Je Remercie

Monique Liebers

Claude Alban

Sassia Hadjadji

Éric Maréchal

Ghina, Rida, Gloria Claudette Abir

Keywords: Carboxy-terminal end of an amino acid chain CDS: Codante sequence Chl: chlorophyll Col-0: Arabidopsis thaliana, ecotype Columbia PAP: promoter::protein pPAP::PAP-GFP: promoter::protein-GFP construct pCaMV35S: 35S: promoter of the cauliflower mosaic virus Arabidopsis, chloroplast biogenesis, albinism, pap7-1 mutant, dual nucleus-plastid localization, proteomics, systems biology, retrograde signaling

J'adresse mes remerciements à Madame Claire Lurin et Monsieur Christophe Laloi d'avoir accepté d'être rapporteurs de mes travaux de thèse. Je remercie, également, Madame Christelle Breton d'accepter de faire partie des membres de jury. Je suis très reconnaissante à Monsieur Laurent Legendre et Monsieur Michel Havaux d'avoir bien voulu suivre mes travaux de thèse pendant les deux comités individuels, et d'accepter également d'examiner ma thèse. Je remercie Monsieur Norbert Rolland pour ses conseils pendant les comités de suivi de thèse. Est-ce que j'ai été capable d'avancer sur ce chemin toute seule ? Non, jamais... A mon encadrant pédagogique, mon "père scientifique", mon "Mentor" ...Thomas Pfannschmidt ! Je vous remercie chaleureusement de m'avoir accueilli au sein de votre équipe et de me considérer comme membre de cette famille scientifique toute unie pour le bien de la science. Je vous remercie mon « chef » pour votre accompagnement, votre pédagogie, vos précieux conseils, votre confiance, votre patience ainsi que votre support dans l'achèvement de ce travail. Vous m'avez montré la voie de la réussite dans la plus grande modestie. Je n'oublie jamais vos phrases en me disant « Tu vas avancer, tu vas arriver… », ou lorsque vous me disiez « Ah, « nice ! », il y a un avancement, tu as commencé à penser comme un vrai chercheur, ne t'inquiète pas tout va bien... » Il y avait toujours de l'espoir après chaque discussion avec vous ! Cher professeur, nos discussions scientifiques vont me manquer énormément. Même si vous étiez loin à distance, vous étiez toujours présent à travers votre sympathie, vos conseils et soutien surtout pendant les périodes difficiles d'échec, du stress de travail, d'angoisse et d'incertitude ... Je vous suis très reconnaissante pour tout ! Monsieur Thomas, ça a été un réel plaisir de travailler en votre compagnie. Merci ! Je tiens aussi à remercier Monsieur Robert Blanvillain pour son aide dans la partie de biologie moléculaire et son support moral surtout pendant les dernières semaines dures de la rédaction! Je remercie également nos collaborateurs à la plateforme d'analyse protéomique attachée au CEA de Grenoble, EDYP, de m'avoir fourni les données protéomiques. Je remercie spécialement Madame Marianne Tardif pour sa serviabilité, ses explications, son temps et son aide dans la rédaction des quelques parties de ma thèse. Je remercie également Sabine Brugière pour ses explications et Yohan Couté. Je remercie infiniment David Cobessi pour sa collaboration mais aussi pour sa serviabilité, ses remarques pertinentes, son aide et ses qualités humaines. Je remercie Stéphane Ravanel et Morgane Minnino pour les figures de l'ultrastructure microscopique et de plusieurs autres supports. Je remercie, également, Boris Hedtke et Bernhard Grimm pour le dosage des pigments, Ben Field pour sa serviabilité et son contribution dans le dosage des quelques composés (non présenté dans le manuscrit), Silva Lerbs-Mache pour ses conseils pertinents en biochimie, Masato Nakai pour l'anticorps YCF1et la compagnie Agrisera qui m'a envoyé des échantillons des anticorps comme un cadeau pour ma motivation. Je tiens à remercier infiniment ma voisine du bureau Livia Merendino pour son encouragement, soutien et surtout sa patience pendant ma première année de thèse. J'aimerais remercier particulièrement Fabien Chevalier pour son aide dans quelques expériences mais surtout pour son écoute quand je pleure, quand je m'énerve et quand je suis triste/heureuse... Merci Fabien! Je remercie Björn Grübler pour ses données de la partie transcriptomique ainsi que pour sa gentillesse et ses interactions scientifiques par mail et Skype. Je remercie Jacques Bourguignon pour sa compréhension et son support infini en me validant mes demandes

