins (Olive et al., 1981). Either with changes in the cationic concentration of the medium in which broken cells of Chlamydomonas were resuspended, or with changes in the light intensity during growth: the larger the EFs particles, the fewer the PFs particles in high salt medium and high light conditions. Thus, the partition coefficient of some proteins—presumably antenna proteins—between the two fracture faces of the stacked membranes varied with the experimental conditions. In agreement with this observation, a C. reinhardtii mutant lacking all classes of Chl a/b containing antennae, the BF4

Chapter 14

Supramolecular Organization of Chloroplast and Thylakoid Membranes

mutant, showed a dramatic loss in PFs particle density, from 6000 particles in WT to 3800 in the mutant, together with a reduction in the size of the EFs particles (Olive et al., 1981). This indicated that Chl a/b proteins were located both as individual particles on PFs and within EFs particles, in association with the PS II reaction centers. Similar effects were reported in barley mutants (Miller et al., 1976; Simpson, 1980), although, in this case, the change in EFs particle size was less marked. This difference reflected the distinct nature of the mutations altering production of the Chl a/b proteins in the various mutants. Whereas the BF4 mutant from C. reinahrdtii has an altered content in all Chl a/b proteins, the chlorina-f2 mutant studied by Simpson (1980) retains normal amounts of the minor antenna complexes. In addition, a maize mutant with normal levels of CP29 and reduced LHCII showed no reduction in the size of EFs particles (Greene et al., 1988). A model thus emerges in which the minor antenna proteins are present together with the PS II reaction centers in the EFs particles, about 12 nm in diameter, while most of the LHCII, although it is functionally associated with the PS II core, is retained on the PFs faces upon freeze-fracture, giving rise to individual LHC particles of 8–10 nm in diameter. In some cases, these LHCII complexes can be retained, upon freeze-fracture, in association with the rest of PS II in the largest EFs particles, 18 nm in diameter. This model is consistent with the findings that CP29 is found more closely (Barbato et al., 1989) and more stably (Dunahay and Staehelin, 1987) associated with the PS II reaction center than LHCII. The presence of the PS II antenna in appressed membranes in State 1 conditions was also demon­ strated by immunocytochemistry. Using antibodies against the polypeptide p 11 (LHCII subunit), 90% of the labeling was found in appressed membranes (Fig. 9 and Table 2) (Vallon et al., 1986).

4. Cytochrome

Complex

The cytochrome complex connects Photosystem II located mainly in the stacked membrane regions to Photosystem I which is restricted to the unstacked membrane regions of the thylakoids membranes. Its presence in both domains has been established in Chlamydomonas by immunogold labeling (Olive et al., 1986, Vallon et al., 1991) and freeze-fracture analysis of mutants lacking the complex (Olive et al., 1986, 1992). A similar conclusion was drawn

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from the freeze-fracture analysis of Chlorella sorokiniana WT and mutant strains (Olive and Wollman, 1987). In two distinct C. reinhardtii mutant strains lacking the complex, a multiplicity of changes was observed both in the stacked and unstacked membrane domains (Olive et al., 1986). There was a decrease in particle densities on PFu, EFs and EFu and a change in particle sizes on PFs, EFs and EFu (Fig. 10). The largest changes were observed on the PFu and EFs faces. These modifications were consistent with the complexes in the stacked and presence of unstacked regions of the thylakoid membranes, some of them in association with the reaction centers PS I and PS II in the largest PFu and EFs particles, respectively (Olive et al., 1986). The presence of 8 nm EF particles corresponding to individual cytochrome complexes present in the stacked membrane regions was further confirmed using a set of double and triple mutants lacking PS II and cytochrome protein complexes (Olive et al., 1992).

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Jacqueline Olive and Francis-André Wollman spinach. Either antibodies unambiguously labeled both the stacked and unstacked regions of the embedded membranes from Chlamydomonas, as illustrated for Cyt f in Fig. 8C, and also from spinach (Olive et al., 1986). Similar conclusions were reached for vascular plant thylakoid membranes by a number of authors using immunocytochemical (Allred and Staehelin, 1985; Goodchild et al., 1985; Shaw and Henwood, 1985) or fractionation (Anderson, 1982; Cox and Anderson, 1982) approaches. In contrast the phase-partitioning technique gave conflicting results, with the cytochrome found only in the stroma lamellae fraction (Henry and Moller, 1981) or in the regions interfacing the stacked and unstacked membranes (Ghirardi and Melis, 1983; Barber, 1984).

5. ATP Synthase The coupling factor complex of the ATP synthase has been shown to be totally excluded from the stacked regions of the thylakoid membranes in C. reinhardtii. This has been demonstrated by an immunocytochemical study, using antibodies directed (Fig. 8D) (Vallon against the and subunits of et al., 1986). These results were confirmed by experiments with spinach membranes where only the stroma and the margins were labeled (Vallon et al., 1986). Previous analysis of vascular plant thylakoid membranes using antibody labeling, enzyme assays and destacking/restacking of thylakoid membranes had also demonstrated that the coupling factor was excluded from grana regions upon membrane stacking (Miller and Staehelin, 1976). The hydrophobic and transmembrane sector of complex has been visualized by the negatively staining them (Staehelin, 1986). When reconstituted into digalactosylipids and freezefractured, they gave rise to 9.5 nm particles falling into the size range of the PFu particles (Mörschel and Staehelin, 1983).

6. The Static Picture

This lateral distribution of cytochrome complexes along the thylakoid membranes was confirmed by immunocytochemistry using antibodies raised against either Cyt f or the Rieske protein from

In summary, the ultrastructural analysis of numerous photosynthesis mutants of Chlamydomonas led to the following conclusions as to the lateral and transverse distribution of the transmembrane protein complexes (Olive and Vallon, 1991) (Table 3). Most of the PS II reaction centers are found in the stacked membrane domains, and are retained on

Chapter 14 Supramolecular Organization of Chloroplast and Thylakoid Membranes

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the exoplasmic faces upon freeze-fracture. The PS I reaction centers, the ATP synthase and the peripheral PS I antenna are located exclusively in the unstacked membrane domain, where they give rise to freeze-fracture particles on the protoplasmic face. The distribution of the PS II peripheral antenna is the same as that of the PS II cores. A part of it partitions to the exoplasmic faces in association with the PS II cores in the same particles while the rest contributes to particle formation on the protoplasmic faces. The cytochrome is present in both stacked and unstacked membranes, mainly on PFu and EFs, in part associated in the same particles with PS II and PS I reaction centers.

B. Transmembrane Organization The vectorial nature of photosynthetic electron transport requires an asymmetric distribution of redox components in the transverse plane of thylakoid membranes. Examples of vectorial processes are proton uptake from the stroma and deposition into the lumen, water-splitting and plastocyanin oxidation which occur only at the lumenal surface whereas reduction and proton-driven ATP synthesis occur only at the stromal surface. It is now recognized that most, and perhaps all, of the intrinsic subunits in thylakoid protein complexes span the membrane and have domains at both the lumenal and stromal surfaces. Intrinsic polypeptides protruding from the membranes have been visualized by the deep-etching technique. The lumenal stacked membrane surface (ESs) contains high density of large protruding particles, each divided into two lobes, whereas the unstacked areas (ESu) show few particles only (Fig. 11). Each particle corresponds to a PS II complex, the protruding elements of which are considered structural equivalents of the OEE subunits making up part of the water splitting enzyme. In the BF25 mutant which lacks most of the OEE subunits, the large ESs particles are absent (O. Vallon, personal communication). Similarly, tetramer-like structures exposed on the lumenal surface were absent in a PS II mutant from tobacco (Miller and Cushman, 1979). In C. reinhardtii, the outer unstacked membrane

surface (PSu) is covered with large particles identified, by analogy with results obtained on higher plants (Miller and Staehelin, 1976; Oleszko and Moudrian­ akis, 1974), as the coupling factor molecules (J. Olive, unpublished). In addition to these large

246 particles, unidentified smaller particles are present which could represent a protruding part of either the LHCI-PS I complex or the cytochrome complex. The two membrane surfaces become accessible to antibodies after cell disruption through a French Press, because of the production of a mixture of inside-out and right-side-out vesicles (Andersson and Akerlund, 1978). When stacked thylakoid membranes are disrupted, they contribute to the formation of inside-out vesicles, which are enriched in PS II, whereas the right-side-out vesicles, originating from the unstacked membranes are enriched in PS I. In C. reinhardtii, cell disruption generates mostly right-side-out vesicles with a minor population of inside-out vesicles. Immunolabeling of these thylakoid membrane vesicles deposited on electron microscope grids can allow the determination of the sidedness of an antibody-binding site. A monoclonal antibody directed against Cyt f, labeled exclusively the insideout vesicles (Fig. 12, O. Vallon, unpublished) indicating that the epitope was accessible on the lumenal surface. This is consistent with the transmembrane organization of the protein, most of which is located in the lumen (Chapter 24, Wollman). The accessibility of an antibody directed against P6 (CP43) has also been observed on one face (undetermined) of the WT membrane vesicles (Vallon, 1986).

IV. Dynamic Aspects of Thylakoid Membrane Organization

A. The Stacking-Destacking Process As mentioned earlier, thylakoid membranes of green algae have well-defined stacked and unstacked domains with distinct protein contents and supramolecular structures, even though their organization is less regular than in vascular plants. Moreover, the ratio of appressed over non-appressed membranes has been shown to vary in mutant strains, depending on the inability of the mutants to synthesize active components of the photosynthetic electron transport chain (Goodenough and Levine, 1969). In particular, in PS I mutants, about 90% of the membranes were appressed versus 65% in WT. By contrast, the ac-115 and ac-141 mutants lacking and Q (the quencher of PS II fluor­ active Cyt escence) contain long, single discs in their chloro-

Jacqueline Olive and Francis-André Wollman

plast (Goodenough and Levine, 1969). The photosynthetic proteins are therefore segre­ gated between these two domains. Barber and coworkers performed an analysis ofthe surface charge density on the thylakoid membranes (Nakatani and Barber, 1980; Barber, 1982) and they reached the conclusion that the unstacked membrane regions carried a higher negative surface charge density than the surfaces in contact in the stacked domains (Barber, 1980; Chow and Barber, 1980; Yamamoto and Ke, 1982). These conclusions were further used in the interpretation of the formation of the two membrane domains, the stacked and unstacked thylakoid membrane regions. Experimentally, it was shown to be a reversible process in vitro: destacking and restacking can be promoted by decreasing then increasing again the concentration of cations. These were proposed to screen the electrostatic surface charges, responsible for repulsive forces, and allow the membranes to stack, due to van der Waals attractive forces. Conversely, removal of cations leads to an increase in repulsive forces due to the net negative charges at the surface of adjacent membranes and to a destacking process. Early studies with Chlamydomonas contributed greatly to the understanding of the relationship between membrane stacking, lateral segregation of complexes and changes in supramolecular organi­ zation. In a pioneering study, Ojakian and Satir (1974) showed by a freeze-fracture analysis of the

Chapter 14 Supramolecular Organization of Chloroplast and Thylakoid Membranes thylakoid membranes from C. reinhardtii that, when destacked by a transfer to zwitterionic buffers at low ionic strength (approximately 50 mM), the lateral segregation of intramembrane particles between grana and stroma membrane regions was lost, due to their intermixing and randomization. Conversely, they showed that segregation of the particles was fully restored when unstacked thylakoids were resuspended in a high salt buffer (more than 3 mM for divalent cations or more than 100 mM for monovalent cations). Similar results were obtained subsequently with vascular plant thylakoid membranes (Staehelin, 1976). The kinetics of light scattering changes which accompany membrane restacking were then studied in C. reinhardtii (Wollman and Diner, 1980). About 80% of the changes in light scatter were completed within 2 s after unstacked membranes were resus­ pended in the cation-containing stacking buffer. In contrast, cation-induced fluorescence changes, which reflect a segregation between PS II and PS I (reviewed in Barber, 1982), showed much slower kinetics, in the time range of several minutes. The segregation of intramembrane particles was not studied in Chlamydomonas. However, similar studies with vascular plant thylakoid membranes showed that complete segregation of the particles requires several minutes and was subsequent to membrane restacking (Staehelin, 1976; Briantais et al., 1983). Thus, thylakoid membrane stacking, which requires screening of surface charges to occur, causes a subsequent lateral segregation of the transmembrane complexes between the two domains, with most of the PS II localized in the stacked domains and PS I in the unstacked domains.

B. State Transition: PhosphorylationDependent Changes As discussed in greater detail by I. Ohad (Chapter 30), plants and algae subjected to light of different spectral quality, undergo state transition, a regulatory mechanism that controls light-energy distribution between the two photosystems (Bonaventura and Myers, 1969; Murata, 1969). It has been suggested that the changes in light energy distribution occur through a lateral displacement of a Chl a/b mobile antenna along the thylakoid membranes (for a review see Allen, 1992). This protein migration would be caused by the reversible phosphorylation of a fraction of the LHCII protein. It was thus proposed that, in

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state 2, phosphorylated antenna proteins move from the Photosystem II-enriched stacked domains to the Photosystem I-enriched unstacked domains. Dephos­ phorylation of the same subunits in state 1 allows the return of these peripheral antenna complexes to the grana regions (Staehelin et al., 1982; Kyle et al., 1983; Barber, 1986). This concept of lateral migration has been studied experimentally in C. reinhardtii, in which particularly extensive state transitions were observed in vivo (Wollman and Delepelaire, 1984). The cells were placed in state 1 or in state 2 and fixed in either state by p-benzoquinone treatment (Bulté and Wollman, 1990). An immunocytochemical study showed extensive changes in the distribution of cytochrome complexes and LHCII between the stacked and unstacked membranes (Vallon et al., 1991). As shown in Table 4, the labeling of LHCII and cytochrome f increased for unstacked membranes in state 2 versus state 1, while the labeling of the core antenna protein P6 (CP43) did not change. Thus, both the lightharvesting antenna and the electron-transport chain undergo a deep reorganization during state transitions. A similar reorganization was observed in maize (Vallon et al., 1991). The driving force for the movement of LHCII is suggested to arise from its change in phosphorylation but there has been no report of reversible phosphory­ lation of the cytochrome complex of C. reinhardtii upon state transitions. However, a cytochrome associated protein of 19.5 kDa (Lemaire et al., 1986) may be part of the process since it has been detected as a phosphoprotein in state 2 (C. de Vitry and F.-A. Wollman, unpublished). In contrast, the minor Chl a/ b antenna protein CP29, which becomes heavily phosphorylated in Chlamydomonas in state 2 (Wollman and Delepelaire, 1984; Bassi and Wollman, 1991) does not undergo lateral displacement upon state transitions.

C. Fusion of Thylakoid Membranes During Sexual Reproduction During the first steps of zygote formation, a number of intracellular rearrangements can be observed including fusion of the nuclei and chloroplasts (Friedman et al., 1968; Cavalier-Smith, 1970, 1975). The question then arises as to how the inner thylakoid membrane system behaves subsequent to chloroplast fusion. Baldan et al. (1991) used two mutant parental strains, one being deficient in the PS I complex and

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Jacqueline Olive and Francis-André Wollman V. Biogenesis

A. Greening: Synthesis of Membrane Complexes

the other in the PS II complex, to follow the possible fusion of their thylakoid membranes upon zygote formation. In order to avoid a genetic comple­ mentation and thus de novo synthesis of the missing chloroplast-encoded polypeptides, the gametes were crossed in the presence of chloramphenicol that inhibits chloroplast translation. Before fusion, the thylakoid membranes originating from the PS II minus-gamete could be distinguished easily from those of the PS I minus-gamete, by their lack in PS II-containing EF particles upon freezefracture as well as by the absence of membrane labeling with antibodies directed against PS II subunits. About 15 h after mating, a complete fusion of the thylakoid membranes from the two parental gametes occurred in the zygote, accompanied by a redistribution of the membrane protein complexes in the fused membranes. The histograms ofthe EFs and EFu particle sizes in the zygote were bimodal with two maxima, each corresponding to that in one type of parental gametes. Also, EF particle densities were intermediate between those recorded in the parental gametes. Immunolabeling of the zygote sections showed that, at an early stage, the thylakoid membranes from the two mutants could still be distinguished on the basis of their differential labeling with antibodies directed against PS II subunits (Fig. 13A). In older zygotes, thylakoid membranes from all regions of the chloroplast were labeled with the antibody, indicating that a redistribution of PS II complexes occurred within the fused membranes (Fig. 13B). Membrane fusion and particle redistribution restored a full functional interaction between PS II and PS I from the two mutant strains as demonstrated by the recovery of linear photosynthetic electron transport in the zygote (Baldan et al., 1991).

