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: 0-306-48204-5 Print ISBN: 0-7923-5174-6

©2004 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow

Print ©1998 Kluwer Academic Publishers Dordrecht

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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: http://kluweronline.com and Kluwer's eBookstore at: 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. Contents

Preface xvii

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1 Introduction to Chlamydomonas 1–11 Elizabeth H. Harris Summary 1 I. Why Chlamydomonas? 1 II. CellArchitecture 3 III. Life Cycle 3 IV. Laboratory strains of Chlamydomonas reinhardtii 4 V. Genetic Analysis 6 VI. Molecular Biology 7 VII. Resources 7 Acknowledgment 8 References 8

2 Perspectives on Early Research on Photosynthesis in Chlamydomonas 13–23 Robert K. Togasaki and Stefan J. Surzycki Summary 13 I. General Background 13 II. The Levine Laboratory in the Early 1960s 14 III. Establishment of Chlamydomonas reinhardtii as a Legitimate Model Organism 15 IV. Development of New Techniques 18 V. Emergence of New Research Targets 19 VI. Old Experiments Becoming Reality 21 Acknowledgment 21 References 22

3 Organization of the Nuclear Genome 25–40 Carolyn D. Silflow Summary 25 I. Introduction and Scope 26 II. General Characteristics of the Nuclear Genome 26 III. Organization of the Genome 26 IV. Characteristics of Chlamydomonas Genes Transcribed by Polymerase II 30 IV. Physical Mapping of the Chlamydomonas Genome 36 VI. Future Prospects 37 Acknowledgments 37 References 37

4 Nuclear Transformation: Technology and Applications 41–61 Karen L. Kindle Summary 42 I. Introduction 42 II. A Brief History of C. reinhardtii Nuclear Transformation 42 III. Selectable Markers 43 IV. Methods for Introducing DNA into the Nuclear Genome of C. reinhardtii 45 V. Reporters and Promoters 46 VI. Characteristics of Transformation Events 48 VII. Insertional Mutagenesis and Gene Tagging 51 VIII. Gene Isolation by Complementation of a Mutant Phenotype 54 IX. Homologous Recombination and Gene Targeting 54 X. The Use of Nuclear Transformation to Study Promoter Function 56 XI. Conclusion 58 Acknowledgments 58 References 58

5 Modes and Tempos of Mitochondrial and Chloroplast Genome Evolution in Chlamydomonas: A Comparative Analysis 63–91 Aurora M. Nedelcu and Robert W. Lee Summary 63 I. Introduction 64 II. Phylogenetic Position of Chlamydomonas 64 III. Monophyletic versus Polyphyletic Origin of Mitochondria and Plastids: The Chlamydomonas Case 65 IV. Evolution of Mitochondrial and Chloroplast Genome Size in Chlamydomonas 69 V. Evolution of Mitochondrial and Chloroplast Genome Organization in Chlamydomonas 79 VI. Evolution of Mitochondril and Chloroplast Gene Structure and Organization in Chlamydomonas 82 VII. Evolution of Mitochondrial and Chloroplast DNA Sequences in Chlamydomonas 85 VIII. Conclusions 87 Acknowledgments 87 References 87

6 Uniparental Inheritance of Chloroplast Genomes 93–113 E. Virginia Armbrust Summary 93 I. Introduction 94 II. Historical Overview of the Uniparental Inheritance of Chloroplast DNA 95 III. Mating-Type Control of Life Cycle Events 98 IV. Protection of Plus Chloroplast DNA 101 V. Zygote Specific Elimination of Minus Chloroplast DNA 103 VI. Regulation of Chloroplast DNA Inheritance 108 VI. Evolution of the Uniparental Inheritance of Organelle Genomes 110 Acknowledgments 110 References 110 7 Replication, Recombination, and Repair in the Chloroplast Genetic System of Chlamydomonas 115–138 Barbara B. Sears Summary 115 I. Introduction 116 II. Replication 116 III. Recombination 123 IV. Repair 130

viii V. Perspectives and Conclusions 133 Acknowledgments 133 References 133

8 Chloroplast Transformation and Reverse Genetics 139–149 Michel Goldschmidt-Clermont Summary 139 I. Introduction 140 II. Delivery of DNA to the Chloroplast 140 III. Selectable Markers and Reporters 140 IV. Fate of Transforming DNA 142 V. Reverse Genetics 147 VI. Conclusion and Perspective 147 Acknowledgments 148 References 148

