Advances in Plant Biology Volume Series Editor John J Harada Davis, USA Advances in Plant Biology provides summaries and updates of topical areas of plant biology This series focuses largely on mechanisms that underlie the growth, development, and response of plants to their environment Each volume contains primarily on information at the molecular, cellular, biochemical, genetic and genomic level, although they will focused on information obtained using other approaches More information about this series at http://www.springer.com/series/8047 Steven M Theg • Francis-André Wollman Editors Plastid Biology 1  3 Editors Steven M Theg Department of Plant Biology Univeristy of California-Davis Davis California USA Francis-André Wollman Physiologie Membranaire et Moléculaire du Chloroplaste Institut de Biologie Physico-Chimique Paris France ISBN 978-1-4939-1135-6 ISBN 978-1-4939-1136-3 (eBook) DOI 10.1007/978-1-4939-1136-3 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014947238 © Springer Science+Business Media New York 2014 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer Permissions for use may be obtained through RightsLink at the Copyright Clearance Center Violations are liable to prosecution under the respective Copyright Law The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made The publisher makes no warranty, express or implied, with respect to the material contained herein Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) Preface Photosynthesis is the process through which the energy inherent in sunlight is captured in the chemical bonds of reduced carbon compounds, thereby providing the food upon which almost all life depends In addition, the production of oxygen as a result of the utilization of water as the ultimate electron donor to the photosynthetic electron transport chain has transformed our atmosphere, allowing for the emergence of oxygenic respiration, without which there would be no human life on Earth Photosynthesis is carried out in plants and algae in chloroplasts Given their central role in energy transduction in the biosphere, chloroplasts have been the focus of attention for generations of scientists This volume brings together many aspects of modern research into plastids relating to their biogenesis, functioning in photosynthesis and utility for biotechnology Plastids had their origins in free living photosynthetic bacteria and took up residence in the primitive eukaryotic cells through endosymbiosis While they have lost most of their DNA to the nucleus, they retain a functioning genome and are capable of a limited but critical amount of semi-autonomous protein synthesis Accordingly, we start this volume with a series of three chapters devoted to the handling of the genetic information contained within the plastid genome and crosstalk between the chloroplast and nucleus as the information encoded in both locations is decoded Following this are five chapters that examine the biogenesis and differentiation of the plastid itself and the sub-structures found at the plastid surface and within the internal thylakoid system Also included here is a treatment of the unusual nonphotosynthetic plastids found within the Apicoplexa, a group of parasitic protists responsible for a number of important human diseases Despite having their own genomes, the vast majority of plastid proteins are synthesized in the cytosol and taken up into and subsequently distributed within the organelle The next six chapters of the volume describe these processes, as well as the roles of molecular chaperones and proteases in protein homeostasis This is followed by three chapters dedicated to critical aspects of chloroplast physiology relating to dissipation of excess light energy, control of electron transport and ion homeostasis Finally, the book ends with two chapters discussing the emerging roles of plastids in biotechnology, one as a platform for synthesis of useful proteins, made v vi Preface desirable because of the superior containment of transgenes within this organelle than when inserted in nuclear genomes, and the other as a source of hydrogen production to be used as biofuel Each of the chapters has been written by leading authorities in their respective research areas Many chapters are the result of collaborations between experts in different laboratories, giving a broader than usual perspective on a given topic