The study ofthe biogenesis of membrane components in C. reinhardtii has been facilitated by the use ofthe yellow mutant y-1, unable to synthesize chlorophyll in the dark, which was isolated by Sager and Palade (1954). When grown heterotrophically in the light, the mutant is indistinguishable from the wild type. When grown in the dark, the chloroplast intralamellar system of y-1 is gradually disorganized and drastically reduced in size (Ohad et al., 1967a). Growth in the dark causes loss of thylakoid membranes by dilution but not dedifferentiation ofthe plastid to a proplastid or etioplast(Ohad et al., 1967a; Hoober e tal., 1991). Dark-grown y-1 cells contain less than 5% of the chlorophyll found in light-grown cells. These darkgrown cells offer an appropriate starting point for examining thylakoid membrane formation. Upon return to light at 25 °C,y-1 cells accumulated chlorophyll, after a lag of 1 to 2 h, and developed thylakoid membranes (Hoober and Stegeman, 1973; see also Chapter 19, Hoober et al.). In contrast, the lag disappeared when cells were placed at higher temperature, 37 to 40 °C (Hoober and Stegeman, 1976; Maloney et al., 1989). Chlorophyll concen­ tration, thylakoid membranes and photosynthetic capacity increased in parallel, at linear rates, over a 6–8 h period (Ohad et al., 1967b; Hoober and Stegeman, 1976) when the cells were exposed to light. During this period, no cell division was observed: all the biosynthetic and morphogenetic activities ofthe greening cell are related to chloroplast differentiation. Initially, the system was constituted of single thylakoid membranes. Membrane appres­ sion was a later event in the process and was not required for detection of photosynthetic activity (Ohad et al., 1967b). The rate of increase in PS I activity initially exceeded that of PS II. Since fluorescence remained low (an indication that the PQ pool was oxidized), this indicates that antenna connection to the reaction centers and electron transport chain between the photosystems were fully functional in these membranes (Hoober et al., 1994). Rapid assembly of thylakoid membranes during greening of y-1 cells provided the opportunity to determine directly the site of formation of the membrane. Remnants ofthe chloroplastic disc system are always present which may act as primordia during the greening process (Ohad et al., 1967b). However,

Chapter 14 Supramolecular Organization of Chloroplast and Thylakoid Membranes

electron micrographs of cells exposed to light for only 5 min. revealed extensive arrays of membrane material extending from the inner envelope membrane, suggesting that thylakoid membranes develop by local expansion and infolding ofthe inner membrane (Hoober et al., 1991, 1994). The y-1 mutant, when allowed to green in the presence of chloramphenicol, an inhibitor of protein synthesis on 70s ribosomes, contained Chl a and Chl b but lacked certain membrane polypeptides (Eytan and Ohad, 1970; Bar-Nun and Ohad, 1977) and photosynthetic electron transport. Miller and Ohad (1978) showed that fewer particles were present on the EF fracture face and that they were significantly smaller (11.5 nm) in presence than in the absence of chloramphenicol. A substantial increase in EF particle number and size was observed upon removal of chloramphenicol, correlated with insertion of newly made PS II centers (Cahen et al., 1977). This observation is consistent with the above-described model that most of the EF particles contained PS II centers associated with some peripheral antenna.

B. Assembly of PS II The assembly of the PS II subunits has been studied in some details at the ultrastructural level in C. reinhardtii. This was achieved by comparing various PS II mutants, whose content in specific residual PS II subunits was known with sufficient accuracy that it could be correlated with structural changes. As described in Chapters 15 (Erickson) and 16 (Ruffle and Sayre), the PS II core complex consists

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of the two large reaction center subunits, D1 and D2, encoded by the psbA and psbD chloroplast genes and two core antenna proteins, P5 (CP47) and P6 (CP43), encoded by the psbB and psbC chloroplast genes. Three nucleus-encoded subunits, involved in oxygen evolution, OEE1, OEE2 and OEE3, associate with the PS II core on the lumenal side of the membranes. A number of smaller subunits, which will not be mentioned here, also participate in the assembly of the fully active PS II oligomeric protein. De Vitry et al. (1989) performed an immuno­ cytochemical study of several PS II mutants which were deficient in the synthesis of only one PS II core subunit, but displayed a pleiotropic decrease of all other transmembrane PS II subunits. However, about 10% ofthe WT amount of P5 and some accumulation of D1 and D2 were revealed in the thylakoid membranes of the mutants lacking P6 by application of immunoblotting methods. Similarly 10% of P6 was accumulated in the mutants lacking D1 or D2 (Fig. 14,A and B)(De Vitry et al., 1989). A P5/D1/ D2 subcomplex and the unassembled P6 subunits were inserted independently in the thylakoid membranes. These two parts of the PS II core each had the ability to segregate independently in the stacked membrane regions of the thylakoid mem­ branes. Interestingly, the OEE subunits accumulated in the lumen despite the absence of PS II cores. They were immunodetected as associated with the thylakoid membranes, but in a loose binding state since they were lost during purification of the membranes. The partially assembled P5/D1/D2 subcomplex and the individual P6 subunit each gave rise to

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particles which could be identified upon freezefracture on the EF faces of the thylakoid membranes from PS II mutants. Their densities were 206 and 240 respectively versus 1500 in WT (Olive particles et al., 1992). Comparison of the respective densities of EFs and EFu particles in PS II single mutants and PS II double mutants, totally devoid of the whole set of PS II core subunits, showed that 78% of the EFs particles and 64% of the EFu particles correspond to PS II cores in the WT. Most ofthe EF particles which remain in the PS II double mutant can be attributed to complex, as indicated by the analysis of a the Cyt triple mutant, deficient in all the PS II subunits and in Cyt (Fig. 15). The PS II subcomplexes identified by freezefracturing (Olive et al., 1992) demonstrated the same lateral segregation in stacked membrane domains, as they did when viewed by immunodecoration (de Vitry et al., 1989). Strangely enough, lateral migration of the PS II subcomplexes from the unstacked membrane regions to the stacked domains was not observed in double mutant strains carrying a single PS II mutation and a mutation preventing production complexes. In these strains, the of cytochrome

Jacqueline Olive and Francis-André Wollman

unstacked domains retained most ofthe PS II subunitcontaining EF particles (Olive et al., 1992). This observation suggests that the mechanism of translocation of neosynthesized PS II subunits to the stacked membrane domains requires either a direct complexes or some interaction with cytochrome elements ofa transition to state 2 that cytochrome mutants, which are blocked in state 1 (Wollman and Lemaire, 1988), cannot perform. This latter hypothesis points to a possible requirement for LHCII reversible phosphorylation in the movement of PS II subunits to the stacked membrane domains.

VI. Conclusion Fractionation of thylakoid membranes, performed by breakage or mild detergent solubilization followed by differential centrifugation or phase partitioning, yielded valuable information on the existence of various domains showing membrane protein heterogeneity (for a review, see Albertsson, 1988). However most of our knowledge on the supra­ molecular organization ofthe photosynthetic proteins comes from the two techniques that we have described in some detail here, the immunocytochemical labeling of membranes and their freeze-fracturing. They allowed the characterization in situ of the structural and biochemical heterogeneity between stacked and unstacked membrane regions. A large part of this experimental effort has focused on Chlamydomonas because of its genetics which opened the way to comparative biochemical and ultrastructural analysis

Chapter 14 Supramolecular Organization of Chloroplast and Thylakoid Membranes of mutant and wild-type strains. Mutant strains, in turn, served as controls for immunocytochemistry and freeze-fracturing. At the present time, a drawback of these approaches is that they are difficult to combine efficiently. Although it has been reported that antibody-coupled to colloidal gold can react with freeze-fracture replicas retaining some biological material (Fujimoto, 1995), the technique has not yet been applied successfully to the direct molecular identification of the freeze-fracture particles observed in the thylakoid membranes. Yet, this identification remains a challenging issue for the next years to those who wish to understand the dynamics of the organization of membrane proteins and also to those who are developing methods to solve their threedimensional structures.

Acknowledgment We thank O. Vallon for critical reading of the manuscript.

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Chapter 14 Supramolecular Organization of Chloroplast and Thylakoid Membranes process in Chlamydomonas. Photosynth Res 39: 15–31 Ish-Shalom D and Ohad I (1983) Organization of chlorophyllprotein complexes of Photosystem I in Chlamydomonas reinhardtii. Biochim Biophys Acta 722: 498–507 Kerby NW and Evans LV (1978) Isolation and partial characterization of pyrenoids from the brown alga Pilayella littoralis. Planta 142: 91–95 Kuchitsu K, Tsuzuki M and Miyachi S (1988) Characterization of the pyrenoid isolated from unicellular green alga C. reinhardtii: Paniculate form of rubisco protein. Protoplasma 144: 17–24 Kyle DJ, Staehelin LA and Arntzen CJ (1983) Lateral mobility of the light harvesting complex in chloroplast membranes controls excitation energy distribution in higher plants. Arch Biochem Biophys 222:527–541 Lacambra M, Larsen U, Olive J, Bennoun P and Wollman FA (1984) Characterization of the thylakoid membranes of wild type and mutants of Chorella sorokiniana. Photobiochem Photobiophys 8: 191–205 Lemaire C, Girard-Bascou J, Wollman FA and Bennoun P complex. Charac­ (1986) Studies on the cytochrome terization of the complex subunits in Chlamydomonas reinhardtii. Biochim Biophys Acta 851: 229–238 Lembi CA and Lang NJ (1965) Electron microscopy of Carteria and Chlamydomonas. Amer J Bot 52: 464–467 Maloney MA, Hoober JK and Marks DB (1989) Kinetics of chlorophyll accumulation and formation ofchlorophyll-protein complexes during greening of Chlamydomonas reinhardtii y­ 1 at 30°C. Plant Physiol 91: 1100–1106 Melkonian M and Robenek H (1980) Eyespot of C. reinhardtii: a freeze-fracture study. J Ultrastruct Res 72: 90–102 Miller KR (1980). A chloroplast membrane lacking Photosystem I. Changes in unstacked membrane regions. Biochim Biophys Acta 592: 143–152. Miller KR and Cushman RA (1979) A chloroplast membrane lacking Photosystem II. Thylakoid stacking in the absence of the Photosystem II particles. Biochim Biophys Acta 546: 481– 497 Miller KR and Ohad I (1978) Chloroplast membrane biogenesis in Chlamydomonas: correlation between the formation of membrane components and membrane structure. Cell Biol Reports 2: 537–549 Miller KR and Staehelin LA (1973) Fine structure of the chloroplast membranes of Euglena gracilis as revealed by freeze-cleaving and deep-etching. Protoplasma 77: 55–78 Miller KR and Staehelin LA (1976) Analysis of the thylakoid outer surface: Coupling factor is limited to unstacked membrane regions. J Cell Biol 68: 30–47 Miller KR, Miller GJ and McIntyre KR (1976) The lightharvesting chlorophyll-protein complex of Photosystem II. J Cell Biol 71: 624–638 Mörschel E and Staehelin LA (1983) Reconstitution of Cyt and ATPsynthase complexes into phospholipid and galactolipid liposomes. J Cell Biol 97: 301–310 Mullet JE, Burke JJ and Arntzen CJ (1980) A developmental study of Photosystem I peripheral chlorophyll proteins. Plant Physiol 65: 823–827 M urata N (1969) Control ofexcitation transfer in photosynthesis. I. Light-induced change of chlorophyll a fluorescence in Porphyridium cruentum. Biochim Biophys Acta 172: 242– 251

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Mustárdy L (1996) Development ofthylakoid membrane stacking. In: Ort DR and Yocum CF (eds) Oxygenic Photosynthesis: The Light Reactions, pp 59–68. Kluwer Academic Publishers, Dordrecht Nakamura K, Bray DF, Costerton JW and Wagenaar EB (1973) Infrastructure of Chlamydomonas eugametos as revealed by freeze-etching: cell wall, plasmalemma and chloroplast membrane. Can J Bot 51: 817–819 Nakatami NY and Barber J (1980) Further studies ofthe thylakoid membrane surface charges by particle electrophoresis. Biochim Biophys Acta 591: 82–91 Ohad I, Siekevitz P and Palade GE (1967a) Biogenesis of chloroplast membranes. I. Plastid dedifferentiation in a darkgrown algal mutant (Chlamydomonas reinhardtii). J Cell Biol 35: 521–552 Ohad I, Siekevitz P and Palade GE (1967b) Biogenesis of chloroplast membranes. II Plastid differentiation during greening of a dark-grown algal mutant (C. reinhardtii). J Cell Biol 35: 553–584 Ojakian G and Satir P (1974) Particle movements in chloroplast membranes: quantitative measurements of membrane fluidity by the freeze-fracture technique. Proc Natl Acad Sci USA 21: 2052–2056 Oleszko S and Moudrianakis EN (1974) The visualization of the photosynthetic coupling factor in embedded spinach chloroplasts. J Cell Biol 63: 936–948 Olive J and Vallon O (1991) Structural organization of the thylakoid membranes: freeze-fracture and immunocyto­ chemical analysis. J Electron Microsc Techn 18: 360–374 Olive J and Wollman FA (1987) Localization of the complex by freeze-fracture analysis of the Chlamydomonas reinhardtii and Chlorella sorokiniana mutants lacking in this complex. In: Biggins, J (ed), Progress in Photosynthesis Res Vol II, pp 325– 328, Martinus Nijhoff Publishers, Dordrecht, The Netherlands Olive J, Wollman FA, Bennoun P and Recouvreur M (1979) Ultrastructure-function relationship in Chlamydomonas reinhardtii thylakoids by means of a comparison between the wild type and the F34 mutant which lacks the Photosystem II reaction center. Mol Biol Rep 5: 139–143 Olive J, Wollman FA, Bennoun P and Recouvreur M (1981) infrastructure of thylakoid membranes in C. reinhardtii. Evidence for variations in the partition coefficient of the lightharvesting complex-containing particles upon membrane fracture. Arch Biochem Biophys 208: 456–467 Olive J, Wollman FA, Bennoun P and Recouvreur M (1983). Localization of the core and peripheral antennae of Photosystem I in the thylakoid membranes of Chlamydomonas reinhardtii. Biol Cell 48: 81–84 Olive J, Vallon O, Wollman FA, Recouvreur M and Bennoun P (1986) Studies on the cytochrome complex. II. Localization of the complex in the thylakoid membranes from spinach and Chlamydomonas reinhardtii by immunocytochemistry and freeze-fracture analysis of mutants. Biochim Biophys Acta 851:239–248 Olive J, Recouvreur M, Girard-Bascou J and Wollman FA (1992) Further identification of the exoplasmic face particles on the freeze-fractured thylakoid membranes: A study using double and triple mutants from Chlamydomonas reinhardtii lacking various Photosystem II subunits and the cytochrome complex. European J Cell Biol 59: 176–186 Rochaix JD, Kuchka M, Mayfield S, Girard-Bascou J and Bennoun