9 Chloroplast RNA Stability 151–163 Jörg Nickelsen Summary 151 I. Introduction 152 II. Cell Cycle Dependent Regulation of Chloroplast RNA Stability 152 III. Nuclear Mutants Affected in Chloroplast RNA Stability 154 IV. Towards a Molecular Model of Chloroplast RNA Stabilization/Degradation 154 V. Conclusions and Perspectives 161 Acknowledgments 161 References 161 10 Chloroplast RNA Synthesis and Processing 165–183 David B. Stern and Robert G. Drager Summary 165 I. Transcription of Chloroplast Genes 166 II. Processing of Chloroplast mRNAs 171 Acknowledgments 177 References 177 11 RNA Splicing in the Chloroplast 183–195 David L. Herrin, Tai-Chih Kuo and Michel Goldschmidt-Clermont Summary 183 I. Introduction 184 II. Group I Introns 184 III. Group II Introns and Trans-Splicing 190 IV. Perspective 193 Acknowledgments 193 References 193

12 Regulation of Chloroplast Translation 197–217 Charles R. Hauser, Nicholas W. Gillham and John E. Boynton Summary 197 I. Introduction 198 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 202 IV. Cis-acting Sequences Involved in Translation Initiation 203 V. Translational Regulation Involves Interactions between cis-Acting Sequences and trans-Acting Factors 205 VI. Ribosomes, Membranes and Tethers 209 VII. Translational Regulation of Complex Assembly 211 VIII. How are the Regulatory Proteins Regulated? 212 IX. Is there Hierarchical Control of Chloroplast mRNA Translation? 213 Acknowledgments 214 References 214

13 Chloroplast Protein Translocation 219–231 Mireille C. Perret, Karen K. Bernd and Bruce D. Kohorn Summary 219 I. Introduction 220 II. Chloroplast Import 220 III. Sorting of Proteins Within the Chloroplast 222 IV. Thylakoid Translocation 223 V. Mutations Affecting Translocation 226 VI. Perspectives 228 Acknowledgments 229 References 229 14 Supramolecular Organization of the Chloroplast and of the Thylakoid Membranes 233–254 Jacqueline Olive and Francis-André Wollman Summary 234 I. Introduction 234 II. Cell and Chloroplast Morphology 235 III. Ultrastructural Organization of Thylakoid Membranes 239 IV. Dynamic Aspects of Thylakoid Membrane Organization 246 V. Biogenesis 248 VI. Conclusion 250 Acknowledgment 251 References 251

15 Assembly of Photosystem II 255–285 Jeanne Marie Erickson Summary 255 I. Introduction 256 II. Developmental Biogenesis of Photosystem II 257 III. Assembly of Photosystem II Complexes 260 IV. Assembly of the Extrinsic Membrane Polypeptides of the PS II Oxygen-Evolving Complex 270 V. Assembly of Manganese: The Catalytic Center of the Oxygen-Evolving Complex 273 Acknowledgments 277 References 277

x 16 Functional Analysis of Photosystem II 287–322 Stuart V. Ruffle and Richard T. Sayre Summary 287 I. Introduction 288 II. The Photosystem II Complex 289 III. The Chloroplast DNA Encoded Small Polypeptides of Photosystem II 308 IV. The Nucleus Encoded Polypeptides of the Photosystem II Complex 311 V. Perspectives 315 Acknowledgments 315 References 315