Résumé Dynamique des protéines dans les premières étapes de la biogenèse du chloroplaste

La biogenèse des chloroplastes à partir des proplastes indifférenciés est un processus qui nécessite un réseau de régulation moléculaire complexe. L'ARN polymérase codée par le plaste (PEP) est un acteur décisif. La PEP est composé de quatre sous-unités codées par les gènes plastidiaux rpo. Lors de l'exposition à la lumière, cette enzyme centrale « PEP » est décorée de 12 protéines associées à la PEP (PAP) et qui sont codées par le génome nucléaire. L'inactivation génétique de l'une de ces protéines PAP bloque la différenciation des plastes et perturbe la biogenèse des chloroplastes conduisant à l'albinisme. Ainsi, le mutant albino d'Arabidopsis pap7-1 a été utilisé comme un outil génétique pour comprendre les premières étapes de la biogenèse des chloroplastes. La protéine PAP7 / pTAC14 (qui a été identifiée comme une sous-unité du complexe PEP) présente une double localisation dans les plastes et le noyau. La complémentation fonctionnelle du mutant pap7-1 avec une construction génétique de la séquence codante du gène PAP7 restaure le mutant albinos et les plantes transgéniques homozygotes développent un phénotype de type sauvage. Cela confirme l'importance de PAP7 dans les stades précoces de la biogenèse des chloroplastes. Néanmoins, les tests d'hémi-complémentation ont montré qu'il était difficile de séparer les fonctions nucléaires et plastidiales. D'autre part, l'absence de PAP7 inhibe la bonne formation de photobodies nucléaires tardifs. Ces données indiquent que la fraction nucléaire de la protéine PAP7 pourrait interagir avec d'autres régulateurs moléculaires afin d'être l'intermédiaire de la signalisation de la lumière via le phytochrome B activé. Cette voie contrôle probablement l'expression des facteurs de transcription nucléaire, tel que GLK1 dont le rôle principal est de contrôler des gènes nucléaires associés à la photosynthèse. Le protéome du mutant pap7-1 suggère que la majorité des protéines a régulée négativement en suivant strictement le profil transcriptomique correspondant. Les plantes albinos ont montré une absence totale d'appareil de photosynthèse, d'accumulation des pigments (chlorophylle et caroténoïde), des lipides de la membrane des thylakoïdes ainsi que certaines hormones. La voie de biosynthèse du tétrapyrrole à partir de laquelle l'hème est produite reste partiellement fonctionnelle. Cependant, la branche magnésium de biosynthèse de la chlorophylle est sélectivement bloquée. En résumé, ces données indiquent l'action d'un signal rétrograde qui empêche l'expression de certains PhANGs et des gènes relatifs. La biosynthèse des caroténoïdes doit être fonctionnelle dans le mutant pap7-1 en raison de l'accumulation normale des gènes de biosynthèse des caroténoïdes et des enzymes correspondantes. Néanmoins, les caroténoïdes étaient absents, probablement en raison de l'absence de photosynthèse qui produit habituellement le précurseur des caroténoïdes, le glycéraldéhyde-3-phosphate. En conséquence, l'acide abscissique ne s'accumule probablement pas. De plus, l'absence des membranes de thylakoïde, les lipides membranaires tels que le MGDG sont absents. Ce dernier explique la régulation négative de la voie de synthèse de l'allène oxyde synthase. Ce qui provoque probablement le blocage de la synthèse d'OPDA (le précurseur plastidial de l'acide jasmonique). A son tour, l'acide jasmonique est connu au son rôle dans la répression de l'élongation d'hypocotyle au début du développement de la plantule. Ce phénotype est observé en absence de PAP7. La présente étude fournit la preuve que PAP7 est un composant principal de l'expression du génome plastidial. En outre, il s'agit d'un composant de signalisation rétrograde qui couple la signalisation de la lumière passant par le phytochrome, avec la biogenèse des chloroplastes ainsi qu'il intègre des boucles de rétroaction métabolique du plaste.