254 P (1989) Nuclear and chloroplast mutations affect the synthesis or stability of the chloroplast psbC gene product in Chlamydomonas reinhardtii. EMBO J 8: 1013–1021 Roberts K, Shaw PJ and Hills GJ (1972) Structure, composition and morphogenesis of the cell wall of C. reinhardtii. I. Ultrastructure and preliminary chemical analysis. J Ultrastruct Res 40: 599–613 Sager R and Palade GE (1954) Chloroplast structure in green and yellow strains of Chlamydomonas. Exp Cell Res 7: 584–588 Sager R and Palade GE (1957) Structure and development of the chloroplast in Chlamydomonas. J Biophys Biochem Cytol 3: 463–487 Sato H, Okada M, Nakayama K and Miyaji K (1984) Purification and further characterization ofpyrenoid proteins and ribulose­ 1 -5-biphosphate carboxylase-oxygenase from the green alga Bryopsis maxima. Plant Cell Physiol 25: 1205–1214. Schötz F, Bathelt H, Arnold CG and Schimmer O (1972) Die Architektur und Organisation der Chlamydomonas-Zelle. Ergebnisse der Elektronen Mikroskopie von Serienschnitten und der daraus resultierenden dreidimensionalen Rekon­ struktion Protoplasma75: 229–254 Shaw PJ and Henwood JA (1985) Immuno-gold localization of cytochrome f, light-harvesting complex, ATP synthase and ribulose 1,5-bisphosphate carboxylase/oxygenase. Planta 165: 333–339 Simpson DJ (1980) Freeze-fracture studies on barley plastid membranes. IV. Analysis of freeze-fracture particle size and shape. Carlsberg Res Commun 45: 201–210 Simpson DJ (1982) Freeze-fracture studies on barley plastid membranes. V. a Photosystem I mutant. Carlsberg Res Comm 47: 215–225 Simpson DJ, Vallon O and Von Wettstein D (1989) Freezefracture studies on barley plastid membranes. VIII. In viridis 115 , a mutant completely lacking Photosystem I, oxygen evolution enhancer 1 (OEE1) and the subunit of cytochrome b-559 accumulate in appressed thylakoids. Biochim Biophys Acta 975: 164–174 Sjolung RD and Smith DD (1974) Freeze-fracture studies of photosynthetically deficient supergranal chloroplast in tissue cultures containing virus-like particles. J Cell Biol 60: 285– 292 Staehelin LA (1975) Chloroplast membrane structure. Intramembranous particles of different sizes make contact in stacked membrane regions. Biochim Biophys Acta 408: 1–11 Staehelin LA (1976) Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro. J Cell Biol 71: 136–158 Staehelin LA (1986) Chloroplast structure and supramolecular organization of photosynthetic membranes. In: Staehelin LA, Arntzen CJ (ed), Photosynthesis III, pp 1–84, Springer-Verlag Berlin Staehlin LA and van der Staay WM (1996) Structure, composition, functional organization and dynamic properties of thylakoid membranes. In: Ort DR and Yocum CF (eds) Oxygenic Photosynthesis: The Light Reactions, pp 11–30. Kluwer Academic Publishers, Dordrecht

Jacqueline Olive and Francis-André Wollman Staehelin LA, Armond PA and Miller KR (1977) Chloroplast membrane organization at the supramolecular level and its functional implications. Brookhaven Symp Biol 28: 278–315 Staehelin LA, Kyle DJ and Arntzen CJ (1982) Spillover is mediated by reversible migration of LHCP between grana and stroma thylakoids. Plant Physiol 69: 69–81 Vallon O (1986) Organisation supramoléculaire des domaines membranaires engagés dans la communication intercellulaire (cristallin de bovidé) et dans la photosynthèse (membrane des thylacoïdes): Étude immunocytochimique. PhD dissertation, Université Paris VI. Vallon O, Wollman FA and Olive J (1985) Distribution of intrinsic and extrinsic subunits of the PS II protein complex between appressed and non-appressed regions ofthe thylakoid membrane: an immunocytochemical study. FEBS Lett 183: 245–250 Vallon O, Wollman FA and Olive J (1986) Lateral distribution of the main protein complexes of the photosynthetic apparatus in C. reinhardtii and in spinach: An immunocytochemical study using intact thylakoid membranes and a PS II enriched membrane preparation. Photobiochem Photobiophys 12: 203– 220 Vallon O, Hoyer-Hansen G and Simpson DJ (1987) Photo­ system II and cytochrome b-559 in the stroma lamellae of barley chloroplasts. Carlsberg Res Commun 52: 405–421 Vallon O, Bulté L, Dainese P, Olive J, Bassi R and Wollman FA complexes (1991) Lateral redistribution of cytochrome along thylakoid membranes upon state transitions. Proc Natl Acad Sci USA 88: 8262–8266 Walne PL and Arnott HJ (1967) The comparative ultrastaicture and possible function of eyespots: Euglena granulata and Chlamydomonas eugametos. Planta 77: 325–353 Wollman FA and Bennoun P (1982) A new chlorophyll-protein complex related to Photosystem I in C. reinhardtii. Biochim Biophys Acta 680: 352–360 Wollman FA and Delepelaire P (1984) Correlation between changes in light energy distribution and changes in thylakoid membrane polypeptide phosphorylation in C. reinhardtii. J Cell Biol 98: 1–7 Wollman FA and Diner BA (1980) Cation control of fluorescence emission light scatter, and membrane stacking in pigment mutants of Chlamydomonas reinhardtii. Arch Biochem Biophys 201: 646–649 Wollman FA and Lemaire C (1988) Studies on kinase-controlled state transitions in Photosystem II and mutants from Chlamydomonas reinhardtii which lack quinone-binding proteins. Biochim Biophys Acta 933: 85–94 Wollman FA, Olive J, Bennoun P and Recouvreur M (1980) Organization of the Photosystem II centers and their asso­ ciated antennae in the thylakoid membranes: A comparative ultrastructural, biochemical and biophysical study of Chlamydomonas wild-type and mutants lacking in Photo­ system II reaction centers. J Cell Biol 87: 728–735 Yamamoto Y and Ke B (1981) Membrane surface electric properties of Triton-fractionated spinach subchloroplast fragments. Biochim Biophys Acta 636: 175–184

Chapter 15 Assembly of Photosystem II Jeanne Marie Erickson

Departments of Biochemistry and Plant Pathology, 1-87 Agriculture Building,

University of Missouri, Columbia, MO 65211, U.S.A.

Summary 255

I. Introduction 256

II. Developmental Biogenesis of Photosystem II 257

A. Plants 257

B. Algae 259

III. Assembly of Photosystem II Complexes 260

A. Migration of Photosystem II in the Thylakoid Membrane: PS II Damage and Repair 260

B. Photosystem II Assembly in Chlamydomonas 260

1. Three Classes of Mutants Defective in the Assembly or Stability of the PS II Core 260

2. Stepwise Assembly of Photosystem II Intrinsic and Extrinsic Membrane Subunits 262

3. Assembly of Nascent D1 and the OEC Polypeptides with Other PS II Subunits 263

4. Assembly of the PS II Redox Components 264

C. Photosystem II Assembly Intermediates: In Organello and In Vitro 265

1. The Synthesis and Fate of PS II Core Subunits 265

2. D2 as a ‘Docking’-Like Protein Required for D1 Synthesis and/or Stability 266

3. Stepwise Assembly of the Photosystem II Polypeptides in Appressed and

267

Non-appressed Thylakoid Membranes 4. Posttranslational Import of Chimeric D1 into Isolated Chloroplasts and Assembly with PS II 268

D. Processing of the D1 Polypeptide Carboxyl-Terminus is Not a Prerequisite for PS II Assembly 268 270

IV. Assembly of the Extrinsic Membrane Polypeptides of the PS II Oxygen-Evolving Complex A. In Organello Assembly of the OEC Polypeptides with the PS II Core Complex 270

B. In Vivo Requirements for the Extrinsic Membrane Polypeptides of the OEC 272

273

V. Assembly of Manganese: The Catalytic Center of the Oxygen-Evolving Complex A. Photoactivation: Assembly of the Functional Manganese Cluster 273

B. A Role for Bicarbonate in Assembly of the OEC 276

C. Is Oxygen Required for Assembly of the OEC? 276

Acknowledgments 277

277

References

Summary Photosystem II (PS II) is a pigment-protein complex located in the chloroplast thylakoid membrane of plants and algae which acts as a light-driven water-splitting enzyme. At least 16 chloroplast- and 11 nucleus-encoded polypeptide subunits, several types of pigments, and lipid, metal and ion cofactors have been identified as constituents of the chloroplast PS II. Water is split in the oxygen-evolving complex (OEC) of PS II, where a catalytic tetranuclear cluster of manganese ions is required for the photodriven extraction of electrons from water and the release of protons and molecular oxygen. The exciton and electron transfer reactions of PS II are determined by the properties of the redox components themselves, as well as the spatial organization of all

J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 255–285. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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Jeanne Marie Erickson

cofactors within the tertiary structure of the protein environment to which they are bound. Correct assembly and conformation of the photosystem polypeptides, pigments and cofactors is critical for photosystem function and stability. Such assembly depends on the concerted expression of both nuclear and chloroplast genes, with gene expression regulated at the transcriptional, translational and posttranslational levels in response to developmental and environmental cues. Details of PS II assembly have been elucidated through in vivo, in vitro and in organello studies. In developing chloroplasts, PS II polypeptides and pigments are synthesized and assemble during light-induced biogenesis of the thylakoid membrane. In mature chloroplasts, photodamage to PS II subunits, particularly the D1 polypeptide of the PS II reaction center, requires the replacement of damaged subunits and reassembly of PS II complexes. This chapter focuses on the assembly and stability of the PS II polypeptides, with particular emphasis on investigations using the unicellular green alga Chlamydomonas reinhardtii. In addition, the light-dependent activation of the PS II OEC is reviewed. This process, termed photoactivation, involves the assembly of the tetranuclear manganese cluster central to OEC function.

I. Introduction Photosystem II (PS II) is a large, multi-subunit pigment-protein complex located in the chloroplast thylakoid membrane of plants and eukaryotic algae. PS II functions as a light-driven water-splitting enzyme, releasing molecular oxygen and hydrogen ions, and providing electrons for the photosynthetic electron transfer chain (reviewed in Rutherford, 1989). At least 27 different polypeptide subunits, several types of pigments, and lipid, metal and ion cofactors have been identified as constituents of the chloroplast PS II and its antenna complex (Fig. 1, Table 1). The nuclear and chloroplast genes encoding PS II subunits, the subunit organization within PS II, and their functions and associated cofactors and pigments are outlined in Table 1. PS II genes and polypeptides have been reviewed extensively (Erickson and Rochaix, 1992; Ikeuchi, 1992; Bricker and Ghano­ takis, 1996; Hankamer et al., 1997; Chapter 16, Ruffle and Sayre). The subunits of the reaction center (RC), the core complex, and the PS II light harvesting complex (LHCII) are intrinsic membrane poly­ peptides, while the subunits of the oxygen-evolving complex (OEC) are extrinsic membrane polypeptides. The D1 and D2 polypeptides of the PS II RC contain and and the redox-active tyrosine residues Abbreviations: ADP – adenosine monophosphate; ATP – adenosine triphosphate; Cyt – cytochrome; D1 – PS II reaction center polypeptide D1; D2 – PS II reaction center polypeptide D2; EPR – electron paramagnetic resonance; LHCII – lightharvesting complex of Photosystem II; OEC – oxygen-evolving – PS II reaction center chlorophyll; PAGE – complex; polyacrylamide gel electrophoresis; PS I – Photosystem I; PS II – Photosystem I I ; – primary quinone acceptor of PS II; – secondary quinone acceptor of PS II; RC – reaction center; redox-active tyrosine 160 of the D2 polypeptide; – redoxactive tyrosine 161 of the D1 polypeptied

bind the redox-active molecules of pheophytin, a non-heme iron, the and plastoquinones, manganese, and the chlorophyll molecules, including P680, essential for PS II photochemistry (Table 1, Fig. 1). As such, these two polypeptides play a central role in PS II assembly and function. The polypeptide subunits of PS II are generally thought to be present in a 1:1 molar ratio (reviewed in Seidler, 1966). However, there is some evidence that a single (i.e., monomer) PS II core complex may subunits contain two copies each of the Cyt (reviewed in Whitmarsh and Pakrasi, 1996), the 22 kDa S polypeptide, PS II-S (Funk et al., 1995), and the extrinsic OEC subunits (Xu and Bricker, 1992; Bricker and Ghanotakis, 1996). Recent reports by Yocum and coworkers (Betts et al., 1996b, 1997) provide the most compelling genetic and biochemical evidence in support of a model with two OEE1 polypeptides per chloroplast PS II RC. Models for the PS II RC have been made (Trebst, 1986; Santini et al., 1994; Svensson et al., 1996; Xiong et al., 1996, 1998) based on the crystal structures of reaction centers isolated from photosynthetic bacteria. A twodimensional crystal structure of the spinach PS II RC has been resolved at the level of 8 Å (Rhee et al., 1997). For further detail on PS II structure and function, the reader is referred to recent reviews (Diner and Babcock, 1996; Satoh, 1996; Barber et al., 1997; Chapter 16, Ruffle and Sayre). Other catalytic proteins, including catalase and polyphenol oxidase (Sheptovitsky and Brudvig, 1997) appear to be associated at substoichiometric levels with PS II membranes, and may play some role in maintaining a chemical environment favorable to PS II function. Assembly of a multisubunit membrane complex such as PS II, which contains at least 16 subunits translated in the chloroplast, and 11 subunits

Chapter 15 Assembly of Photosystem II

translated in the cytoplasm, imported into the chloroplast and localized to the thylakoid membrane or thylakoid lumen, is clearly a complicated process. Assembly can be affected by levels of transcription and translation, protein processing and modification, import of metal cofactors, synthesis of pigment cofactors, temperature, light quantity, and light quality. The focus of this chapter is the assembly of the PS II core complex and the process of photoactivation, i.e., the light-dependent assembly of the catalytic manganese cluster of the OEC. Emphasis will be given to assembly of PS II polypeptides in the chloroplast thylakoid membrane of plants and of the unicellular green alga, Chlamydomonas reinhardtii, which provides an excellent model system for the study of photo­ synthesis (Rochaix, 1995). Other aspects of the assembly of thylakoid membrane complexes, including LHCII assembly, have been reviewed (Cohen et al., 1995; Webber and Baker, 1996;

257

Chapters 19, Hoober et al.; 25, Strotmann et al.; 17, Webber and Bingham; 24, Wollman).

II. Developmental Biogenesis of Photosystem II

A. Plants In most plants, chlorophyll biosynthesis and plastid development are light-dependent processes that occur during seedling development or during greening of dark-grown, etiolated plants (Mullet, 1988; Barkan et al., 1995). Development of etioplasts into chloroplasts is accompanied by the biogenesis and organization of the thylakoid membrane, including the synthesis and assembly of PS II components. PS II biogenesis is dependent on processes directed by both the nuclear and plastid genomes (Taylor, 1989; Rochaix, 1992, 1996; Mayfield et al., 1995;

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Jeanne Marie Erickson

Chapter 15 Assembly of Photosystem II Roell and Gruissem, 1996; Goldschmidt-Clermont, 1998; Chapter 12, Hauser et al.; Chapter 10, Stern and Drager). The biogenesis of PS II in greening etioplasts has been studied in many plant systems (Schuster et al, 1985; Sutton et al., 1987; Hird et al., 1991; Meierhoff and Westhoff, 1993; Palomares et al., 1993). Mullet and coworkers analyzed mRNAs and translation products labeled in plastids isolated from etiolated and greening barley seedlings (Klein and Mullet, 1987), or labeled in plastid polysomal fractions separated by sucrose density gradient centrifugation (Klein et al., 1988). These studies showed that psbA transcription and translation are light-regulated; during greening the number of membrane-bound and soluble polysomes increases, and psbA mRNA becomes associated with chloroplast polysomes. Subsequent in vitro translation experi­ ments using lysed barley etioplasts have shown that accumulation of the PS II chlorophyll-binding proteins CP47, CP43 and D1 is dependent on de novo chlorophyll biosynthesis (Eichacker et al., 1990; Franck et al., 1997), Moreover, chlorophyll increases the stability of CP43 and D1 (Mullet et al., 1990), and ribosome ‘pausing’ during Dl translation increases during chloroplast development and may improve the efficiency of chlorophyll binding to nascent D1 (Kim et al., 1994b). Interestingly, the D2 core polypeptide is the only major PS II core subunit that accumulates in dark-grown etioplasts (Gamble et al., 1988). Greening studies with the barley nuclear mutant viridis-115 show that the stability of the CP47, CP43, D1 and D2 polypeptides is reduced after 16 h illumination, although the OEE1 and OEE3 polypeptides accumulate normally, suggesting that a nuclear gene product somehow stabilizes the intrinsic PS II core polypeptides (Gamble and Mullet, 1989). D1 translation initiation, elongation and ribosome pausing are not significantly altered in the vir-115 mutant, but D1 translation intermediates normally detected are lacking. This suggests that the wild-type nuclear viridis-115 gene may encode a factor which stabilizes D1 intermediates, possibly during assembly of D1 with chlorophyll (Kim et al., 1994c). Whether chlorophyll associates with D1 intermediates cotranslationally and affects D1 synthesis is not yet clear. Although chlorophyll did not appear to influence the extent of D1 elongation in barley translational runoff assays, full-length D1 translated on polysomes accumulated only in the presence ofchlorophyll, and

259 chlorophyll was seen to greatly increase the stability of D1 after release from ribosomes (Kim et al., 1994a). These results reaffirm the role of chlorophyll in a posttranslational stabilization of D1.