17 Structure and Function of Photosystem I 323–348 Andrew N. Webber and Scott E. Bingham Summary 324 I. Introduction 324 II. Structure of Photosystem I 325 III. Nature and Function of Electron Transfer Cofactors 328 IV. Antenna Structure and Function 332 V. Function of Photosystem I Subunits 333 VI. Biogenesis of Photosystem I 341 Acknowledgments 344 References 344

18 Reexamining the Validity of the Z-Scheme: Is Photosystem I Required for Oxygenic Photosynthesis in Chlamydomonas? 349–362 Kevin Redding and Gilles Peltier Summary 349 I. The Z-Scheme of Oxygenic Photosynthesis and Alternative Schemes 350 II. Electron Transport in the Absence of PS II 351 III. Photosynthesis in the Absence of PS I 354 IV. Putative Electron Transport Pathways Outside of the Z-Scheme 357 V. Thermodynamic Considerations 358 VI. Evolutionary Considerations 358 VII. Conclusions 359 Acknowledgments 359 References 360

19 Assembly of Light-Harvesting Systems 363–376 J. Kenneth Hoober, Hyoungshin Park, Gregory R. Wolfe, Yutaka Komine and Laura L. Eggink Summary 363 I. Thylakoid Biogenesis in Chlamydomonas 364 II. Analysis of LHCII Assembly 366 III. Site of Assembly of LHCII During Initial Greening 368 IV. Conclusions 371 Acknowledgments 373 References 373

xi 20 Pigment Biosynthesis: Chlorophylls, Heme, and Carotenoids 377–414 Michael P. Timko Summary 378 I. Introduction 378 II. Tetrapyrroles and Their Derivatives—An Overview 378 III. Formation of ALA 379 IV. The Pathway from ALA to Protoporphyrin IX 383 V. The Magnesium Branch—Chlorophyll Formation 388 VI. The Iron Branch—Formation of Heme 397 VII. Light and Metabolic Regulation of Chlorophyll Formation 398 VIII. Carotenoids 401 Acknowledgments 406 References 406

21 Glycerolipids: Composition, Biosynthesis and Function in Chlamydomonas 415–431 Antoine Trémolières Summary 415 Introduction 416 II. Glycerolipid and Fatty Acid Composition of Chlamydomonas 417 III. Lipid Metabolic Pathway in Chlamydomonas spp. 422 IV. In vivo Modifications of Lipid Composition in Chlamydomonas 425 V. Mutants Affected in Lipid Composition 426 VI. Involvement of Lipids in the Functional Organization and the Biogenesis of the Photosynthetic Apparatus 428 Acknowledgments 429 References 429

22 In vivo Measurements of Photosynthetic Activity: Methods 433–449 Pierre Joliot, Daniel Béal and René Delosme Summary 433 I. Introduction 434 II. Kinetic Analysis of the Fluorescence Yield 436 III. Fluorescence Emission Spectra at Low Temperature 439 IV. Delayed Fluorescence Measurements 439 V. Oxygen Measurements 439 VI. Absorption Spectroscopy 440 VII. Photoacoustic Measurements 443 VIII. Conclusion and Perspectives 445 Appendix A: Estimation of the Signal-to-Noise Ratio in Fluorescence Measurements 446 Appendix B: Flash Spectrophotometer 446 Acknowledgment 448 References 448 23 New Digital Imaging Instrument For Measuring Fluorescence and Delayed Luminescence 451–458 Pierre Bennoun and Daniel Béal Summary 451 I. Introduction 452 II. Setup for Fluorescence and Delayed Luminescence Video Imaging 452

xii III. Digital Fluorescence Imaging Related to Photosynthetic Electron Transfer 452 IV. Digital Fluorescence Imaging Related to the Permanent Thylakoid Electrochemical Gradient 453 V. Digital Delayed Luminescence Imaging Related to Light-Induced and Permanent Thylakoid Electrochemical Gradient 455 Acknowledgments 457 References 457 24 The Structure, Function and Biogenesis Of Cytochrome Complexes 459–476 Francis-André Wollman Summary 460 I. General Traits 460 II. Biochemical and Structural Studies 461 III. Functional Studies 463 IV. The pet Genes 466 V. Biogenesis and Assembly 467 VI. Concluding Remarks 472 Acknowledgments 474 References 474