Mots-clés: Arabidopsis, biogenèse des chloroplastes, albinisme, mutant pap7-1, double localisation noyau-plaste, protéomique, biologie des systèmes, signalisation rétrograde Chapter 1

State of the art

Introduction

Ce travail a été réalisé au sein du laboratoire de Physiologie Cellulaire et Végétale (LPCV) au centre du Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA) de Grenoble. Dans l'équipe «Nucleo-plastidic Interaction -Chloroplast biogenesis and redox control», qui s'intéresse à l'étude de la transcription du génome plastidial et de la biogenèse de chloroplaste. Mon projet de thèse a été effectué sous la direction du Pr. Thomas Pfannschmidt et co-encadré par le Dr. Robert Blanvillain.

Le projet est financé par le Labex GRAL afin de mener des études de protéomiques détaillées sur un modèle d'arrêt de développement chloroplastique entre l'obscurité et la lumière. La cellule végétale contient de nombreux plastes contenant chacun plusieurs copies identiques du chromosome de l'organite. Chez Arabidopsis thaliana, le génome plastidial existe sous deux formes:

1-une première forme d'ADN double brin circulaire dont la taille est comprise entre 120 et 160 kpb et code pour environ 120 protéines (Jarvis and Lopez-Juez, 2013;[START_REF] Sato | Complete structure of the chloroplast genome of Arabidopsis thaliana[END_REF][START_REF] Lilly | Cytogenomic Analyses Reveal the Structural Plasticity of the Chloroplast Genome in Higher Plants[END_REF].

2-Une deuxième forme linéaire plus ou moins concaténées [START_REF] Bendich | Circular Chloroplast Chromosomes: The Grand Illusion[END_REF].

L'ADN plastidial est organisé en nucléoïdes. Ces nucléoïdes sont localisés au niveau de la membrane des thylacoïdes [START_REF] Sato | Organization, developmental dynamics, and evolution of plastid nucleoids[END_REF]. Ils contiennent plusieurs molécules d'ADN qui sont associées à des protéines de différentes origines et de différentes fonctions dont certaines interviennent dans le développement plastidial [START_REF] Pfalz | Essential nucleoid proteins in early chloroplast development[END_REF].

La localisation de l'ADN plastidial a été étudiée lors de la transition induite par la lumière des proplastes en chloroplastes [START_REF] Pogson | Genetic dissection of chloroplast biogenesis and development: an overview[END_REF]. Ce processus est caractérisé par la formation des membranes des thylakoïde et d'un verdissement rapide des jeunes chloroplastes.

Les plastes ont leur propre machinerie génétique impliquant deux types d'ARN polymérase, des facteurs de transcription nucléaires et des sous-unités protéiques liées à ces polymérases.

1-L'ARN polymérase de type phagique (NEP) composée de deux enzymes monomériques codées par le génome nucléaire appelé RPOTp et RPOTmp [START_REF] Shiina | Plastid RNA polymerases, promoters, and transcription regulators in higher plants[END_REF][START_REF] Liere | The transcription machineries of plant mitochondria and chloroplasts: Composition, function, and regulation[END_REF][START_REF] Börner | Chloroplast RNA polymerases: Role in chloroplast biogenesis[END_REF], Pfannschmidt et al., 2015).