In each case, readers will find well-crafted chapters containing information and insights for both novices and experts alike We are grateful to our many friends and scholars who contributed these outstanding chapters The breadth of their knowledge and clarity of their writing have made for a unique and readable volume bringing together many disparate but interconnected topics relating to plastid biology We are also indebted to those at Springer, especially Kenneth Teng and Brian Halm, who oversaw this project in its final stages of production Davis, CA, USA Paris, France Steven M Theg Francis-André Wollman Contents Part I  Genetic Material and its Expression Chloroplast Gene Expression—RNA Synthesis and Processing����������    3 1  Thomas Börner, Petya Zhelyazkova, Julia Legen and Christian Schmitz-Linneweber Chloroplast Gene Expression—Translation������������������������������������������   49 2  Jörg Nickelsen, Alexandra-Viola Bohne and Peter Westhoff 3  The Chloroplast Genome and Nucleo-Cytosolic Crosstalk������������������   79 Jean-David Rochaix and Silvia Ramundo Part II  Plastid Differentiation An 4  Overview of Chloroplast Biogenesis and Development������������������  115 Barry J Pogson and Veronica Albrecht-Borth 5  Dynamic Architecture of Plant Photosynthetic Membranes����������������  129 Helmut Kirchhoff 6 Plastid Division�����������������������������������������������������������������������������������������  155 Jodi Maple-Grødem and Cécile Raynaud 7 Stromules��������������������������������������������������������������������������������������������������  189 Amutha Sampath Kumar, Savithramma P Dinesh-Kumar and Jeffrey L. Caplan 8 The Apicoplast: A Parasite’s Symbiont��������������������������������������������������  209 Lilach Sheiner and Boris Striepen vii viii Contents Part III  Biogenesis of Chloroplast Proteins Mechanisms of Chloroplast Protein Import in Plants������������������������  241 9     Paul Jarvis and Felix Kessler Protein Routing Processes in the Thylakoid����������������������������������������  271 10    Carole Dabney-Smith and Amanda Storm 11  Protein Transport into Plastids of Secondarily Evolved Organisms��������������������������������������������������������������������������������  291   Franziska Hempel, Kathrin Bolte, Andreas Klingl, Stefan Zauner and Uwe-G Maier Processing and Degradation of Chloroplast Extension Peptides�������  305 12    Kentaro Inoue and Elzbieta Glaser Molecular Chaperone Functions in Plastids����������������������������������������  325 13    Raphael Trösch, Michael Schroda and Felix Willmund 14 Plastid Proteases�������������������������������������������������������������������������������������  359   Zach Adam and Wataru Sakamoto Part IV  Chloroplast Photophysiology Photoprotective Mechanisms: Carotenoids�����������������������������������������  393 15    Luca Dall’Osto, Roberto Bassi and Alexander Ruban Regulation of Electron Transport in Photosynthesis��������������������������  437 16    Giles N Johnson, Pierre Cardol, Jun Minagawa and Giovanni Finazzi Ion 17  homeostasis in the Chloroplast������������������������������������������������������  465   Marc Hanikenne, Marík Bernal and Eugen-Ioan Urzica Part V  Chloroplast Biotechnology Synthesis of Recombinant Products in the Chloroplast���������������������  517 18    Ghislaine Tissot-Lecuelle, Saul Purton, Manuel Dubald and Michel Goldschmidt-Clermont Hydrogen and Biofuel Production in the Chloroplast������������������������  559 19    Yonghua Li-Beisson, Gilles Peltier, Philipp Knörzer, Thomas Happe and Anja Hemschemeier Index . 587 Contributors Zach Adam  The Robert H Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University, Rehovot, Israel Veronica Albrecht-Borth  Australian National University, Canberra, Australia Roberto Bassi  Dipartimento di Biotecnologie, Università di Verona, Verona, Italy María Bernal  Plant Nutrition Department, Estación Experimental De Aula Dei, Consejo Superior de Investigaciones Científicas (CSIC), Zaragoza, Spain Department of Plant Physiology, Ruhr University Bochum, Bochum, Germany Alexandra-Viola Bohne  Molekulare Pflanzenwissenschaften, Biozentrum LMU München, Planegg-Martinsried, Germany Kathrin Bolte  Laboratory for Cell Biology, Philipps University of Marburg, Marburg, Germany Thomas Börner  Institute of Biology, Humboldt University Berlin, Berlin, Germany Jeffrey L