25 Assembly and Function of the Chloroplast ATP Synthase 477–500 Heinrich Strotmann, Noun Shavit and Stefan Leu Summary 477 I. Introduction 478 II. Structure of 478 III. Molecular Genetics of 482 IV. Mechanism of 487 V. Regulation of 492 VI. Conclusions 494 References 494

26 Molecular Aspects of Components of the Ferredoxin/Thioredoxin Systems 501–514 Jean-Pierre Jacquot, Mariana Stein, Stéphane Lemaire, Paulette Decottignies, Pierre Le Maréchal and Jean-Mark Lancelin Summary 501 I. Introduction 502 II. Ferredoxin Dependent Systems 505 III. Thioredoxin Dependent Systems 508 IV. Conclusion 511 Acknowledgment 512 References 512

27 Genetic Engineering of Rubisco 515–527 Robert J. Spreitzer Summary 515 I. Introduction 516 II. Chloroplast Genetic Screening and Selection 518

xiii III. Directed Mutagenesis and Chloroplast Transformation 521 IV. Rubisco Nuclear Mutants 523 V. Conclusion and Perspective 524 Acknowledgments 524 References 524

28 Acquisition. Acclimation to Changing Carbon Availability 529–547 Martin H. Spalding Summary 529 I. Introduction 530 II. Photosynthetic Carbon Assimilation 530 III. Induction of the CCM and Related Adaptations to Limiting 539 Acknowledgments 544 References 544

29 Regulation of Starch Biosynthesis 549–567 Steven G. Ball Summary 549 I. Starch: Structure and Function 550 II. The Starch Pathway 554 III. The Genetics of Starch Biosynthesis 559 IV. A Model Explaining the Biogenesis of the Plant Starch Granule 563 V. Future Prospects 564 Acknowledgments 565 References 565

30 State Transition and Photoinhibition 569–596 Nir Keren and Itzhak Ohad Summary 569 I. Introduction 570 II. State Transition: The Phenomenon 574 III. Light Stress: Photoinhibition and Recovery 578 IV. Concluding Remarks and Perspectives 590 Acknowledgments 590 References 590

31 Synthesis of Metalloproteins Involved in Photosynthesis: Plastocyanin and Cytochromes 597–611 Sabeeha Merchant Summary 598 I. Introduction 598 II. Copper-Responsive Synthesis of Plastocyanin and Cytochrome 600 III. Genetic Analysis of Chloroplast Metalloprotein Assembly 605 IV. Conclusions 608 Acknowledgments 608 References 609

32 Responses to Deficiencies in Macronutrients 613–635 John P. Davies and Arthur R. Grossman Summary 614 I. Introduction 614

xiv II. Nutrients in the Environment 615 III. Specific Responses 617 IV. Common Responses 622 V. Model Integrating the Responses to Nutrient Deprivation 626 VI. Regulation of the Responses to Nutrient Deprivation 627 VII. Identification of Mutants Deficient in the Acclimation to Nutrient Deprivation 629 Acknowledgments 630 References 630

33 Nitrogen Assimilation and its Regulation 637–659 Emilio Fernández, Aurora Galván and Alberto Quesada Summary 638 I. Introduction. Pathways for Nitrogen Assimilation in Chlamydomonas 638 II. Assimilation of Ammonium 639 III. Assimilation of Amino Acids 642 IV. Assimilation of Purines 643 V. Assimilation of Nitrate and Nitrite 645 VI. Concluding Remarks 654 Acknowledgments 655 References 655 34 Mitochondrial Genetics 661–674 Claire Remacle and René F. Matagne Summary 661 I. Introduction 662 II. Mitochondrial Genome 662 III. Mitochondria and the Electron Transport Chain 664 IV. Mutations Affecting the Mitochondrial Genome 665 V. Transmission of Mitochondrial Genes in Meiotic Zygotes 668 VI. Transmission of Mitochondrial Genes in Vegetative Zygotes and Mapping of Mitochondrial Mutations by Recombinational Analysis 669 VII. Mitochondrial Transformation 671 Acknowledgments 672 References 672