2 -L'ARN polymérase de type procaryotique codée par le plaste (PEP). C'est une polymérase multimérique constituée de quatre sous-unités (α, β, β' et β'').

Le développement de chloroplastes fonctionnels dépend de l'action coordonnée des deux ARN polymérases, nucléaire et plastidial. Par la suite, il est important de distinguer entre les trois catégories de gènes plastidiaux classés selon la structure de leurs promoteurs [START_REF] Allison | Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants[END_REF][START_REF] Hajdukiewicz | The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids[END_REF][START_REF] Ishizaki | A nuclear-encoded sigma factor, Arabidopsis SIG6, recognizes sigma-70 type chloroplast promoters and regulates early chloroplast development in cotyledons[END_REF]:

• Classe I: les gènes codés exclusivement par la PEP (gènes de photosynthèse tels que les gènes de PSI, PSII et ATP synthase)

• Classe II: les gènes exprimés à la fois par la NEP et la PEP (par exemple: atpB, atpI, ndhB, ndhF, clpP, YCF1, rr16, rps16, rpl33, rps33, rps18;[START_REF] Trifa | The nuclear RPL4 gene encodes a chloroplast protein that co-purifies with the T7-like transcription complex as well as plastid ribosomes[END_REF][START_REF] Fan | The Arabidopsis PEX12 gene is required for peroxisome biogenesis and is essential for development[END_REF].

• Classe III: les gènes codés exclusivement par la NEP (rpo, accD, ycf1, ycf2, clpP;[START_REF] Mullet | Dynamic Regulation of Chloroplast Transcription[END_REF].

La reconnaissance séquence-spécifique des promoteurs du plastome par l'appareil de transcription est assurée par six facteurs sigma codés par des gènes nucléaires. A la lumière, le coeur enzymatique de la PEP est entouré de douze PEP-associated proteins (PAPs). Ces protéines jouent un rôle essentiel dans l'expression des gènes de type II dépendant de la PEP en jouant un rôle dans le maintien du complexe PEP et la transcription. Au niveau macroscopique, l'absence de l'une des douze protéines PAPs, sauf PAP4 et PAP9, bloque la biogenèse des chloroplastes dans les stades précoces du développement, la membrane de thylakoïde ne se forme pas, les plantes développent un phénotype albinos et ne sont viables que sur un milieu gélosé riche en saccharose. Donc les protéines PAP jouent un rôle essentiel dans les premières étapes de biogenèse de chloroplaste [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF][START_REF] Pfannschmidt | Plastidial retrograde signalling -a true "plastid factor" or just metabolite signatures[END_REF]Pfannschmidt et al., , 2015;;[START_REF] Pfalz | Essential nucleoid proteins in early chloroplast development[END_REF][START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF][START_REF] Gao | Regulatory role of Arabidopsis pTAC14 in chloroplast development and plastid gene expression[END_REF][START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF][START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. Des études récentes ont été menées sur plusieurs protéines PAP tel que PAP2, PAP5, PAP8 et PAP7. Néanmoins les études faites ont été focalisées sur des analyses phénotypiques, transcriptomiques (RT-PCR, étude de la totalité de plastome, analyse de macroarray et microarray…). Aucune étude protéomique détaillée n'a été utilisée sur le modèle des plantes paps mutantes.

Mon projet de thèse consiste principalement à caractériser le profile protéomique d'un mutant génétique de l'une des protéines PAP, de couleur blanc albinos, incapable d'effectuer la photosynthèse et de différencier des chloroplastes. Nous avons choisi de travailler sur le mutant pap7-1 suite à un enchainement d'étude du transcriptome complet (ARNm) déjà effectué et publié au sein du notre équipe [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF].