Caplan  Department of Plant and Soil Sciences, Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA Pierre Cardol  Laboratoire de Génétique des Microorganismes, Institut de Botanique, Université de Liège, Liège, Belgium Carole Dabney-Smith  Department of Chemistry and Biochemistry, Miami University, Oxford, OH, USA Luca Dall’Osto  Dipartimento di Biotecnologie, Università di Verona, Verona, Italy Savithramma P Dinesh-Kumar  Department of Plant Biology and The Genome Center, College of Biological Sciences, University of California, Davis, CA, USA Manuel Dubald  Bayer CropScience, Morrisville, NC, USA ix 574 Y Li-Beisson et al Fig 19.3   Pathways of fatty acid synthesis and lipid assembly as targets for genetic engineering studies The scheme of the subcellular organization of lipid metabolic pathways is based on that of plants, unless specific experimental evidence is provided for algal species Names of enzymes are in italic, and those enzymes described in this chapter are highlighted in red Lipid-X means that the exact substrate for this enzyme is unknown ACCase acetyl-CoA carboxylase, ACP acyl carrier protein, CoA coenzyme A, DAG diacylglycerol, DGAT diacylglycerol acyltransferase, FAS fatty acid synthase, ER endoplasmic reticulum, FAT fatty acyl-ACP thioesterase, G3P glycerol3-phosphate, GPAT glycerol-3-phosphate acyltransferase, LACS long chain acyl-CoA synthetase, LPA lysophosphatidic acid, LPAT lysophosphatidic acid acyltransferase, MLDP major lipid droplet protein, PA phosphatidic acid, PDAT phospholipid:diacylglycerol acyltransferase, PAP phosphatidic acid phosphatase, TAG triacylglycerol 19.4.4 Triacylglycerol Biosynthesis The best known TAG biosynthetic pathway involves the sequential acylation of sn-glycerol-3-phosphate (G3P) with three acyl-CoAs catalyzed by three distinct a ­ cyltransferases (Fig. 19.3) It is initiated by G3P acyltransferase (GPAT) to produce lysophosphatidic acid (LPA), which is then further acylated by LPA acyltransferase (LPAT) to form phosphatidic acid (PA) Phosphatidic acid phosphatase (PAP) catalyzes the removal of the phosphate group from PA to generate sn-1,2-diacylglycerol (DAG), the central intermediate of all glycerolipids The last and committed step to oil synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) This enzyme has ­ been subjected to intensive studies, including overexpression, directed evolution and quantitative trait loci mapping [18, 139, 172] In Chlamydomonas, homology searches identified five type-2 DGATs (encoded by DGTT1–5) and one type-1 DGAT (DGAT1) DGTT1 exhibits increased transcript abundance in N-starvation conditions, and it has been demonstrated to be able to complement a yeast quadruple mutant deficient for i TAG synthesis [14] Engineering strategies ­nvolving overexpression of DGTT1 alone or in combination with other enzymes might be a possible way to increase oil content 19  Hydrogen and Biofuel Production in the Chloroplast 575 An alternative reaction important for oil synthesis is catalyzed by phos­ pholipid:diacylglycerol acyltransferase (PDAT) contributing to TAG synthesis using phosphatidylcholine as an acyl donor and sn-1,2-diacylglycerol as an acyl acceptor [28, 171] Contrary to all three acyltransferases described above, PDAT does not require acyl-CoA as donor Therefore, its reaction is often termed acyl-CoA independent pathway PDAT has been well characterized in both plants and yeast [28, 171] A homolog of this enzyme is present in Chlamydomonas and has lately been shown to be important for TAG accumulation as insertional null mutants ( pdat1–1 and pdat1–2) accumulate 25 % less TAG compared to the parent strain [14] This evidence for a trans-acylation pathway in TAG synthesis in Chlamydomonas was corroborated by the observation that cell lines carrying PDAT-directed amiRNA silencing constructs accumulate up to 30 % less TAG compared to the wild-type strain [168] However, as pdat mutants exhibit reduced, but not abolished TAG accumulation, DGTT1 must also contribute to oil synthesis [14] The same overlapping function of PDAT and DGAT has been demonstrated in the model plant Arabidopsis thaliana [171] in which minor reductions in oil content could be observed in either of the single mutants, whereas the double mutation is embryo-lethal As well as their acylation to glycerol, fatty acyl chains are modified by fatty acidmodifying enzymes including