B. Algae In several organisms, including green algae, chlorophyll synthesis can occur via light-dependent and light-independent pathways, such that wild-type cells are green in the dark (Chapter 20, Timko). Chlamydomonas mutants lacking the light-independent pathway for chlorophyll synthesis have been isolated (Ohad et al., 1967; Bennoun et al., 1995), are ‘yellow in the dark’, and serve as model systems for studying PS II biogenesis during greening (Wettern et al., 1983). The light-dependent assembly of PS II has also been studied in a Scenedesmus obliquus mutant lacking light-independent carotenoid biosynthesis (Humbeck et al., 1990). In a study of wild-type and greening C. reinhardtii y-1 mutants, Malnoë et al. (1988) have shown that immunodetectable levels of the intrinsic PS II core polypeptides D1, D2, CP47 and CP43, as well as the LHCII apoproteins, are similar in light- and darkgrown wild-type cells, but are barely detectable in the dark-grown, chlorophyll-lacking y-1 mutant. In contrast, levels of the OEEC polypeptides are comparable, in light and dark conditions, in both the wild-type and mutant strains, demonstrating that the OEC polypeptides can accumulate independently of the PS II intrinsic core. Synthesis of D1 and D2 is enhanced immediately by light in wild-type cells, and enhanced with a lag in y-1 cells, suggesting that light-induced plastid development must precede lightactivated translation of D1 and D2. A 2 h lag for both accumulation of chlorophyll and accumulation of the D1, D2, CP47, CP43 and LHCII polypeptides during greening suggests that chlorophyll may stabilize these chlorophyll apopolypeptides, as is the case in greening barley (Mullet et al., 1990). Since synthesis of D1 and D2 in wild-type cells is increased in the light, while the abundance of these polypeptides appears constant, the rate of degradation of these polypeptides is assumed to be higher in the light (Malnoë et al., 1988), as seen previously in both Chlamydomonas (Wettern and Ohad, 1984; Section III.A) and in Spirodela oligorrhiza (Fromm et al., 1985). Thus, PS II biosynthesis is affected by both light itself and by the light-dependent stages of plastid development.

260 III. Assembly of Photosystem II Complexes

A. Migration of Photosystem II in the Thylakoid Membrane: PS II Damage and Repair Thylakoid membrane structure and function has been studied extensively, both through electron microscopy freeze-fracture analysis (Staehelin and van der Staay, 1996) and through genetic and biochemical dissection of thylakoid protein components (Cohen et al., 1995; Webber and Baker, 1996; Chapter 11, Olive and Wollman). The nature and function of the thylakoid membrane is determined to a large extent by its glycerolipid, pigment and prenylquinone composition (Douce and Joyard, 1996; Chapter 21, Trémoliéres). In vascular plants and green algae, the thylakoid is characterized by the presence of highly organized helical stacks of membrane, called the appressed membranes or granal lamellae, interspersed by single strands of membrane called non-appressed mem­ branes or stromal lamellae. The terminal membranes of each appressed membrane stack are exposed to the chloroplast stroma and have biochemical properties similar to non-appressed thylakoid membranes. Functional heterogeneity in PS II, between ‘reducing’ and ‘non-reducing’ PS II centers, also called and centers, respectively, has been noted (Guenther et al., 1990; Melis, 1991; Lavergne and Briantais, 1996). Structural heterogeneity is seen also in the segregation of most PS II into the appressed membranes, with only 10–20% of PS II centers found in the non-appressed membranes (Andersson and Anderson, 1980); PS I is found primarily in nonappressed membranes. Such segregation of PS II and PS I may be facilitated by the membrane stacking (Mustárdy, 1996), and may optimize positioning of the two photosystems for efficient energy transfer. Subsequent studies have confirmed this spatial distribution of PS II between the granal and stromal lamellae, and shown that PS II in the non-appressed membranes is depleted of the LHCII-inner antenna complexes, CP26 and CP29 (Vallon et al., 1986, 1987; Bassi et al., 1988; Barbato et al., 1992). A model has emerged in which functional PS II in the appressed membrane is ‘damaged’, dissociates from the LHCII antenna complexes, and migrates to the non-appressed membrane (Mattoo and Edelman, 1987; Adir et al., 1990; Guenther and Melis, 1990; Barbato et al., 1992). There, damaged subunits (primarily D l ) are removed from the inactive PS II

Jeanne Marie Erickson complexes and replaced by newly synthesized subunits. The ‘repaired’ PS II then migrates back to the appressed membrane where it associates with the PS II antenna and is again photoactive (for recent reviews Andersson and Aro, 1997; Barber et al., 1997; Chapter 30, Keren and Ohad). Damage to PS II occurs as a consequence of normal PS II function and is exacerbated during photoinhibitory conditions (Prásil et al., 1992; Aro et al., 1993; Anderson et al., 1997). The D1 reaction center polypeptide appears to be damaged the most (Mattoo et al., 1984; Ohad et al., 1984). D1, and to a lesser extent the D2 polypeptide, are the PS II components with the highest turnover rate (Schuster et al., 1988). Phosphorylation of PS II polypeptides may stabilize PS II in the appressed membranes and protect D1 from degrada­ tion (Ebbert and Godde, 1996; Andersson and Aro, 1997) but perhaps not from damage (Gal et al., 1997). While functional PS II in the granal lamellae may exist as a dimer, the inactive PS II that migrates to the stromal lamellae for repair is most likely a monomer (Santini et al., 1994; Lavergne and Briantais, 1996; Hankamer et al., 1997). In mature chloroplasts, D1 synthesis takes place primarily to replace photodamaged D1 subunits. Hence, most of the PS II ‘assembly’ occurring during steady-state photosynthetic function in mature plant cells may be a specific replacement of the D1 polypeptide rather than the de novo assembly of all newly synthesized PS II subunits, as occurs during greening. The process of photoinhibition is reviewed by Karen and Ohad (Chapter 30).

B. Photosystem II Assembly in Chlamydomonas

1. Three Classes of Mutants Defective in the Assembly or Stability of the PS II Core Proper subunit stoichiometries of the individual polypeptides of soluble, multisubunit protein complexes may in part be obtained by the stabilization of assembled subunits and the proteolysis of unassembled subunits, as shown for C. reinhardtii ribulose-bisphosphate carboxylase by Schmidt and Mishkind (1983). Similar results have been seen with the thylakoid membrane complexes, including the ATP synthase complex (Merchant and Selman, complex (Chapter 24, Wollman), 1984), the Cyt and Photosystem I (PS I) (Takahashi et al., 1991). The PS II intrinsic core polypeptides provide no exception to this rule. However, increasing evidence

Chapter 15

Assembly of Photosystem II

shows that the cell nucleus controls many levels of chloroplast gene expression, and that nuclear control over the temporal and spatial synthesis of polypeptides of multisubunit complexes ultimately controls the coordinate expression and fate of individual subunits (Rochaix, 1996; Rodermeletal., 1996; GoldschmidtClermont, 1998). There appear to be at least three classes of C. reinhardtii PS II assembly/stability mutants that lack one of the intrinsic membrane PS II subunits. In the first group, the absence of one subunit results in the rapid degradation of the other core subunits, the absence of assembled PS II, and obligate heterotrophic growth (reviewed in Rochaix and Erickson, 1988). PS II intrinsic membrane subunits are synthesized but fail to accumulate in the absence of either the D1 polypeptide (Bennoun et al., 1986; de Vitry et al., 1989), the D2 polypeptide (Erickson et al., 1986; Kuchka et al., 1988, 1989; de Vitry et al., 1989), the CP47 apoprotein (P5), (Jensen et al., 1986; Monod et al., 1992), or the CP43 apoprotein (P6), (de Vitry et al., 1989; Rochaix et al., 1989; Zerges et al., 1997). Such a destabilization of PS II is not unique to algae; a similar lack of the other PS II core subunits is seen in a nuclear maize mutant in which the D1 and CP43 polypeptides are rapidly degraded (Leto et al., 1985). The extrinsic membrane OEC subunits and the LHCII subunits appear to accumulate normally in the absence of the PS II core (Greer et al., 1986; Kuchka et al., 1988; de Vitry et al., 1989). The phosphorylated form of D2 (Delepelaire, 1984) is not found in this class of mutants (de Vitry et al., 1989), indicating that D2 phosphorylation may follow core assembly or require PS II function. In addition to the posttranslational destabilization of PS II intrinsic subunits synthesized in the above mutants, there is some evidence for the translational regulation of Dl by D2 (Erickson et al., 1986; de Vitry et al., 1989) and for a tight coupling of D1 and P5 translation (Jensen et al, 1986; de Vitry et al., 1989 and Summer et al., 1997), such that D2 may regulate synthesis of both Dl and P5. Alternatively, interaction with D2 may stabilize a newly synthesized Dl polypeptide (Wu and Kuchka, 1995). A second class of C. reinhardtii PS II mutants includes those obligate heterotrophs missing the psbH or psbK chloroplast gene products. In these mutants, the remaining PS II subunits are degraded more slowly than in the first class of mutants; small amounts of PS II complexes are formed, but are not sufficient to support photoautotrophic growth. Takahashi et al.

261 (1994) showed that disruption of Chlamydomonas psbK did not affect synthesis of the other PS II core subunits, but significantly reduced accumulation of PS II subunits as assessed by pulse chase experiments and immunoblotting. The psbK deletion mutants accumulated 620 nm) were identical to vascular plant storage starches. 6) The chain-length distribution ofAp was shown to be trimodal and identical to vascular plant storage starches. 7) The proton and carbon NMR spectra ofAp and Am were identical to the vascular plant storage starches. 9) The wide-angle X-ray diffraction pattern of wild type C.reinhardtii starches were of the Atype confirming the identity with cereal endosperm starches. 10) The physicochemical properties of the algal starches assayed by DSC (Differential Scanning Calorimetry) were analogous to higher plant storage starches. C. reinhardtii after 5 days of nitrogen starvation is displayed in Fig. 2A and compared to log phase-grown nitrogen-supplied algae (Fig. 2B). It is evident that drastic modifications in the flow of carbon metabolism have occurred. In nitrogensupplied medium, in the presence of acetate and light, log phase cells accumulate modest amounts of starch around the pyrenoid and in the stroma. The bulk of the carbon is directed to the building of the new cellular material required for the rapid cell division that occurs under these optimal growth conditions. Cell division provides thus a powerful photosynthate sink yielding a net outflow of carbon from the plastid to the cytosol. During nitrogen

Steven G. Ball

554

amylose. Moreover the remaining amylopectin displayed an altered chain length distribution consisting of a relative increase both in short (50) chains. Very recently, Van den Koornhuyse et al. (1996) have shown that starches extracted from mutant cells defective for the supply of the ADP-glucose substrate displayed during nitrogen starvation a structure identical to that of the polysaccharide purified from wild-type nitrogen-supplied cultures. Therefore storage starch becomes a phenocopy of the transient form of polysaccharide whenever the supply of substrate is lowered. We believe that these structural differences have important functional implications with respect to crystallinity of the polysaccharides and therefore to the availability of the carbon stores through degradation. It is possible that switches from one structure to another may be triggered by modifying simply the substrate supply without the need to induce a specific subset of enzyme activities. The required changes will be obtained through the differences in affinities ofthe various starch synthases of distinct structural specificity for their common ADP-glucose substrate. Similar differences were found between the structures of transient and storage starches in higher plants. In pea embryos, mutations leading to a decrease in ADP-glucose supply were also found to substantially decrease the relative amount of amylose (for review, see Smith et al., 1997). II. The Starch Pathway

A. An Overview of Starch Metabolism starvation cellular material such as proteins, RNAs or photosynthetic pigments are degraded and converted into storage molecules such as starch or lipids. There is therefore a net inflow of carbon from the cytoplasm into the non photosynthetic plastid. These two extreme physiological conditions mimic the differences that are witnessed in vascular plants between the plant leaf chloroplast and the non photosynthetic amyloplast from storage organs. Libessart et al. (1995) have witnessed drastic differences in the structure and composition of starches extracted from nitrogen-supplied and nitrogen-starved Chlamydomonas cells. By comparison with starved cells (storage starch), starch extracted from nitrogen-supplied cultures (transient starch) contained very little low molecular weight

Our present state of knowledge concerning starch metabolism is summarized in Fig. 3. It is presently clear that starch biosynthesis has evolved from the preexisting cyanobacterial glycogen synthesis pathway. Before we detail various aspects of green algal starch biosynthesis it is necessary to give a brief account of the basic reactions involved. These consist of synthesis of the ADP-glucose substrate, transfer of glucose to the growing polysaccharide chain (elongation) and branching: 1) Plastidial phosphoglucomutase (Pgm, EC 5.4.2.2) Glucose-6-P

Glucose-1-P.