35 Chlororespiration, Sixteen Years Later 675–683 Pierre Bennoun Summary 675 I. Introduction 676 II. The Thylakoid Electrochemical Gradient Present in the Dark 676 III. Reduction of Plastoquinone in the Dark 678 IV. Oxidation of Plastoquinol in the Dark 680 V. Conclusion 680 Cautionary Note 682 Acknowledgments 682 References 682

36 Perspectives 685–703 Lauren J. Mets and Jean-David Rochaix Summary 685 I. Introduction 686 II. The Niche of Chlamydomonas in Photosynthesis Research 687

xv III. Forefront Problems in Photosynthesis and Organelle Research 696 Acknowledgments 700 References 700

Index 705

xvi Preface

The Molecular Biology of Chloroplasts and a genetic dissection of photosynthesis was first Mitochondria in Chlamydomonas is the seventh recognized by Paul Levine. Together with his volume to be published in the series Advances in coworkers, he initiated a long-range genetic approach Photosynthesis of Kluwer Academic Publishers which proved to be highly successful. It provided (Series Editor: Govindjee). Volume 1 dealt with The genetic support for the linear Z scheme of Molecular Biology of Cyanobacteria; Volume 2 with photosynthesis and led to the identification of new Anoxygenic Photosynthetic Bacteria; Volume 3 with components of the photosynthetic electron transfer Biophysical Techniques in Photosynthesis; Volume 4 chain such as the Rieske protein ofthe cytochrome with Photosynthesis and the Environment; and complex. Volume 6 with Lipids in Photosynthesis: Structure, During the past 20 years, the powerful techniques Function and Genetics. of molecular biology and genetics, and the The main goal of this book is to provide a development of methods for efficient nuclear and comprehensive overview of current research with chloroplast transformation of C. reinhardtii have the green alga Chlamydomonas on chloroplast and greatly enhanced the potential of this organism as an mitochondrial biogenesis and function, with special experimental system for studying chloroplast emphasis on the assembly and structure-function biogenesis. This has led to impressive advances in relationships ofthe constituents ofthe photosynthetic our understanding of the regulation of chloroplast apparatus. The editors have encouraged the gene expression and it has provided important new contributors of this volume to emphasize the insights into the complex cooperative interplay particular features of Chlamydomonas that make between the chloroplast and nuclear compartments this unicellular organism uniquely suited for study- in the assembly of the photosynthetic apparatus. At ing photosynthesis and its multiple regulatory the same time, the ability to manipulate the chloroplast mechanisms operating under various environmental genome with surgical precision has opened the door and stress conditions. A second, but equally important for a detailed structure-function analysis of aim is to show that current research in photosyn- photosynthetic complexes in vitro, and thanks to the thesis is multidisciplinary and combines molecular refinements and new developments in spectroscopic genetics, biochemical, biophysical andphysiological and fluorescence techniques, also in vivo. We feel approaches. Although Chlamydomonas has also strongly that abook on these recent exciting advances proven to be apowerful system forunderstanding the in research on photosynthesis in Chlamydomonas is structure, function and assembly of flagella, this timely and important. topic is not covered in the book. The first part of the book provides a general Chlamydomonas research would nothave reached introductionto Chlamydomonas (Chapter 1, Harris), its present status without the pioneering studies of a historical chapter on early research on photo- the late Ruth Sager and of Paul Levine. Organellar synthesis in this organism (Chapter 2, Togasaki and genetic analysis of this alga started over 40 years ago Surzycki) and chapters on nuclear genome organi- when Ruth Sager discovered that during crosses zation (Chapter 3, Silflow), nuclear transformation certain traits were transmitted uniparentally to the (Chapter 4, Kindle), mitochondrial and chloroplast progeny from the mating-type plus parent, but not genome evolution (Chapter 5, Nedelcu and Lee), from the mating-type minus parent. These uniparental chloroplast uniparental inheritance (Chapter 6, traits were shown later to be specified by the Armbrust), chloroplast DNA metabolism (Chapter 7, chloroplast genome. Sager also found that, in rare Sears) and chloroplast transformation and reverse cases, the uniparental traits ofboth parents could be genetics (Chapter 8, Goldschmidt-Clermont). The inherited and that the analysis of their segregation second part includes several chapters on chloroplast pattern during crosses could be used to construct a gene expression: RNA stability (Chapter 9, genetic map. The potential ofusing C. reinhardtii for Nickelsen), RNA processing (Chapter 10, Stern and