desaturases, epoxidases, elongases, and hydroxylases Desaturases catalyze the reduction of a C-C bond to form a C=C bond in an existing acyl chain [135] The number of double bonds in a fatty acid molecule plays a determinant role in its final utility For example, biodiesel containing a too high proportion of saturated fatty acids turns to gel even at ambient temperatures On the other hand, when too many unsaturated fatty acids are present, the biodiesel will have a good cold flow but will be prone to oxidation Desaturases have long been used as targets to engineer fatty acid compositions in higher plants [79, 95, 135] Four desaturases have been characterized in Chlamydomonas [23, 82, 128, 169] and many more have been identified based on sequence homology searches Molecular manipulation of these desaturases constitutes a promising way to engineer fatty acid composition in Chlamydomonas 19.4.5  Accumulation of Oil Bodies After a certain amount of TAGs has accumulated in specific domains of the ER or the plastid, oil bodies or lipid droplets bud off and form distinct subcellular organelles Oil bodies are spherical organelles consisting of a neutral lipid core enclosed by a membrane lipid monolayer coated with proteins [74] Oil body biogenesis and its associated proteins have been well studied in yeast [26, 27], as well as in plant oilseeds [73] Only recently, compositions of lipid body-associated proteins 200 proteins have been identified have been analyzed in Chlamydomonas and >  28 kDa is the most abundant of these and was thus [107, 113] One protein of ~  named major lipid droplet protein (MLDP) MLDP has been postulated to play a similar structural role as oleosin in oilseeds Besides MLDP, numerous metabolic a enzymes (­ cyltransferases, lipases) or trafficking proteins are also present, indicating the dynamic nature of Chlamydomonas oil bodies The knowledge about oil 576 Y Li-Beisson et al body-associated proteins provided by these studies represents a rich source for the exploration of oil accumulation mechanisms in general, and also elucidates biotechnological targets For example, either N- or C- terminal fusion of a desired protein to MLDP could potentially direct it to oil bodies, as has been demonstrated for oleosins [10] One unique feature of Chlamydomonas oil bodies is that they are not only present in the ER (as is true for most organisms studied), but also in the plastid [40, 55] This has been shown by Transmission Electron Microscope (TEM) and is further supported by the strong enrichment in C16 fatty acids at the sn-2 position in both TAGs and chloroplast membrane lipids (but not in extra-plastidial lipids) This finding has implications for our overall understanding of the subcellular organization of glycerolipid metabolism and of the specificities of key lipid metabolic enzymes involved A plastid TAG synthesis pathway could provide additional advantages because engineering of lipid metabolic pathways could be achieved via a synthetic biology approach based on manipulation of the plastid genome Unlike the still-problematic transgene expression in the C reinhardtii nuclear genome [131], it is a well-established technique in the plastid genome and transgene expression can reach very high levels (over 70  % of total protein) [114] Transgenes can be delivered to the plastid genome via biolistic bombardment and they are integrated by homologous recombination [31, 105] Successful introduction of a 50 kb DNA fragment into the plastid genome of tobacco has been reported [1] This opened up the possibility of introducing several genes simultaneously in the plastid genome using a synthetic biology approach 19.4.6  Transcriptional Regulation of TAG Biosynthesis WRINKLED1 (WRI1), belonging to the APETALA2-ethylene responsive elementbinding protein (AP2-EREBP), family is the only transcription factor identified in regulation of fatty acid synthesis in Arabidopsis [7, 21] and maize [137] It has also been implicated in regulating oil synthesis in other species such as oil palm [13] Overexpression of WRI1 leads to a large increase in seed oil content in maize [137] and in tubers [69] No WRI1 homolog could be identified in the genome of Chlamydomonas, but comparative transcriptomic studies have led to identification of two regulatory proteins, NRR1–1 (nitrogen responsive regulator) [14] and an stressinduced lipid trigger [167] Overexpression or silencing