Notre stratégie a été basée sur l'utilisation des plantes pap7-1, âgées de 7 jours et cultivées sur un milieu MS supplémenté de 1% saccharose à la lumière. L'extrait totale en protéines de ces plantes a été utilisé pour déterminer le profile protéique et le comparer avec celui des plantes sauvages cultivées soit à la lumière, soit à l'obscurité. Le choix de ces deux types de contrôles a pour but de déterminer l'effet de la photomorphogenèse et de la signalisation lumineuse sur la régulation des protéines dans le modèle d'arrêt de développement plastidial. Une étude protéomique globale a consisté en l'utilisation de trois types d'extraits totaux de protéines en triplicats biologiques. Ensuite le profile protéique a été comparé avec un profile transcriptomique déjà existant [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. Les résultats protéomiques ont été validés par des données d'immuno-détection. Finalement différents régulateurs moléculaires, protéiques et métaboliques ont été définis. Principalement les protéines de la photosynthèse, de la voie de synthèse des pigments (chlorophylles et caroténoïdes, les protéines plastidiales et les enzymes impliqués dans les voies de biosynthèse de l'acide abscissique et de l'acide jasmonique. L'étude détaillée de ces groupes protéiques fonctionnels a été effectué grâce à leur rôle potentiel dans l'albinisme et la biogenèse des chloroplastes.

J'ai également mené une approche de génétique et biologie moléculaire dans le but de comprendre la fonction et la double localisation de la protéine PAP7. Nous avons essayé de comprendre et de confirmer expérimentalement les prédictions de localisation de la protéine PAP7 dans les plastes et le noyau. De plus, nous avons testé l'hypothèse d'un rôle de PAP7 dans la voie de signalisation des phytochromes par l'analyse de la formation des tâches de phytochrome B et à la dégradation des PIF dans le noyau (Ce rôle de certains PAP a été montré pour PAP5/HEMERA [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF] et par notre équipe pour PAP8 (Liebers et al., en revision)). PAP7 est-elle nécessaire pour le maintien du complexe PEP-PAP? PAP7 interagit-elle avec d'autres PAPs dans le noyau ? Est-ce que PAP7 est impliquée dans la signalisation rétrograde ? Le syndrome albino en lien avec la perte de fonction du gène PAP7 procure un outil d'investigation intéressant pour comprendre les effets d'un blocage génétique de la biogenèse du chloroplaste. En outre, en l'absence de cible et d'activité enzymatique révélée, la fonction moléculaire de PAP7, bien que prédite par le domaine méthyl-transférase et le domaine d'interaction avec des protéines, reste mystérieuse. Nous avons généré un ensemble de lignées transgéniques, notamment dans le fond mutant pap7-1 afin de tester plusieurs hypothèses sur la distribution subcellulaire de la protéine par le suivi d'étiquette fluorescente ou bien l'analyse fonctionnelle de complémentation du mutant.

Une troisième partie du projet a été effectué dans le but mieux comprendre la nature de la voie du signal rétrograde impliquée dans la biogenèse des chloroplastes. Pour cela, deux inhibiteurs de la différenciation des chloroplastes ont été utilisés. Le norflurazon, pour inhiber la voie de la biosynthèse des caroténoïdes, et la lincomycin, pour inhiber la traduction plastidiale. Les deux inhibiteurs sont connus pour induire des plantes albinos comparables à celles qui sont générées par l'effet du défaut génétique du mutant pap7-1. Une analyse phénotypique, biochimique et d'accumulation de protéines a été réalisée afin de vérifier le rôle de ces inhibiteurs dans la création d'un feedback négatif sur les gènes associés à la photosynthèse (PhANG) et dans le blocage ultérieur du développement du plastide. En comparant ces données aux résultats des autres parties, nous pourrons mieux comprendre la biogenèse des chloroplastes.