xvii Drager), splicing (Chapter 11, Herrin et al.) and Chapter 35 (Bennoun) discusses the current models translation (Chapter 12, Hauser et al.). Protein of chlororespiration. The last Chapter (36, Mets and targeting in the chloroplast is discussed in Chapter 13 Rochaix) offers a perspective on research on (Perret et al.). The third part includes articles on the photosynthesis with Chlamydomonas. biosynthesis and function of thylakoid membranes We thank the authors for their invaluable (Chapter 14, Olive and Wollman), Photosystem II contributions which we hope will make this book (Chapter 15, Erickson; Chapter 16, Ruffle and Sayre), very useful forresearchers and students interested in Photosystem I (Chapter 17, Webber and Bingham; photosynthesis and organellar biology in Chlamy- Chapter 18, Redding and Peltier), LHCII (Chapter domonas. The book is also intended for a wide 19, Hoober et al.), pigments (Chapter 20, Timko), audience, but is specifically designed for advanced glycerolipids (Chapter 21, Trémolières), the undergraduateand graduatestudentsandresearchers cytochrome complex (Chapter 24, Wollman), the in the fields of biochemistry, molecular biology, ATP synthase (Chapter 25, Strotmann et al.), physiology, biophysics, plant biology, phycology and ferredoxin and thioredoxin (Chapter 26, Jacquot et biotechnology. We also hope that this book will al.) and of ribulose 1,5 bisphosphate carboxylase- stimulate scientists outside the Chlamydomonas oxygenase (Chapter 27, Spreitzer). In addition, research community to use this organism for their Chapters 22 (Joliot et al.) and 23 (Bennoun and Beal) studies. describe new and powerful techniques used for We wish to express our gratitude to Larry Orr for measurements of photosynthetic activity in vivo. his patience with inexperienced editors, generous These techniques are particularly suited for help and remarkable efficiency, to Govindjee for his Chlamydomonas. The fourth part includes chapters continued interest and for his many helpful on uptake (Chapter 28, Spalding) and starch suggestions, and to Nicolas Roggli and Michael biosynthesis (Chapter 29, Ball). Several articles are Hippler for their help in designing the cover graphics. devoted to the responses of Chlamydomonas to Finally, we hope that by showing the extraordinary various stress conditions, such as high light power and uniqueness of Chlamydomonas as a (Chapter 30, Keren and Ohad), copper deficiency research tool in photosynthesis and by documenting (Chapter 31, Merchant) and macronutrient depri- the fast pace of progress achieved in the past years vation (Chapter 32, Davies and Grossman). Nitrogen with this unicellular organism, this book will help assimilation and its regulation is discussed in promote Chlamydomonas as the ‘green yeast’ among Chapter 33 (Fernández et al.). Chapter 34 (Remacle the plant research community. and Matagne) describes mitochondrial genetics and Jean-David Rochaix Michel Goldschmidt-Clermont Sabeeha Merchant

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