of the genes encoding these proteins led to altered cellular oil content, but the exact mechanism and downstream target(s) of these proteins remain to be tested 19.5 Closing Remarks Plastids are the power house of all photosynthetic cells Photosynthesis converts the abundant energy of the sun into high-energy electrons and chemical energy equiva­ lents Accordingly, chloroplasts of microalgae are sources of valuable compounds 19  Hydrogen and Biofuel Production in the Chloroplast 577 such as molecular hydrogen, starch and lipids Recent studies of H2 and lipid m ­ etabolic pathways in microalgal models have led to significant advances in our understanding of the molecular and biochemical mechanisms [66, 94, 104] Currently, the vast majority of studies on microalgal biofuel are focused on understanding and boosting the generation of H2 and the accumulation of TAGs [20, 66, 83, 84] In our view, generation of oil is only the first step toward the engineering of algal cell factories Production of value-added fatty acid-derived molecules such as alkanes, free fatty acids, wax esters and fatty alcohols will constitute the next major step At the moment, significant effort has been put on analyzing the H2 and lipid metabolism of the model microalga C reinhardtii However, the genomes of around ten microalgal species have been sequenced so far, and many more are currently being sequenced Intensive efforts are underway to develop molecular genetic tools for Chlamydomonas and other algae For example, the occurrence of homologous recombination in Nannochloropsis sp has been reported [85] This development, together with our knowledge gained through examining model systems, should aid in the master design of an ideal algal cell factory for the production of 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normal pollen and seed development Plant Cell 21:3885–3901 172 Zheng P et al (2008) A phenylalanine in DGAT is a key determinant of oil content and composition in maize Nat Genet 40:367–372 Index 3’ untranslated region (3’UTR),  532 5´ untranslated regions (5’UTRs),  532 5′ untranslated regions (5′UTRs),  52 A Abiotic stress responses,  197, 198 Actin, 196 Alzheimer’s disease (AD),  312 Aminoglycoside adenyl transferase (aadA),  52, 536, 537, 539, 543 Aminoglycoside phosphotransferase (aphA-6),  537, 539 Anion,  467, 486 Apicoplast,  211, 213–217, 219–221, 224–228, 295 B Biofuels,  450, 546, 560, 570 Biolistic transformation,  525 Biotic stress responses,  198, 199 C Carotenoids,  118, 122, 200, 228, 403, 405, 407, 415, 418, 488 xanthophyll cycle,  419–421 Cation,  250, 298, 467 Chaperones,  94, 102, 121, 124, 251, 253, 326, 327, 330, 340, 342, 344, 367 Chaperonin,  166, 330, 334 isoforms and complex compositions, 327–329 Chlamydomonas,  4, 7, 10, 15, 18, 28, 30, 102, 451–453, 456, 475, 480–483, 485 Chloramphenicol acetyl transferase (cat),  537 Chloroplast biogenesis,  8, 19, 25, 71, 102, 116–120, 123–125, 344, 368, 370, 476 Chloroplast editing,  24 Chloroplast envelope,  19, 93, 96, 124, 160, 168, 195, 245, 257, 276, 307, 476–479, 486, 493, 523 Chloroplast genome,  13, 24, 52, 54, 80, 120, 242, 250, 307, 535, 541 Chloroplast nucleases,  26 Chloroplast promoter,  Chloroplast protein import,  242, 243, 245, 248, 249, 252, 253, 255, 259, 338, 345, 474 Chloroplast RNA,  degradation, 29 polymerase,  19, 100 processing, 29 Chloroplast RNA-binding proteins,  4, 5, 23, 62 Chloroplast Sec,  257, 272, 274, 278 Chloroplast splicing,  15, 18, 19 Chloroplast SRP,  274 Chloroplast Tat (cp Tat),  278, 280 Chloroplast transcription,  5, 83, 84, 101, 125, 524 Chloroplast translation,  26, 27, 50–52, 58, 60, 62, 68, 95 regulatory principles of,  62, 64 spatial organization of,  70, 71 Chloroplast translational apparatus,  constituents of,  54 Chromalveolates,  214, 456 Co-chaperone,  164, 166, 167, 252 Complex plastids,  218, 258, 293, 295, 298, 299 evolution of,  292 D Degradation,  4, 24, 26, 28, 92, 120, 122, 139, 141, 306, 314, 326, 360, 368, 376–378, 487 RNA cleavage and,  25 S.M Theg, F.