Plastid origin: The endosymbiosis theory

Green, vascular plants are composed of eukaryotic cells that contain plant-specific organelles, the plastids. Plastids can differentiate into several forms, such as chloroplasts, which are considered as the most popular one (Schimper, 1885). This PhD thesis focusses on the different stages of chloroplast biogenesis and the ways of plastid-nucleus communication that are involved. In 1885, Schimper hypothesized that chloroplasts evolved from the symbiosis of microorganisms [START_REF] Palenik | The genomics of symbiosis: Hosts keep the baby and the bath water[END_REF]. Mereschkowsky emphasized in 1905 the theory of endosymbiosis linking the evolution of chloroplasts to similarities with cyanobacteria (Martin and Muller., 1998). He described plastids as reduced forms of cyanobacteria acting as "small, green slaves" in the cell [START_REF] Martin | Annotated English translation of Mereschkowsky's 1905 paper 'Über Natur und Ursprung der Chromatophoren imPflanzenreiche[END_REF][START_REF] Dyall | Ancient invasions: from endosymbionts to organelles[END_REF]. Gray and Lang confirmed in 1998 that the mitochondrion is the result of a symbiosis between an αproteobacterium and an archeal host cell. This generated a proto-mitochondrion around 1.5 billion years ago. A similar scenario explaining the origin of the chloroplast was launched later. Apparently, plastids are the result of a symbiosis between a non-photosynthetic eukaryotic cell containing fully evolved mitochondria and a photosynthetic cyanobacterium, which occurred about 1.2 billion years ago resulting in an autotrophic, eukaryotic cell, which is able to perform photosynthesis [START_REF] Dyall | Ancient invasions: from endosymbionts to organelles[END_REF][START_REF] Adams | Evolution of mitochondrial gene content: gene loss and transfer to the nucleus[END_REF].

Consequently, mitochondria evolutionary appeared before the plastids, but historically plastids were discovered before mitochondria because of their larger size and their green color.

Plastids are separated from the rest of the cell by two membranes: the outer membrane is of eukaryotic origin and the inner membrane derived from the cyanobacterial ancestor (Fig. 1) [START_REF] Dyall | Ancient invasions: from endosymbionts to organelles[END_REF][START_REF] Yoon | A molecular timeline for the origin of photosynthetic eukaryotes[END_REF][START_REF] Kutschera | Endosymbiosis, cell evolution, and speciation[END_REF][START_REF] Parfrey | Estimating the timing of early eukaryotic diversification with multigene molecular clocks[END_REF].

Figure 1. Endosymbiosis theory and plastid origin -

The figure depicts the essential steps of organelle evolution. On top a rough time scale of events occurring during Earth history. Below the evolutionary branches for the corresponding evolution of mitochondria and plastids [START_REF] Dyall | Ancient invasions: from endosymbionts to organelles[END_REF].