Chapter 29

Starch Biosynthesis

555 Starch synthases transfer glucose from the glycosylnucleotide ADP-glucose to the nonreducing end of a growing linked glucan. These enzymes catalyze an elongation step and cannot prime the reaction. Starch synthases can be found either predominantly bound to the granule or in the soluble phase. It is now accepted that the major granule-bound starch synthase is the major, if not only, amylose biosynthetic enzyme. Three distinct types of starch synthases have been found in those species of vascular plants that were characterized in sufficient detail. Their relative contributions to amylopectin synthesis remain unknown. 4) Branching enzyme (EC 2.4.1.18)

While in bacteria phosphoglucomutase cannot be considered a step specific to glycogen synthesis only, the conversion of glucose-6-P to glucose-1P by Pgm is the sole documented function for the plastidial form of the plant enzymes and can thus be considered as the first step ofthe starch pathway. 2) ADP-glucose pyrophosphorylase (AGPase, EC 2. 7. 7. 27). Glucose-1-P + ATP

ADP-glucose + PPi

ADP-glucose pyrophosphorylase catalyzes the rate-limiting step of starch and bacterial glycogen synthesis. The bacterial enzyme is a homotetramer made of subunits ranging in the 50 to 55 kDa range. 3-PGA is the most efficient allosteric activator of the cyanobacterial AGPase while orthophosphate is the most potent inhibitor. The higher plant enzymes are heterotetramers harboring two distinct but related types of subunits in the same size ranges as those of the bacterial enzymes. The so-called small subunits are proposed to be the catalytic subunits while the large subunits are proposed to modulate the sensitivity to allosteric effectors. As with the cyanobacterial enzyme the plant AGPase is exquisitely sensitive to the 3-PGA to orthophosphate ratio. 3) Starch synthase (EC 2.4.1.21) ADP-glucose +

ADP +

linked glucan ofat least 12-16 residues Linear branched glucan with one branch point. These enzymes share many common features with other starch hydrolytic enzymes. The vascular plant enzymes display the barrel enzyme core structure which is common to amylases, debranchhydrolases. ing enzymes and many other Two distinct families are found in vascular plants. Mutations in the structural gene for the A (type II) family have been reported to alter amylopectin synthesis and lead to the high amylose phenotype. No functions have yet been found for enzymes of the B (type I) family. Starch catabolism in plants involves three distinct pathways including hydrolases, phosphorylases and lyases. The relative importance of the three pathways in the different tissues is still a matter of debate (for a review see Steup, 1988). Starch hydrolases transfer a glucosyl group to water by a general acid catalysis mechanism. Amylases are generally classified as according both to the optical properties or EC of cleavage products and to the endo ( 3.2.1.1) or exo-amylase ( EC 3.2.1.2) type of reaction. Because of the endo-type attack and its unique ability to work on the granule itself, is able to rapidly depolymerize starch and thus paves the way for complete degradation of the polysaccharide through the production of soluble Amylases unlike several other hydrolases linkage. are unable to cleave the Starch phosphorylase (E.C. 2.4.1.1) catalyzes the transfer of glucose from the non-reducing end of an

556 glucan to orthophosphate yielding glucose-1 -phosphate. Starch lyases (EC 4.2.2.-) have presently only been described in red algae and fungi (Yu and Pedersen, 1993; Yu et al., 1997). These enzymes produce 1,5-anhydrofructose from the non glucan. Neither reducing end of an lyases nor phosphorylases can degrade the linkage or bypass the branch point. Therefore complete degradation always does require to some extent the concerted action of hydrolytic enzymes linkage. able to cleave the

B. Enzymes of Starch Metabolism in Chlamydomonas While in depth characterizations ofpartially purified ADP-glucose pyrophosphorylase and starch synthase from Chlorella pyrenoidosa (Preiss and Greenberg, 1967; Sanwal and Preiss, 1967), as well as of phosphorylase and ADP-glucose pyrophosphorylase from Chlorella vulgaris (Nakamura and Imamura, 1983, 1985), had been previously reported, the first characterizations of enzymes of starch metabolism in C. reinhardtii can be traced back to 1984 (Levi and Gibbs, 1984). In this study phosphorylase, amylase, limit dextrinase (EC 3.2.1.3) and maltase ( EC 3.2.1.20), four enzymes thought to be involved in starch catabolism were detected in synchronously grown (12-h light/dark cycle) C. reinhardtii cells. Limit-dextrinase can be defined as linkage. an enzyme that can only hydrolyze the Limit-dextrinases and isoamylases are collectively known as debranching enzymes. Limit-dextrinase, also known as plant pullulanase can digest pullulan, a bacterial polysaccharide made of a regular succession of maltotriose chains linked together by linkages at the ends ofeach maltotriosyl residue. Isoamylase cannot debranch tightly spaced branches and thus will not hydrolyze pullulan. However it will cleave more efficiently both glycogen and amylopectin. The activities of both phosphorylase and amylase were characterized in greater detail and followed through the cell cycle. Levi and Gibbs (1984) were able to monitor a four to five-fold increase in both activities between the midlight and middark periods. Both enzymes had broad pH optima ranging between 6.5 to 7.5. However the activity of only the amylase displayed a sharp decrease above 7.6. This could be correlated with the in vivo stromal pH shifts from 7 to 8 known to occur during a transition from dark to light. These observations are consistent with

Steven G. Ball a function of both enzymes in starch degradation. The partially purified amylase was further characon the grounds of the kinetics terized as an of amylopectin degradation. Moreover the enzyme was shown to be heat labile at 55 °C (5 min), inhibited by 2 mM N-ethylmaleimide (NEM) and insensitive to 10 mM EDTA inhibition. These properties are shared with the spinach leaf amylase but differ from those of the enzymes involved in the degradation of storage starch during germination. An enzyme of similar properties was also partially purified by Mouille et al. (1996a) and shown to be a 53 kDa starch hydrolase. Moreover Mouille et al. (1996a) were able to identify the nature of the oligosaccharides produced by the enzyme. These consisted ofvery short highly branched glucans less than 12 glucose residues in overall size. Further characterization of the C. reinhardtii phosphorylase was performed by Ball et al. (1991) who reported a strong mixed type of inhibition of the enzyme by ADP-glucose (50% inhibition by 7 assayed at 1.3 mM Pi). This inhibition, which was also found in Chlorella vulgaris by Nakamura and Imamura (1983), distinguishes the algal phosphorylases from their land plant counterparts. Strong inhibition of phosphorylases by the substrate of starch synthesis also argues that the enzyme is involved solely in polysaccharide degradation. Soluble and granule-bound starch synthases were reported initially in C.reinhardtii by Kuchitsu et al. (1988) who noted a three to five-fold increase in total grown soluble starch synthase activity in 4% cells by comparison to air-adapted cultures. Two soluble starch synthases (SSSI and SSSII) were subsequently partially purified and characterized by Fontaine et al. (1993), Maddelein et al. (1994) and Buléon et al. (1997). Molecular masses of 115 and 75 kDa were respectively measured for SSSI and SSSII by zymogram in denaturing conditions and western blotting. SSSI favored elongation ofglycogen over amylopectin while SSSII displayed opposite preferences. Both enzymes were unable to prime the reaction even in the presence of 0.5 M citrate while SSSII was clearly inhibited and SSSI activated twofold under these conditions. This behavior distinguished the algal synthases from the vascular plant enzymes. Moreover to date no traces could be found of a third soluble starch synthase in C. reinhardtii cultures (Buléon et al., 1997). GBSSI from C. reinhardtii was characterized as a 76 kDa protein with N-terminal sequences similar to those

Chapter 29

Starch Biosynthesis

reported for higher plant enzymes (Delrue et al., 1992). The granule-bound enzyme displayed a 4 mM for ADP-glucose, a five to ten-fold higher value than those reported for the soluble enzymes, and unlike higher plant enzymes could not use UDPglucose. The optimal pH of both granule-bound and soluble enzymes is markedly alkaline (above 9) which fits the biosynthetic function of these enzymes. Branching enzymes were initially detected by Ball et al. (1990, 1991). However the first significant characterizations of branching enzymes in C. rein­ hardtii were reported by Fontaine et al. (1993). Two branching enzymes (BEI and BEII) were detected following anion-exchange chromatography in C. reinhardtii. Both ofthese activities branched potato amylose and produced a polysaccharide with a of 540 nm. However BEII turned out to branch amylose faster than BEI. While the numbering ofthe enzyme peaks reflect their elution order on the columns, their characterization is presently insufficient to relate them precisely to the vascular plant enzymes ofthe A or B families. However biochemical results obtained to date are sufficient to imply the existence of at least two types of enzyme activities. ADP-glucose pyrophosphorylase was reported and characterized in partially purified extracts by Ball et al. (1991) and was subsequently purified to homogeneity by Iglesias et al. (1994). The enzyme was shown to be a heterotetramer composed of two 53 kDa and two 51 kDa subunits which both crossreacted with antiserum directed against the spinach leaf enzyme (Iglesias et al., 1994). The enzyme was found to be activated 22 fold by 3-PGA and inhibited by orthophosphate. The activity is thus tightly regulated by the 3-PGA/Pi ratio as is the case for most ADP-glucose pyrophosphorylases from both vascular plants and cyanobacteria. Cytosolic and plastidic phosphoglucomutase activities of similar properties were also reported in C. reinhardtii (Klein, 1986). It is therefore presently clear from all these enzymological studies that all the critical activities reported to be involved in higher plant starch synthesis are found in C. reinhardtii in the same number of isoforms and with similar biochemical properties. These observations further strengthen the case of C. reinhardtii as an ideal microbial model system to understand the biogenesis of the higher plant starch granule.

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C. Compartmentation of Starch Metabolism in Chlamydomonas A detailed compartmentation study was reported for enzymes of glycolysis and of the oxidative pentosephosphate pathway (Fig. 4) (Klein, 1986). For the activities of the first part of the glycolytic chain (from fructose-6-phosphate to triose phosphate), over 90% of the activity was shown to be associated with the plastid. For the enzymes required for the conversion of 3-PGA to pyruvate, over 95% of the activity was associated with the cytosolic fractions. Moreover 70% of both glucose-6-phosphate dehydrogenase and gluconate-6-phosphate was found in the plastid (Klein, 1986). Hexokinase activity was also detected in C. rein­ hardtii (Levi and Gibbs, 1984). While the compartmentation of the hexokinase was not studied, C. reinhardtii’s inability to grow on glucose supplied in the medium as a carbon source together with the plastid location of the upper glycolytic pathway and the presence of a plastid-located starch degradation pathway strongly suggest the need for a plastidic form of the enzyme. Two hexokinases were found associated to purified chloroplasts by Singh et al. (1993), both in spinach and Chlamydomonas. The major form was found in the chloroplast stroma while the minor form was assigned to the cytoplasmic side of the outer plastid membrane. Moreover the entry ofglucose into the plastid was largely dependent on the exogenous supply ofATP. According to Singh et al. (1993), this could be explained by the presence of a hexose-phosphate translocator that would be supplied with glucose-6-P by the outer membrane hexokinase. Phosphoglucoisomerase and phosphoglucomutase which are needed to metabolize glucose-1-P produced by starch phosphorolysis were found both inside and outside the chloroplast. As to the enzymes of starch metabolism per se, extra-plastidic forms of or phosphorylase were not detected (Levi and Gibbs, 1984; Klein, 1986). These observations are at variance with those reported for higher plant leaves but remain in perfect agreement with those reported for Dunaliella marina, another unicellular green alga of the order Volvocales (Kombrink and Wöber, 1980). In the case of D. marina compartmentation studies coupled to detailed activity and zymogram analyses of starch synthases, branching enzymes, phosphorylases and ADP-glucose pyrophosphorylase establish the starch pathway as exclusively intra-

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plastidic. In C. reinhardtii classical algal transit peptide sequences were indeed encoded by the ADPglucose pyrophosphorylase large subunit cDNA (Van den Koornhuyse et al., 1996). The picture that is slowly emerging for unicellular green algae is that of an entirely plastidic location of starch metabolism. This was further confirmed by Kreuzberg et al. (1987) and by a recent study of the compartmentation of metabolite pools (Klock and Kreuzberg, 1990). The compartmentation of glycolysis displayed in Fig. 4 requires the presence of a phosphate translocator to shuttle triose-P and 3-PGA from the plastid to the cytosol. Such a translocator has indeed been found (Klein et al., 1983) in C. reinhardtii. Moreover metabolites exported by intact purified chloroplasts after 15 min illumination in the presence were conclusively shown to consist of of labeled 23% 3-PGA, 15% DHAP, 20% hexose monophosphates and 13% glycolate (Klein, personal communication). Therefore in addition to triose-P transported through the phosphate translocator, hexose phosphates can also be shuttled between the two cellular compartments.

Steven G. Ball

D. Regulation of Starch Metabolism Contradictory results have appeared concerning polysaccharide content or the rates of starch accumulation during the cell cycle of synchronized microalgal cultures. While the contradictions may be due to species or strain specific differences, they most probably originate from the very different techniques that were used to measure these rates. Of particular relevance are the observations made by Klein (1987) which point to the existence of two maxima, at the very beginning and end of the light phase, in the starch content of synchronized (12 h light/ 12 h dark) C. reinhardtii cells. While, as was found by several others, the starch slowly decreased at night, an even sharper decline was observed at the middle of the light phase. This decrease coincides with the minimum observed in independent measures of starch synthesis rates. In fact net degradation was observed when measuring these rates despite the presence of an overall constant rate of ´ fixation in the light phase. These results strongly suggest cell cycle control of starch degradation during the light phase.

Chapter 29 Starch Biosynthesis Spudich and Sager (1980) in an older report had also noted that starch degradation measured in darkness was greatly stimulated if the cells switched to the dark were taken at mid-G1 at the middle of the light phase. It would be of great interest to correlate these observations with measures of key enzyme and most activities such as phosphorylase, of all ADP-glucose pyrophosphorylase. The evidence obtained by Levi and Gibbs (1984) seems to rule out and phosphorylase activities an increase in at the middle of the light phase. This analysis should also take into account the evolution of the plastidic pools of orthophosphate, hexose monophosphates, ADP-glucose and 3-PGA during the light phase. It must be stressed that Stitt and Heldt (1981) as well as Kruger et al.(1983) have found similar results for isolated spinach chloroplasts. In C.reinhardtii, a net decrease in ADP-glucose concentration would rapidly trigger degradation by relieving the inhibition of phosphorylase by the nucleotide sugar. Several studies have appeared concerning starch breakdown under anaerobiosis in darkness (Klein and Betz, 1978; Gfeller and Gibbs, 1984; Kreuzberg and Martin, 1984). Of interest is the oscillatory fermentation and starch degradation that was noted and characterized by Kreuzberg and Martin (1984). After a switch to anaerobiosis and darkness the rate of starch degradation decreases and increases (oscillates) in a highly regular fashion. A 59 min mean period of starch degradation was measured with phosphofructokinase as a possible rate-limiting step. Because they do not detect significant amounts of glucose in their extracts, Kreuzberg and Martin (1984) believe that the major route for starch degradation during anaerobiosis in darkness requires phosphorylase followed by glycolysis. Ofcourse one cannot rule out a hydrolytic onset of starch degradation followed by phosphorolysis ofthe soluble dextrin. Degradation of starch in darkness through either glycolysis or the pentose phosphate pathway both yield reducing equivalents that have to be reoxidized within the plastid. While both chlororespiration and the activity of a plastidic malate/oxaloacetate shuttle have been proposed as possible mechanisms for regeneration ofoxidized NADP for continuous starch breakdown, Klöck and Kreuzberg (1987) suggest the involvement of a plastidic glycerol-3-phosphate dehydrogenase that reduces one DHAP into glycerol3-P for each 3-PGA generated through starch breakdown. Klöck et al. (1989) further prove that starch degradation from isolated chloroplasts in the

559 dark does not require oxygen. It must be stressed that most studies dealing with starch degradation deal more with establishing the routes of fermentation from starch and their regulation, which are out of the scope of this review, rather than the mechanisms of starch mobilization per se. The identity of the enzymes responsible for breakdown of the crystalline granules, the nature of the dextrin produced and the key metabolic signals triggering starch degradation have, in fact received very little attention. The study of granule degradation and its regulation in C. reinhardtii could prove invaluable to understand the mechanisms at work for starch degradation in vascular plants.

III. The Genetics of Starch Biosynthesis

A. The Iodine Screen for Starch Defective Mutants Nitrogen starved Chlamydomonas cells, especially when supplied with acetate, loose the bulk of their photosynthetic pigments and accumulate both lipids and starch (Ball et al., 1990; Ball et al., 1991; Libessart et al., 1995). Cells plated on solid media with acetate but limiting nitrogen appear as pale yellow patches prior to the first round of iodine vapor spraying. The fixed cells destain rapidly and bleach completely within a few hours. The white fixed patches can be kept indefinitely and restained at will with iodine vapors. The color appearing then is that of the pure iodine-polysaccharide interaction with very little or no interference due to other cellular material (see linked Color Plate 7). Iodine interacts with glucans by inserting in the hydrophobic cavity of the linear glucan helices. However these helices can be generated only if the chains are sufficiently long. This explains why iodine will not interact with linear chains under 12 glucose residues in length at 20 °C. Above this threshold value the interaction will yield complexes whose stability will depend on length (Banks et al, 1971). Moreover the color of the iodine glucan complex will evolve from a weak brown to a strong dark green color for the chains with more than 100 glucose residues. This explains why the iodine-polysaccharide complexes of glycogen (with very short chains), amylopectin (with medium size chains) and amylose (with extra-long chains) will stain respectively brown, purple and green. The wave-length of the maximal absorbence of the iodine-polysaccharide complexes

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(the ) will range respectively between 450–490 nm (glycogen) to 530-560 nm (amylopectin) and 600-650 nm (amylose). The mix of the purple amylopectin with the greenish amylose yields the dark blue color that typifies starch. It so happens that the starch content of 5 to 10 d-old nitrogen-starved algae is optimal to detect both large and minute differences in color among thousands to millions of surviving colonies on a few petri dishes. Because a direct screen with iodine cannot be applied easily in higher plants, Chlamydomonas ranks first as to the ease with which mutants defective for the starch pathway can be detected. In vascular plants mutant screens rely mostly on selection of kernels or seed with abnormal shape or texture. A detailed account of the elegant work performed on the maize mutants defective for starch biosynthesis can be found in Nelson and Pan (1995). Subsequently it was found that starchless algae are perfectly viable thus facilitating even more the genetic dissection ofstarch metabolism. Four distinct phenotypes can be revealed in C. reinhardtii. Nitrogen-starved cell patches will stain yellow if the amount ofpolysaccharide falls beneath 5% of the wild-type content. Purple-red cell patches will appear upon loss ofamylose. A relative decrease in amylopectin content will yield the high-amylose olive-green phenotype. The dark blue phenotype characterizes the wild-type references.