-A Wollman (eds.), Plastid Biology, Advances in Plant Biology 5, DOI 10.1007/978-1-4939-1136-3, © Springer Science+Business Media New York 2014 587 588 Development,  7, 10, 12, 52, 95, 103, 117, 178, 202, 224, 311, 469, 484, 489 plastid, 338 Disaggregation,  331, 342–344 E Endosymbiosis,  54, 211, 219, 332 F Fatty Acid Synthase I (FASI),  223, 224 Filamenting temperature-sensitive mutant Z (FtsZ),  124, 157–159, 162, 163, 166, 172, 173, 175, 179 Fluorescent proteins,  195, 198, 201 Folding,  19, 121, 298, 329, 416 general protien,  334 Functional genomics,  carotenoids,  415, 416 G Gene gun,  93 Grana thylakoid,  130–132, 135, 137, 139, 142, 143, 146 Green Fluorescent Protein (GFP),  310 H Heat shock proteins (HSPs),  198, 255, 309, 326 Helical repeat proteins,  62, 71 Heme,  92–94, 222, 487, 495 biosynthesis,  225, 226, 469 Herbicide tolerance,  537, 543 Heteroplasmic,  521, 531, 539 Homologous recombination,  520, 521, 543, 576 Homoplasmic,  521, 525, 537, 539 Human PreP homologue (hPreP),  312 Hydrogen,  482, 560 Hypoxia, 491 I Import,  118, 121, 125, 215, 247 stages of,  243 Inducible expression,  341, 535 Inter-organelle communication,  200 Isoprenoid precursors,  99 L Lipids,  118, 223, 415, 440, 570, 577 M Macromolecular crowding,  142–144, 146 Macronutrients,  calcium, 474–476 chloride,  486, 487 Index magnesium,  469, 473, 474 nitrogen, 484–486 phosphorus, 479–481 potassium, 476–478 sodium,  478, 479 sulphur,  482, 483 Malaria,  210, 227, 541 Maternal inheritance,  520, 544 Metabolic engineering,  536, 542, 546 Metal,  226, 455, 467 Microalgae,  51, 440, 450, 451, 454, 455, 457, 545, 546, 563, 570, 576 Micronutrients, 467 copper, 490–493 iron, 487–490 manganese, 493 zinc, 494 Microtubule, 124 Min,  158, 159, 162, 179 Mineral nutrition,  179 Molecular chaperone,  165, 251, 326, 327, 335, 342, 378 N Neomycin phosphotransferase (nptII),  537 Non-photochemical quenching (NPQ),  397, 408, 442, 449 Nutrient starvation,  452 O Oral vaccines,  518 Organellar peptidasome,  310 P Photoinhibition,  336, 371, 374–376, 410, 421, 456, 488 Pitrilysin, 311 Plant development,  68, 166, 179, 251, 255, 311, 315, 337 Plastid,  dividing rings,  168 Plastid biogenesis,  123, 124, 219 Plastid division ring,  156 Plastid(s),  4, 5, 8, 11, 56, 82 Plastome,  10, 12, 519, 521, 531, 539 target loci in,  531, 532 Polycistronic unit,  520, 531, 536, 542 PPR proteins,  20, 23, 24, 28, 62, 87, 120 Presequence Protease, PreP,  310 Promoter,  NEP, 9–12 PEP,  7, Proteases,  139, 168, 312, 335, 342, 360, 361, 374, 376, 380 deg, 369 Protein arrays,  144, 145, 147 Index Protein import,  121, 167, 214, 217, 220, 250, 253, 310, 313, 365 in plastids,  337, 338 Protein routing in chloroplasts,  272, 274–280, 282–284 Protein synthesis,  50, 53, 56, 58, 59, 62, 70, 118, 211, 213, 257, 368, 482, 518 Protein targeting,  121, 257, 344 Protein translocation,  195, 219, 279, 293, 297 Protein transport,  217, 284, 292, 297, 313 non-canonical,  258, 259 R Redox control,  65, 97, 253, 441, 443 Regulatory processes,  62, 326, 441 Repressible chloroplast gene expression,  in Chlamydomonas,  99–102 Retrograde signaling,  80, 91, 93, 95, 96, 99, 102, 103, 377, 469, 472 RNA polymerase,  4, 5, 7, 8, 10, 12, 82, 97, 520 S Secondary endosymbiosis,  214, 292, 293, 295 Selectable marker,  251, 531, 532, 537, 543 Senescence,  346, 372, 376 Shine-Dalgarno sequences,  56, 58, 533 Signaling,  80, 86, 178, 202, 472, 481, 484 Stress response,  98, 99 Stromule,  190–193, 197 589 Supramolecular level,  142, 148 Sustainable energy,  570 T Targeting sequences,  371, 521 Thylakoid membrane (TM),  9, 56, 57, 66, 70, 102, 121, 130–134 Thylakoid protein translocation \t See Chloroplast protein import,  122 Thylakoids,  70, 121, 133, 136, 141, 148, 254, 297, 313, 407, 418 TOC/TIC machinery,  298, 307 Toxoplasma,  210, 213, 217, 221, 224, 225, 228 Transcription,  4–6, 8–10 nuclear,  119, 120 Transformation,  519, 521, 525, 537, 539, 542 Translation factors,  57, 533, 534 Translocon,  121, 216, 219, 253, 259, 297, 298 Transport,  118, 121, 122, 130, 137, 198, 200, 202, 283 of metabolites,  228, 229 T-zones,  30, 66, 70 U Uniparental inheritance,  524, 544 V Virus,  191, 198 ... rps12 int.1, ycf3 int.1, clpP int.1 psbH petB int atpF int., trnK int., trnA int., trnI int., trnV int., rpl2 int., rps12 int.2 ycf3 int.2 rps12 int.1 trnG-UCC int psaA int1 and int2 Albino, pale... ycf3 int.2, clpP int1, petD, ndhA, ndhB pet Dint., trnG int., rps16 int., rpl16 int., ycf3 int.1, clpP int.1, rpoC1 int., ndhA int rps12 int.1; petB int., ndhB int., ndhA int., ycf3 int.1 trnL int.,... binding domains, including the CRM domain found in ribosome-assembly factors [16], the abundant RRM domain [ 257 ], the mTERF domain [92], and the organelle-specific PPR domain [19, 52 , 55 , 1 35] In