B. Mutants Defective for the Supply of ADPglucose Mutations in three different loci (STA1, STA5 and

Steven G. Ball

STA6) lower the supply of ADP-glucose in the chloroplast. The X-ray induced sta1-1 (Ball et al., 1991) and the insertion alleles sta1-2::ARG7 and sta1-3::ARG7 (van den Koornhuyse et al., 1996) all lead to identical phenotypes (Table 2). Mutants of STA1 accumulate from 1 to 8% of the wild-type starch amount. Moreover ADP-glucose pyrophosphorylase has a lower response to 3-PGA. While the purifiedADP-glucose pyrophosphorylase displays a maximal 22-fold activation by 3-PGA (Iglesias et al., 1994), the mutant enzyme either partially purified (Ball et al., 1991), or purified to near homogeneity (van den Koornhuyse et al., 1996), shows at most a two-fold enhancement. Ofparticular relevance is the fact that the kinetics of the mutant enzyme and its sensitivity to orthophosphate are unaffected in the absence of 3-PGA (Ball et al., 1991; van den Koornhuyse et al., 1996). In addition, the total amount of activity measured for the mutants ranges between 50 to 100% ofthe wild-type. Therefore the phenotype of the mutants can be solely attributed to the lack of 3-PGA activation of the enzyme and brings in vivo demonstration of the importance of allosteric regulation of the enzyme. We believe that it is the remaining sensitivity of the mutant enzyme to the orthophosphate inhibition which is responsible for the collapse of starch synthesis. Van den Koornhuyse et al. (1996) have cloned and sequenced a cDNA corresponding to the large subunit of ADP-glucose pyrophosphorylase. The cDNA was used as probe to establish that STA1 indeed encoded an ADP-glucose pyrophosphorylase subunit gene (van den Koornhuyse et al., 1996). Recently insertional mutagenesis yielded a novel mutant

Chapter 29

Starch Biosynthesis

containing less than 0.1% total storage polysaccharide making it and the Arabidopsis thaliana adg1 defective plants (Lin et al., 1988) the most severely impaired starch mutants isolated to date (C. Zabawinski, personal communication). The sta6-1::ARG7 carrying mutant grew well but turned out to be sterile and paralyzed. The starchless mutants were completely devoid of ADP-glucose pyrophosphorylase activity. PEG induced fusions between the wall defective original mutant strain and protoplasts generated from a sta1-1 carrying strain yielded fully wild-type pseudo-diploids establishing trans complementation of all defects. The fusion product proved fertile in crosses and was used to produce triploid zygotes. Upon meiosis the aneuploid progeny was used to analyze the segregation of the defects. This analysis coupled to Southern blotting using the large ADP-glucose pyrophosphorylase subunit cDNA probe established the presence of 2 genes required for AGPase activity. STA1 is now proven to encode the large 53 kDa regulatory subunit. The sta1-1 allele was also proved either to be a major translocation or, more likely, a pericentric inversion ofa large chromosomal segment. The inversion (or translocation) occurred in the middle of the enzyme coding sequence. Therefore the phenotype of sta1-1 is explained by the absence of the large subunit and the formation either of fully active monomers or more likely of a homotetramer ofsmall subunits. This was confirmed by the isolation of insertional mutants with the very same phenotype (van den Koornhuyse et al., 1996). The most likely hypothesis to explain the extremely severe phenotype caused by the sta6-1::ARG7 allele is that it encodes the small 51 kDa catalytic subunit of the enzyme. Interestingly it is clear from the triploid zygote genetic analysis that sterility segregated independently from both STA6 and STA1. Sterility therefore does not come as a consequence ofimpaired mobility and loss of starch. Indeed Fletchner and Cirino (1992) had previously demonstrated that photosynthetic mutants with low starch storing capacity did not differ in their mating efficiency when compared to other such mutants with normal or high starch storing abilities. Paralysis however could still define a major pleiotropic effect due to the absence ofstarch. Interestingly the flagellae ofstrains carrying the sta6-1::ARG7 allele appeared normal under the microscope while motility could not even be rescued through the supply of acetate in the medium.

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A spontaneous mutant displaying a low starch phenotype was isolated by Bulté and Wollman. The mutant carried a single genetic defect at the STA5 locus and was subsequently found to be selectively defective for the major Chlamydomonas phosphoglucomutase (van den Koornhuyse et al., 1996). The phenotype seemed however less severe than that of the corresponding Arabidopsis (Caspar et al., 1985; for review see Caspar, 1994) and Nicotiana sylvestris (Hanson and McHale, 1988) mutants. Since Klein (1986) showed that the major C. reinhardtii phosphoglucomutase was compartmented in the chloroplast and since the whole starch pathway seems to be confined to the plastid (Kombrink and Wöber, 1980), we conclude that it is the plastidic form of the enzyme that is missing in the mutant. Mutants carrying sta5-1 accumulate from 4 to 20% of the normal starch amounts (van den Koornhuyse et al., 1996). We believe this reflects the activity of the minor cytosolic phosphoglucomutase and strongly argues that glucose-1-P is transported through the plastid membranes (Fig. 4). As mentioned above, sta1-1 and sta5-1 both display modified structures of their residual starches without showing any modification ofall other major enzymes involved in starch biosynthesis. We thereby conclude that ADP-glucose supply tightly controls the polysaccharide’s structure and propose that this occurs through differential effects on the balance of the active starch synthases. It is known, for instance, that GBSSI has a five-fold higher for ADP-glucose than the soluble starch synthases. Modifications in the ADP-glucose pool could then alter the structure because ofthe specialized functions displayed by the different starch synthases in the building ofdifferent granule substructures (van den Koornhuyse et al., 1996)

C. Mutants Defective for Elongation Mutants defective for amylose biosynthesis appear as red staining cell patches on solid nitrogen sprayed with iodine (Table 2). All mutants selected map to a single locus (STA2) for which 26 distinct UV and two insertional mutants were generated (Delrue et al., 1992; Maddelein et al., 1994). All mutants are characterized by an identical phenotype. They accumulate wild-type amounts of starch lacking both amylose and a moderately branched fraction that was named Ap II (amylopectin type II). This fraction is clearly related to the intermediate material that is

562 found in wild-type starches ofhigher plants. Moreover a small but significant difference was found in the long chain content ofthe major amylopectin fraction (Ap I). It was therefore concluded that STA2 controls both the biosynthesis ofamylose and the fine structure of amylopectin. Delrue et al. (1992) described two mutations that yielded allele-specific modifications of both the molecular mass of the major protein associated with the granule and of the for ADP-glucose of the major granule-bound starch synthase activity. Moreover the major 76 kDa protein bound to the granule displayed the classical N-terminal KTGGL sequence reported for starch synthases. Insertional mutants carrying the sta2-27::ARG7 allele were further shown to lack both GBSSI and the major 76 kDa starch-bound protein. Taken together these observations make a compelling case for STA2 being the structural gene for GBSSI. X-ray mutagenesis has yielded three distinct alleles of a single locus (STA3) that leads to 40 to 80% decrease in starch amounts (Fontaine et al., 1993). The residual starch is enriched in amylose and yields the characteristic olive-green phenotype of the highamylose class of mutants (Table 2). The overall defect thus consists ofa 90% decrease in amylopectin synthesis. The remaining amylopectin has a completely modified structure. The chain-length distribution of amylopectin shows a prominent maximum at d.p. 6 (degree of polymerization of 6) and a strong decrease in those chains ranging in size between 8 to 40 glucose residues (Fontaine et al., 1993). These make the bulk of the so-called amylopectin clusters (see Fig. 1 panels C and D). Since the clusters harbor the glucan double helices responsible for the crystallinity ofstarch we suspected that the basic organization ofamylopectin was altered in the mutants. Wide-angle X-ray diffraction experiments confirm these suspicions (Buléon et al., 1997). The overall crystallinity in sta3 mutants has virtually collapsed and switched to the B-type of crystalline lattice. This is correlated with an unusual melting behavior ofthe glucan double helices during DSC (Differential Scanning Calorimetry) experiments suggestive of the presence of shorter double helices. Moreover the shape and size distribution of the granules are altered. These profound and diverse modifications can be traced back to one single enzymological defect. All mutants were shown to lack SSSII (Soluble Starch Synthase II) activity. Fontaine et al. (1993) also

Steven G. Ball found a two-fold decrease of the activity in heterozygous mutants which suggests thatSTA3 could be the structural gene for the enzyme. Maddelein et al. (1994) conducted a detailed investigation of mutants defective at the STA3 and STA2 loci. Double mutant strains witnessed a collapse of starch biosynthesis despite the presence of fully active SSSI which accounts for half of the total soluble starch synthase activity. This suggests that SSSI cannot fully sustain amylopectin synthesis on its own. Moreover the 5% residual polysaccharide found in the double mutants was deeply modified. In fact, its structure does not truly qualify as starch but still adopts a dense macrogranular organization with altered shapes and sizes. The chain-length distribution of the double mutant polysaccharide still harbors the prominent maximum at d.p 6, but now consists solely of very short glucans. The X-ray diffraction pattern has switched from the B to the C type and has regained some crystallinity due to the absence of the amylose fraction. The results of Maddelein et al. (1994) also confirm the suspected function ofGBSSI in the synthesis of the long glucans associated with (and covalently bound to) amylopectin.

D. Mutants with Unidentified Enzymological Defects Another high amylose, X-ray generated mutant displayed a significant (from 40 to 60%) reduction in starch amount together with a strong and intriguing modification of the chain-length distribution of the residual starch (Libessart et al., 1995). The phenotype was clearly seen on storage starch but was hard to distinguish from wild-type transient starch when the polysaccharide was extracted from nitrogen-supplied mutant cells. Despite intensive efforts we were unable to find the enzymological defect in mutants of the STA4 locus. Further characterization will have to await tagging of STA4 by insertional mutagenesis. Equally intriguing is the recent finding of low starch mutants in the homothallic Chlamydomonas monoica reported by Rickoll et al. (1997). The authors convincingly showed the presence of a mutation in a single mendelian gene (STA-1, not to be confused with the STA1 locus from C. reinhardtii). Electron micrographs failed to reveal standard starch granules in the mutants. However the defective algae still contained a quite significant amount of starch with a more or less normal structure and buoyant density. We have found the major biosynthetic enzymes in

Chapter 29 Starch Biosynthesis Chlamydomonas monoica but have presently not uncovered the biochemical defect. We do not yet know how the material we have extracted relates to the amorphous material seen on the electron micrographs reported by Rickoll et al. (1997). If this material is starch then the C. monoica mutants will be the first oftheir kind reported in the plant-kingdom and therefore deserve particular future attention. STA-1could thus be quite uniquely involved in starch granule morphogenesis.

E. Mutants Defective for Pre-Amylopectin Trimming Insertional mutagenesis has generated a new class of mutants initially displaying both the low starch (staining yellow with iodine) and the high-amylose (staining olive-green with iodine) phenotypes. Mutants of STA7 (Mouille et al., 1996a) and STA8 (Mouille et al., 1996b) both lead to the production of a water-soluble polysaccharide (WSP). This fraction amounted to 5% ofwhat would have been the amount of starch in a wild-type strain. The water-soluble linked and polysaccharide consisted of branched glucans with a chain-length distribution, an iodine interaction ( of 490 nm) and NMR spectra analogous to animal, fungal or bacterial glycogen. The polysaccharide was thus named phytoglycogen by analogy with the maize su1 (sugary 1) mutation which leads to the accumulation of a similar fraction (Sumner and Somers, 1944; for review see Nelson and Pan, 1995). The phytoglycogens from sta7 and sta8 mutants differ with respect to their chain-length distributions. In addition to phytoglycogen, sta8 mutants accumulate high amylose (60%) granular starch amounting to 20% of the starch content from a wild-type strain. The high amylose phenotype is expected for a mutation acting predominantly on amylopectin synthesis. The sta7 mutants accumulated up to 0.5% granular material. This material consisted of virtually pure amylose with a maximum of 1% linkages. Again in sta7 mutants we suspect that the major defect remains in the amylopectin pathway and that the collapse of amylose synthesis comes as an aftermath of the disappearance of the minimum amount of granular structure required for GBSSI binding. The phenotype of sta8 mutants resembles that of the su1 mutation harbored by sweet corn while that of sta7 containing mutants remains to be described in higherplants. Sweet corn was shown to be defective

563 for a debranching enzyme activity (Pan and Nelson, 1984; James et al., 1995). Enzymological characterization ofthe 7 distinct sta7 alleles have uncovered a selective defect in a 88 kDa debranching enzyme that behaved mostly as an isoamylase and displayed very weak pullulanase activity. No other enzymological defect could be scored in the mutants thus establishing that polysaccharide debranching is mandatory to obtain significant amylopectin synthesis. While sta8 containing mutants displayed wild-type levels of the 88 kDa debranching enzyme, the purification behavior ofthe enzymes on columns was reported to be modified (Mouille et al., 1996b). This suggests that the product of the STA8 locus might be a subunit or other enzyme interacting with the 88 kDa debranching enzyme. The astonishing results obtained during the characterization of STA7 have laid the foundations of a model explaining the biosynthesis of plant amylopectin (Ball et al. 1996). In this model debranching enzymes are considered as trimming enzymes involved in ordering the branches produced in an intermediate of synthesis that was named preamylopectin (see below).

IV. A Model Explaining the Biogenesis of the Plant Starch Granule Until recently researchers have believed that the intrinsic properties of starch synthase and starch branching enzymes would be sufficient to explain starch granule biogenesis. Yet the branching enzymes expressed in vivo in E. coli, even in the presence of glycogen or starch synthases, are unable to yield the asymmetrical pattern ofbranches responsible for the continuous growth ofamylopectin (Guan et al., 1995). By opposition, a symmetrical pattern of branching will lead to an upper limit to the size that the unit granule will be able to reach before steric hindrance impairs further growth. Mathematical modeling predicts that the maximal diameter for a symmetrically branched polysaccharide with a 1 to 12 branching ratio would be 25 nm which is precisely what is measured for the unit particles of glycogen. There seems to have been a tremendous drive to overcome this limit in the plant kingdom. As usual this has not been in a logical fashion by de novo design of a novel optimal pathway, but rather as painting (the old canvas being cyanobacterial glycogen synthesis) by adding an additional color

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touch in the perpetual search for harmony. This was achieved as often is the case by putting old tools (debranching enzyme previously used in bacterial glycogen degradation) to new uses (glucan trimming) and subsequently optimized to retrieve the energy ‘lost’ by the ordering steps. The model proposed in Fig. 5 (Ball et al., 1996) for plant amylopectin synthesis explains how the asymmetrical distribution of branches amylopectin could be generated. The model is built by taking into account the phenotypes of the C. reinhardtii sta3 and sta7 mutations. Briefly, a previously synthesized amorphous lamella containing tightly spaced branches is being elongated by a specific soluble starch synthase (SSSII in Chlamydomonas). No branching occurs at this first step. We explained this (Ball et al., 1996) by assuming that branching is prevented because the branching enzymes need a minimal length for the glucan to adapt in their catalytic site. As soon as this length is attained, unordered branching and elongation (by SSSI) occur simultaneously. At the same time debranching enzymes will trim down the structure into the next amorphous lamella. This model was named the discontinuous synthesis model for amylopectin synthesis. It explains the amylopectin cluster constant size throughout the plant kingdom (Jenkins et al., 1993) and relates the size of the crystalline lamella and therefore of the amylopectin cluster to the minimal requirements of the branching enzymes. Quite interestingly it was subsequently found that maize BEI and BEII require respectively a minimum number of 16 and 12 glucose residues for branching

to occur. A glucan of 12 residues is of the very same size as that measured for the crystalline lamella of maize amylopectin. It is comforting to realize that mutants of maize defective for BEII and thus retaining only BEI witness an increase of the size of this lamella from that which characterizes a d.p. 12 glucan to that of d.p. 16 (Jenkins and Mc Donald, 1996). V. Future Prospects The reasons for the appearance of starch in photosynthetic eukaryotes remain mysterious. Did starch assume early on a function specific to these organisms that could not be taken in charge by glycogen? The size and density of starch granules make these compounds the fastest sedimenting structures. This property could have been put to use for gravity sensing. Indeed the starch-statolith theory of geotropism in higher plants states that it is sedimentation of the starch-filled amyloplast that induces the curving response in roots. Evidence for or against this hypothesis have accumulated over the years. Bean (1977) has noted a negative (positive being downwards) geotactic behavior in Chlamydomonas consisting of an upward swimming pattern in darkness. Since C. reinhardtii is not reported to use gas vesicles as floating devices, the alga must have some gravity sensing mechanism perhaps related to the one used by land plants for oriented cell growth. It will thus be of great interest to examine the geotactic behavior of the starch defective mutants.

Chapter 29 Starch Biosynthesis However, this study will be complicated by the paralysis noted for the sta6-1::ARG7 carrying starchless mutant. An inverse relation was indeed observed between chemical energy storage and cell motility. Paralyzed mutants containing defects in PF-1, PF-2, PF-7 and PF-18, were shown to accumulate a substantially higher level of starch (Hamilton et al., 1992). Whether paralysis comes as a direct consequence of sta6-1::ARG7 will need further work to prove that insertional mutagenesis has not lead to the deletion of a neighboring PF cistron. The most exciting short-term prospects will definitely consist of unraveling the glucan trimming pathway involved in amylopectin synthesis. We strongly believe that more genes involved in this pathway will be identified in Chlamydomonas. Finally the iodine-spray screening procedure can also be adapted to select for strains carrying mutations for starch overproduction (STO) or breakdown (STB). Characterization of such loci has only just begun.

Acknowledgments Research is a collective human effort. The author would thus like to thank all researchers mentioned in this work for their contribution to the understanding of starch biosynthesis in Chlamydomonas. The author would also like to acknowledge the help and insights of Dr. Uwe Klein and Karen Van Winkle-Swift for disclosing and discussing their unpublished results.

References Ball SG (1995) Recent views on the biosynthesis of the starch granule. Trends Glycosci Glycotechnol 7: 405–415 Ball SG, Dirick L, Decq A, Martiat JC and Matagne RF (1990) Physiology of starch storage in the monocellular alga Chlamydomonas reinhardtii. Plant Sci 66: 1– 9 Ball S, Marianne T, Dirick L, Fresnoy M, Delrue B, and Decq A (1991) A Chlamydomonas reinhardtii low-starch mutant is defective for 3-phosphoglycerate activation and orthophosphate inhibition of ADP-glucose pyrophosphorylase. Planta 185: 17–26 Ball S, Guan HP, James M, Myers A, Keeling P, Mouille G, Buléon A, Colonna P and Preiss J (1996) From glycogen to amylopectin: a model explaining the biogenesis of the plant starch granule. Cell 86: 349–352 Banks W, Greenwood CT, and Khan KM (1971) The interaction of linear amylose oligomers with iodine. Carbohydr Res 17: 25–33.

565 Buléon A, Gallant DJ, Bouchet B, Mouille G, D’Hulst C, Kossmann J and Ball S (1997) Starches from A to C: Chlamydomonas reinhardtii as a model microbial system to investigate the biosynthesis of the plant amylopectin crystal. Plant Physiol 115: 949–957 Caspar T (1994) Genetic dissection of the biosynthesis, degradation, and biological functions ofstarch. In: Meyerowitz EM and Sommerville C (eds). Arabidopsis, pp. 913–936, Monograph 27. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Caspar T, Huber SC and Somerville C (1985) Alterations in growth, photosynthesis, and respiration in a starchless mutant of Arabidopsis thaliana (L.) deficient in chloroplast phosphoglucomutase activity. Plant Physiol 79: 11–17 Craigie JS ( 1974) Storage products. In: Stewart WDP (ed) Algal Physiology and Biochemistry, pp 206–235. Blackwells, Oxford Delrue B, Fontaine T, Routier F, Decq A, Wieruszeski JM, van den Koornhuyse N, Maddelein, ML, Fournet B and Ball S (1992) Waxy Chlamydomonas reinhardtii: Monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase accumulate a structurally modified amylopectin. J Bacteriol 174: 3612–3620 Fletcher V and Cirino R (1992) The effect of growth regimen, heat shock and intracellular starch storage on the mating efficiency of Chlamydomonas reinhardtii. Br Phycol 27: 3–9 Fontaine T, D’Hulst C, Maddelein ML, Routier F, MariannePepin T, Decq A, Wieruszeski, JM, Delrue B, van den Koornhuyse N, Bossu JP, Fournet B and Ball SG (1993) Toward an understanding of the biogenesis of the starch granule. Evidence that Chlamydomonas soluble starch synthase II controls the synthesis of intermediate size glucans of amylopectin. J Biol Chem 268: 16223–16230 Gfeller R and Gibbs (1984) Fermentative metabolism of Chlamydomonas reinhardtii. I. Analysis of fermentative products from starch in dark and light. Plant Physiol 75: 212– 218 Guan HP, Kuriki T, Sivak M and Preiss J (1995) Maize branching enzyme catalyzes synthesis of glycogen-like polysaccharide in glgB-deficient Escherichia coli. Proc Natl Acad Sci USA 92: 964–967 H a m i l t o n BS, Nakamura K and Roncari DAK (1992) Accumulation of starch in Chlamydomonas reinhardtii flagellar mutants. Biochem Cell Biol 70: 255–258 Hanson KR and McHale NA (1988) A starchless mutant of Nicotiana sylvestris containing a modified plastid phosphoglucomutase. Plant Physiol 88: 838–844 Hirst E, Manners D, and Pennie IR(1972) Part XXI – The molecular structure of starch-type polysaccharide from Haematococcus pluvialis and Tetraselmis carteriiformis. Carbohydr Res 22: 5–11 Iglesias AA, Charng YY, B a l l S and Preiss J (1994) Characterization of the kinetic, regulatory and structural properties of ADP-glucose pyrophosphorylase from Chlamy­ domonas reinhardtii. Plant Physiol 104: 1287–1294 Imberty A, Buléon A, Tran V and Pérez S (1991) Recent advances in knowledge of starch structure. Starch/Stärke 43: 375–384 James MG, Robertson DS and Meyers AM (1995) Characterization of the maize gene sugary, a determinant of starch composition in kernels. Plant Cell 7: 417–429 Jenkins PJ and Donald AM (1995) The influence of amylose on starch granule structure. Int J Biol Macromol 17: 315–321

566 Jenkins PJ, Cameron RE and Donald AM (1993) A universal feature in the starch granules from different botanical sources. Starch/Stärke 45: 417–420 Klein U (1986) Compartmentation of glycolysis and of the oxidative pentose-phosphate pathway in Chlamydomonas reinhardtii. Planta 167: 81–86 Klein U (1987) Intracellular carbon partitioning in Chlamy­ domonas reinhardtii. Plant Physiol 85: 892–897 Klein U and Betz A (1978) Fermentative metabolism of hydrogenevolving Chlamydomonas moewusii. Plant Physiol 61: 953– 956 Klein U, Chen C andGibbs M (1983) Photosynthetic properties ofchloroplasts from Chlamydomonas reinhardtii. Plant Physiol 72: 488–491 Klöck G and Kreuzberg K (1987) Sn-glycerol-3-phosphate is a product of starch degradation in isolated chloroplasts of Chlamydomonas reinhardtii. Z Naturforsch 42c: 567–569 Klöck G and Kreuzberg K (1991) Compartmented metabolite pools in protoplasts from the green alga Chlamydomonas reinhardtii: Changes after the transition from aerobiosis to anaerobiosis in the dark. Biochim Biophys Acta 1073: 410415 Klöck G, Sueltmeyer DF, Fock HP and Kreuzberg K (1989) Gas exchange in intact isolated chloroplasts from Chlamydomonas reinhardtii during starch degradation in the dark. Physiol Plant 75: 109–113 Kombrink E and Wöber G (1980) Identification and subcellular localization of starch-metabolizing enzymes in the green alga Dunaliella marina. Planta 149: 130–137 Kreuzberg K (1984) Starch formation via a formate producing pathway in Chlamydomonas reinhardtii, Chlorogonium elongatum and Chlorella fusca. Physiol Plant 61: 87–94 Kreuzberg K and Martin W (1984) Oscillatory starch degradation and fermentation in the green alga Chlamydomonas reinhardtii. Biochim Biophys Acta 799: 291–297 Kreuzberg K, Klöck G and Grobheiser D (1987) Subcellular distribution of pyruvate-degrading enzymes in Chlamydomonas reinhardtii studied by an improved protoplast fractionation procedure. Physiol Plant 69: 481–488 Kruger NJ, Bulpin PV and ap Rees T (1983) The extent of starch degradation in the light in pea leaves. Planta 157: 271–273 Kuchitsu K, Tsuzuki M and Miyachi S (1988) Changes of starch localization within the chloroplast induced by changes in concentration during growth of Chlamydomonas reinhardtii: Independent regulation of pyrenoid starch and stroma starch. Plant Cell Physiol 29: 1269–1278 Levi C and Gibbs M (1984) Starch degradation in synchronously grown Chlamydomonas reinhardtii and characterization of the amylase. Plant Physiol 74: 459–463 Libessart N, Maddelein ML, van den Koornhuyse N, Decq A, Delrue B and Ball SG (1995) Storage, photosynthesis and growth: The conditional nature of mutations affecting starch synthesis and structure in Chlamydomonas reinhardtii. Plant Cell 7: 1117–1127 Lin TP, Caspar T, Somerville C and Preiss J (1988) Isolation and characterization of a starchless mutant of Arabidopsis thaliana (L.) Heynh. lacking ADP-glucose pyrophosphorylase activity. Plant Physiol 86: 1131–1135 Maddelein ML, Libessart N, Bellanger F, Delrue B, D’Hulst C, van den Koornhuyse N, Fontaine T, Wieruszeski JM, Decq A and Ball SG (1994) Toward an understanding of the biogenesis

Steven G. Ball of the starch granule: Determination of granule-bound and soluble starch synthase functions in amylopectin synthesis. J Biol Chem 269: 25150–25157 Manners DJ (1989) Recent developments in our understanding of amylopectin structure. Carbohydr Polymers 11: 87–112 Meeuse BJD (1962) Storage products. In: Lewin RA (ed) Physiology and Biochemistry of Algae, pp 289–311, Academic Press, New York and London Meeuse BJD and Kreger DR (1959) X-ray diffraction of algal starches. Biochim Biophys Acta 35: 26-30 Meeuse BJD, Andries and Wood (1960) Floridean starch. J Exp Bot 11: 129–140 Mouille G, Maddelein ML, Libessart N, Talaga P, Decq A, Delrue B and Ball S (1996a) Phytoglycogen processing: A mandatory step for starch biosynthesis in plants. Plant Cell 8: 1353–1366 Mouille G, Colleoni C, Maddelein ML, Libessart N, Decq A, Delrue B and Ball S (1996b) Glucan trimming a novel mechanism that explains the asymmetric distribution of branches in amylopectin. In: Nakamura Y (ed) Regulation and Manipulation of Starch and Sucrose Metabolism in Plants, pp 27–32. National Institute of Agrobiological Resources, Tsukuba. Nakamura Y and Imamura M (1983) Characteristics of a glucan phosphorylase from Chlorella vulgaris. Phytochemistry 22: 835–840 Nakamura Y and Imamura M (198 5) Regulation ofADP-glucose pyrophosphorylase from Chlorella vulgaris. Plant Physiol 78: 601–605 Nelson OE and Pan D (1995) Starch synthesis in maize endosperms. Annu Rev Plant Physiol Plant Mol Biol 46:475– 496 Olaitan SA and Nothcote DH (1962) Polysaccharides of Chlorella pyrenoidosa. Biochem J. 82: 509–519 Pan D and Nelson OE (1984) A debranching enzyme deficiency in endosperms of the Sugary-1 mutants of maize. Plant Physiol 74: 324–328 Pino-Plumed MD, Villarejo A, de los Rios A, Garcia-Reina G concentrating mechanism and Ramazano v Z (1996) The in a starchless mutant of the green unicellular alga Chlorella pyrenoidosa. Planta 200: 28–31 Preiss J and Greenberg E (1967) Biosynthesis ofstarch in Chlorella pyrenoidosa: I purification and properties of the adenosine diphosphoglucose: glucosyl transferase from Chlorella. Arch Biochem Biophys 118: 702–708 Preiss J and Sivak MN (1996) Starch synthesis in sinks and sources. In: Zamski E and Schaffer AA (eds) Photoassimilate Distribution in Plants and Crops: Source-Sink Relationships, pp 63–96. Marcel Dekker Inc., New York Ramazanov Z, Rawat M, Henk MC, Mason CB, Matthews SW concentrating and Moroney JV (1994) The induction of the mechanism is correlated with the formation of the starch sheath around the pyrenoid of Chlamydomonas reinhardtii. Planta 195: 210–216 Rickoll W, Rehkopf D, Dunn C, Van Winkle-Swift, K (1998) The sta-1 mutation prevents assembly of starch granules in nitrogenstarved cells and serves as a useful morphological marker during sexual reproduction in Chlamydomonas monoica (Chlorophyceae). J Phycol 34: 147–151 Robin JP, Mercier C, Charbonnière R and Guilbot A (1974) Lintnerized starches. Gel filtration and enzymatic studies of

Chapter 29 Starch Biosynthesis insoluble residues from prolonged acid treatment of potato starch. Cereal Chem 51: 389–406 Sanwal GG and Preiss J (1967) Biosynthesis of starch in Chlorella pyrenoidosa: II regulation of ATP: glucose-1-phosphate adenyl transferase (ADP-glucose pyrophosphorylase) by inorganic phosphate and 3-phosphoglycerate. Arch Biochem Biophys 119: 454–469 Singh KK, Chen C, Epstein DK and Gibbs M (1993) Respiration of sugars in spinach (Spinacia oleracea), maize (Zea mays), and Chlamydomonas reinhardtii F-60 chloroplasts with emphasis on the hexose kinases. Plant Physiol 102: 587–593 Smith A, Denyer K and Martin C (1997) The synthesis of the starch granule. Ann Rev Plant Physiol Plant Mol Biol 48: 67– 87 Spudich JL and Sager R (1980) Regulation of the Chlamydomonas cell cycle by light and dark. J Cell Biol 85: 136–145 Steup M (1988) Starch degradation. In: Preiss J (ed) The Biochemistry of Plants. A Comprehensive Treatise, Vol 14, pp 255–296. Academic Press, San Diego Stitt M and Heldt HW (1981) Simultaneous synthesis and degradation of starch in spinach chloroplasts in the light. Biochim Biophys Acta 638: 1–11 Sumner JB and Somers GF (1944) The water soluble polysaccharide of sweet corn. Arch Biochem 4: 4–7

567 Süss KH, Prokhorenko I and Adler K (1995) In situ association of Calvin cycle enzymes, ribulose-l,5-biphosphate carboxylase/ oxygenase, ferredoxin reductase, and nitrite reductase with thylakoid and pyrenoid membranes of Chlamydomonas reinhardtii chloroplasts as revealed by immunoelectron microscopy. Plant Physiol 107: 1387–1397 Van den Koornhuyse N, Libessart N, Delrue B, Zabawinski C, Decq A, Iglesias A, Preiss J and Ball S (1996) Control of starch composition and structure through substrate supply in the monocellular alga Chlamydomonas reinhardtii. J Biol Chem 271: 16281–16287 Villarejo A, Martinez F, Pino-Plumed MD and Ramazanov Z concentrating mechanism in (1996) The induction of the a starchless mutant of Chlamydomonas reinhardtii. Physiol Plant 99: 293–301 Yu S and Pedersen M (1993) Alpha-1,4-glucan lyase, a new class of starch/glycogen degrading enzyme. I. Efficient purification and characterization from red seaweeds. Biochim Biophys Acta 1156: 313-320 Yu S, Chrisyensen TM, Kragh KM, Bojsen K and Marcussen J (1997) Efficient purification, characterization and partial amino acid sequencing of two alpha-1,4-glucan lyases from fungi. Biochim Biophys Acta 1339: 311-320

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Chapter 30

State Transition and Photoinhibition

Nir Keren and Itzhak Ohad

Minerva, Avron Even-Ari Center for Photosynthesis Research,

Silberman Institute of Life Sciences, Department of Biological Chemistry,

The Hebrew University of Jerusalem, Jerusalem, 91904 Israel

Summary I. Introduction A. Photosynthetic Electron Flow in C. reinhardtii B. Light Energy Absorption and Dissipation C. Chlorophyll a Fluorescence Measurements II. State Transition: The Phenomenon A. Relation Between the Plastoquinone Pool Redox Balance and State Transitions B. Redox Related Activation of the LHCII-Kinase: Role of Cytochrome bf C. Phosphorylation-Dependent Reversible Association of LHCII with PS II III . Light Stress: Photoinhibition and Recovery A. Mechanisms of PS II Photoinactivation 1. Acceptor-Side Photoinhibition 2. Photoinactivation by Limiting Light Intensities 3. Donor-Side Photoinhibition 4. UV Induced Photodamage B. D1 Cleavage as a Result of the Different Photoinhibitory Treatments 1. Role of the Binding Site in D1 Protein Degradation in vivo C. Degradation of Other PS II Proteins 1. The Nature of the Protease D. The Recovery Process 1. Synthesis and Reassembly of PS II Proteins 2. The Role of Chlorophyll a and

in the Reassembly of PS II IV. Concluding Remarks and Perspectives Acknowledgments References

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Summary Optimal utilization of absorbed light energy and avoidance of oxidative damage induced by excessive excitation (photoinhibition) constitute a major problem for photosynthetic oxygen evolving cells. Adaptation to transient changes in light absorption and energy utilization is achieved by regulation of photochemistry, and both radiative and nonradiative energy dissipation. Balancing the absorbed energy distribution between PS II and PS I is achieved by redox dependent, reversible phosphorylation of the LHCII antennae, resulting in their reversible coupling with the photosystem cores (state transition). This process optimizes linear electron flow and the cyclic electron flow dependent ATP synthesis. Back electron flow, recombination of the PS II primary charge separated pair, and formation of and occur at all light intensities. Generation of active oxygen species causes oxidative damage and induces degradation of PS II-proteins, particularly the J.-D. Rochaix, M. Goldschmidt-Clermont and S. Merchant (eds): The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas, pp. 569–596. © 1998 Kluwer Academic Publishers. Printed in The Netherlands.

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PS II-D1 protein. Excessive excitation induces a stepwise inactivation of PS II, affecting primarily its acceptor side components, thereby promoting charge recombination and oxidative damage. Infrequent excitation of PS II (‘low light’), promotes charge recombination driven by the equilibrium between the oxidized states In this process of the PS II donor side manganese cluster and the reduced semiquinone acceptor are regenerated and their recombination results in oxidative damage exposing the PS II proteins to proteolytic cleavage. De novo protein synthesis and reassembly of PS II compensate for photoinactivation and degradation of the D1 protein. This process involves dissociation of the residual PS II core proteins from the antenna complex, lateral migration from the appressed to the non-appressed lamellae and reassembly with precursor pD1 protein, newly synthesized by polyribosomes bound to non-appressed lamellae. Processing of pD1, light dependent reactivation of the donor side Mn cluster, lateral migration of the reactivated PS II core to the appressed lamellae, and its reassociation with LHCII regenerates active PS II units and complete the PS II photoinactivation and repair cycle. Thus, photoinactivation and repair of PS II activity are regulated at the metabolic and molecular levels and are modulated by environmental changes.

I. Introduction The ongoing effort to understand, in molecular terms, the response of the photosynthetic eukaryotic cell to a wide range of light intensities under various ambient temperature and nutrient conditions involves extended physiological, biochemical, biophysical and molecular studies, which have been reviewed periodically in the past (Barber and Andersson, 1992; Prasil et al., 1992; Ohad et al., 1994; Gal et al., 1997). Many of these studies were reinforced by using C. reinhardtii as a model organism. The classical genetic approach, combined with site-directed mutagenesis and transformation techniques allows one to obtain engineered mutations in the chloroplast encoded genes (see Chapters 4, Kindle; 8, Goldschmidt-Clermont). Thus C. reinhardtii provides an ideal vehicle for the study of the mechanism of cellular responses to changes in the ambient light intensity and quality. These studies address, among others, the following questions: what are the primary targets for light stress (i.e. the

chlorophyll-protein complexes) and what are the resulting changes in their properties; what is the machinery responsible for alleviation of excess energization; what is the mechanism of light-induced damage and repair; how are the irreversibly altered proteins or protein complexes specifically degraded and replaced; how are these processes regulated and at what levels of macromolecular metabolism, protein synthesis, processing and assembly of the membrane integral protein-pigment complexes. Photosynthetic cells can respond to short term changes in the environmental light conditions or adapt to long term variations (Melis et al., 1996). The aim of this chapter is to review in general lines the knowledge obtained by analyzing the response of the unicellular green alga C. reinhardtii to short term, limiting or excessive illumination, with particular emphasis on the modulation of energy distribution between the two photosystems (state transition) and the process of photoinhibition and recovery of photosynthetic activity.

A. Photosynthetic Electron Flow in C. reinhardtii Abbreviations: – singlet and triplet chlorophyll; DBMIB – 2,5–dibromo–3–methyl–6–isopropyl–p–benzoquinone; DCIP – 2,3 dichlorophenol indophenol; DCMU – diuron, 3–(3´4–dichlorophenyl)–1,1–dimethyl urea; DMBQ – 2,6-dimethyl benzoquinone; DPC – 1,5-diphenyl carbazide; – singlet and triplet oxygen respectively; OEE – the oxygen evolving enhancer proteins; – oxidized and excited sate of the primary electron donor of RCII, respectively; Pheo – pheophytin, the primary electron acceptor of RCII; – the first and second electron acceptors of PS II; RCII – photochemical reaction center II; – mixed RCII populations containing Mn clusters that have lost either 1 and 2, or 2 and 3 electrons respectively; –oxidized states of the PS II Mn cluster that have lost either2 or 3 electrons respectively; Yz – tyrosine 161 of the D1 protein, the electron donor to

The composition of the photosynthetic apparatus in C. reinhardtii in terms of the major thylakoid membrane complexes, their subunit composition, function and macro-molecular organization into appressed and non-appressed membrane domains, is comparable to that of the green plant chloroplast (Ohad et al., 1967; Harris, 1989; Chapter 14, Olive and Wollman). The so-called ‘Z- scheme’ that describes the path of electron flow from the water oxidation complex associated with Photosystem II (Chapter 16, Ruffle and Sayre) via the plastoquinone pool, cytochrome bf complex (Chapter 24, Wollman),

Chapter 30 State Transition and Photoinhibition plastocyanin (Chapter 32, Merchant), Photosystem I to NADP reduction (Chapter 17, Webber and Bingham; Nechushtai et al., 1996), provides an accepted and well-demonstrated basis for the bioenergetics of photosynthesis in this organism. The ATP-synthase of C. reinhardtii is similar to that of higher plants (Chapter 25, Strotmann et al.). Genetic and biochemical evidence indicate the presence of an additional membrane complex, the NADPH-dehydrogenase accounting for the phenomenon ofchlororespiration in C. reinhardtii (Godde and Trebst, 1980; Godde, 1982; Chapter 18, Redding and Peltier). The light harvesting antennae system of C. rein­ hardtii PS II has been characterized (Bassi and Wollman, 1991), and includes the chlorophyll a/b LHCII complex comprised offive major polypeptides (Bassi and Wollman, 1991). The antenna also include the CP43 and CP47 chlorophyll a binding proteins of the PS II core. PS I harvests light through the chlorophyll bound to the PsaA and PsaB core proteins and the Chl a/b LHC-I antenna (Chapter 17, Webber and Bingham; Ish-Shalom and Ohad, 1983; Nechushtai et al., 1996).

B. Light Energy Absorption and Dissipation The absorbed light energy is used for photochemistry or dissipated via heat or fluorescence emission processes (Govindjee, 1995; Lavergne and Briantais, 1996). The rate of the photochemical activity depends on the light intensity as well as on the internal state of the intermediate electron carrier pools and the final acceptors. Therefore, the term ‘excess light’, used to describe excitation overload of the photosynthetic apparatus, is a relative term that should be discussed in conjunction with the algal metabolic state. Non-radiative dissipation (heat emission) can be performed by both reaction center and antenna complexes, and the rate of this process can be modulated by transient changes in the membrane carotenoid composition due to de-epoxidation of and increase in the xanthophyll content of the membranes (Demmig-Adams and Adams, 1996). For efficient utilization of the absorbed light, both photosystems should be excited to generate equal rates of electron flow and thus balance the reduction of the PQ pool with its oxidation. Increase nm, absorbed primarily in the light intensity at by the PS II light harvesting complex LHCII,

571 containing a large relative amount ofChl b, or increase in the light intensity absorbed preferentially by PS I to 740 nm will create an imbalance resulting i in the dissipation of the excess absorbed light energy by the respective photosystem. Imbalance between the excitation of the photosystems may occur not only as result of the changes in the light quality but also due to changes in the availability of electron acceptors for each of the two photosystems. Dissipation of excess energy by increase in the fluorescence emission occurs mostly in PS II. At cryogenic temperatures, fluorescence emission increases considerably also in PS I (Gershoni et al., 1982). Light, while providing the energy required to extract electrons from water, to generate ATP and reduce carbon and nitrogen, also activates regulatory mechanisms for the optimization of the energy utilization and protective mechanisms aimed to alleviate over-excitation even at relatively low photon flux densities. The term ‘fluorescence quenching’ is widely used to indicate that processes such as photochemistry or heat dissipation compete with fluorescence emission. Energy dissipation in the reaction centers and antenna by mechanisms that are not related to the photochemical processes, are generally termed non-photochemical quenching (Krause and Weis, 1991; Lavergne i and Briantais, 1996). The fluorescence yield can be quenched (i. e. increase in heat dissipation) by energization of the membrane due to increase in Quenching of antenna fluorescence is based on reversible covalent modifications of the thylakoid membrane carotenoids, whereby consecutive de-derived violaxanthin epoxidations of the increase the content of antheraxanthin and zeaxanthin (xanthophyll cycle). This reductive deepoxidation process requires NADPH (DemmigAdams and Adams, 1996) and is triggered by an induced by excessive illumincrease in the ination (Endo and Asada, 1996). The antenna xanthophylls participate in the quenching ofantenna chlorophyll triplet states. In addition, antheraxanthin and zeaxanthin enhance thermal dissipation of the excited singlet state of antenna chlorophylls (Crofts andYerkes, 1994; Gilmore et al., 1995; Horton et al., 1996). The thermal dissipation of the absorbed light energy within the antenna reduces energy transfer to PS II and lowers the fluorescence emission as well as the formation of harmful triplet chlorophyll in RCII (Vass and Styring, 1993).

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Grossman and coworkers have isolated a nuclear C. reinhardtii mutant, npq1 and used the lor1 mutant to construct a double mutant npq1/lor1. The npq1 mutant is impaired in the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin. The lor1 mutant is impaired in the conversion of lycopene to -carotene, lutein and loroxanthin. Each of the above mutants could grow phototrophically and did not exhibit light sensitivity, although both exhibited a reduction in the NPQ response. However, the npq1/ lor1 double mutant was light sensitive and exhibited a drastically reduced NPQ (Niyogi et al., 1997a,b). These findings represent direct evidence for the involvement of the xanthophylls in the non-radiative dissipation of excess energy as well as the fact that xanthophylls derived from -carotene can substitute for those derived from -carotene. The enzymes involved in this process are membrane bound and can be activated in isolated thylakoid membranes, in the presence of NADPH. The possibility that the enzymes are closely associated with the PS II antenna has been proposed for vascular plants (Gruszecki and Krupa, 1993). Presently it is not known whether the xanthophyll cycle enzymes in C. reinhardtii are associated with a specific membrane complex. The term also includes energy dissipation in the

Nir Keren and Itzhak Ohad

reaction centers. The nature ofthis process is thought to be related to either pH dependent release of ions from the membrane-bound water-splitting complex, (Krieger and Weis, 1992) or to the generation of centers (Schreiber and Neubauer, 1990). The fluorescence yield is also modulated by changes in energy transfer from the LHCII-antenna to the PS II reaction centers and redistribution of absorbed energy between the two photosystems. The dissociation of the LHCII-antennae due to phosphorylation of the LHCII proteins diminishes the PS II absorption cross-section and thus reduces the total energy absorbed and the ensuing fluorescence. This process is termed ‘state transition’ and the lowering ofthe PS II fluorescence emission rate in this case is referred to as (Horton and Hague, 1988) This phenomenon will be dealt with in more detail in Section II. Distinction between the different mechanisms of energy dissipation can be monitored by kinetic fluorescence measurements. High energy quenching ( ) in the reaction centers is a fast process occurring in the millisecond to second time range. The xanthophyll cycle and state transition are enzymatic processes which occur in the minute time range. The

Chapter 30

State Transition and Photoinhibition

phosphorylation of LHCII related to the state transition in C. reinhardtii cells and thylakoids is achieved in about 10–15 min of light exposure (Owens and Ohad, 1982). It is important to note that nonradiative processes account for most of the energy dissipation. Fluorescence quenching by membrane energization ( ) has a smaller contribution to light energy management, relative to the state transition component, in algae as compared to higher plants (Falkowski et al., 1986; Schreiber et al., 1995).

C. Chlorophyll a Fluorescence Measurements Fluorescence dynamics phenomena can be monitored by measurements ofchlorophyll fluorescence in vivo, a fast, non-invasive method for evaluation of photosynthetic electron transfer and energy distribution. In this section the main methods used for photosynthetic fluorescence measurements and the parameters derived from them will be described briefly. For information on the in vivo measurements of photosynthetic activity, see Chapter 22 (Joliot et al.). Measurements of fluorescence rise kinetics (fluorescence induction) are performed using a ) and continuous weak actinic light ( measuring the emitted fluorescence above The fluorescence induced by the rapid onset of the continuous illumination preceded by dark-adaptation of intact cells (time required for the light ‘on’ condition, about 1 ms) is shown in Fig. lA.A fast rise in the fluorescence parallels the rise of the actinic light intensity with the opening of the shutter. This initial fluorescence measured immediately (>1 ms) after the complete opening of the shutter, is an estimate of the fluorescence level. This fluorescence is ascribed to competition with energy trapping by active centers as well as to active PS II centers that are closed due to the presence of in its reduced state in darkness and to irreversibly nonfunctional centers (Lavergne and Briantais, 1996). The level of measured as in Fig. 1A may be higher (