During this evolutionary process, the integration of cyanobacteria into the host cell was accompanied by a reduction in the genomes of the endosymbiot (Glockner et al., 2000;[START_REF] Martin | Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus[END_REF][START_REF] Palenik | The genomics of symbiosis: Hosts keep the baby and the bath water[END_REF][START_REF] Ankele | In vivo visualization of Mg-protoporphyrin IX, a coordinator of photosynthetic gene expression in the nucleus and the chloroplast[END_REF]. Many plastidic genes have been lost (Gabaldon and Huynen, 2003), some have been retained in the plastid genome, but the majority of cyanobacterial genes have been transferred to the nuclear genome by massive horizontal gene transfer [START_REF] Palenik | The genomics of symbiosis: Hosts keep the baby and the bath water[END_REF][START_REF] Martin | Gene transfer from organelles to the nucleus: how much, what happens, and Why?[END_REF]. Thus, these cellular organelles became genetically semi-autonomous (Fig. 2). Nowadays, plastids have lost their genetic autonomy and the majority of their proteins are encoded by the nucleus, translated in the cytoplasm and then imported into the plastid [START_REF] Bruce | The paradox of plastid transit peptides: conservation of function despite divergence in primary structure[END_REF]Jarvis and Soll, 2002). Plastids still possess their own genome (called a "plastome") with a size of about 120 to 200 kb [START_REF] Sugiura | The chloroplast genome[END_REF]. It codes for a highly conserved set of 100 to 130 proteins that are components mainly involved in ribosomal structure and function (tRNAs and rRNAs), the photosynthetic apparatus and the transcription of plastid genes. In Arabidopsis thaliana the size of the plastome is about 154.5-kb and codes for about 75-80 proteins [START_REF] Timmis | Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes[END_REF]. In addition a 18% of the nuclear genome of around 25,000 genes encodes about 2,500 to 4,500 plastidial proteins [START_REF] Soll | Protein import into chloroplasts[END_REF]) that are largely from cyanobacterial origin [START_REF] Abdallah | A prediction of the size and evolutionary origin of the proteome of chloroplasts of Arabidopsis[END_REF][START_REF] Martin | Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus[END_REF][START_REF] Kleffmann | The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions[END_REF]. According to [START_REF] Martin | Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus[END_REF], half of these cyanobacterial genes encode proteins that cannot return back to the chloroplast and perform novel functions in the cytosol. About 95% of the chloroplast proteins are encoded by the nucleus [START_REF] Martin | Gene transfer from organelles to the nucleus: how much, what happens, and Why?[END_REF]. The rest of proteins are encoded by the plastome and have not been transferred [START_REF] Martin | Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus[END_REF][START_REF] Strand | Chloroplast to nucleus communication triggered by accumulation of Mg-protoporphyrinIX[END_REF]. The evolutionary reasons for retaining these genes were heavily discussed and a final explanation is not yet agreed on [START_REF] Abdallah | A prediction of the size and evolutionary origin of the proteome of chloroplasts of Arabidopsis[END_REF][START_REF] Allen | Why chloroplasts and mitochondria retain their own genomes and genetic systems: Colocation for redox regulation of gene expression[END_REF].

Nevertheless, many of these components are necessary for plastid transcription/translation and photosynthesis and thus absolutely essential for plastid development and plant survival [START_REF] Martin | Gene transfer from organelles to the nucleus: how much, what happens, and Why?[END_REF][START_REF] Sakamoto | Chloroplast biogenesis: control of plastid development, protein import, division and inheritance[END_REF][START_REF] Timmis | Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes[END_REF][START_REF] Sugiura | The chloroplast genome[END_REF].

Because of the dual localization of genes for protein components in mitochondria and plastids a tight communication between the endosymbiote and the nucleus of the host cell was essential to establish and maintain endosymbiosis.

Communication between plastids and nucleus

The mutual communication between cellular organelles is essential for plant growth and development (Martin et al., 2016;[START_REF] Pogson | Plastid signalling to the nucleus and beyond[END_REF]. The majority of the proteome of semiautonomous organelles such as plastids is encoded by the nuclear genome, translated to the cytosol. These proteins contain an N-terminal chloroplast transit peptide allowing them to be imported into chloroplasts (Bruce, 2000).

The maintenance of plastid protein complexes requires a direct flow of information from the nucleus to the plastids called anterograde signaling, which is required for a proper function of the organelle (Dietzel et al., 2008;[START_REF] Pfannschmidt | Plastidial retrograde signalling -a true "plastid factor" or just metabolite signatures[END_REF].

However, retrograde signal is described as a "backward" flow of information that includes information emitted by the chloroplast to the nucleus [START_REF] Pfannschmidt | Plastidial retrograde signalling -a true "plastid factor" or just metabolite signatures[END_REF]Jarvis and Lopez-Juez, 2013). These signals indicate the functional and developmental status of the plastid (Dietzel et al., 2008;[START_REF] Pfannschmidt | Plastidial retrograde signalling -a true "plastid factor" or just metabolite signatures[END_REF]. This mutual communication coordinates the expression of plastid proteins encoded in the nuclear genome and the plastome.

However, plastids destroyed by genetic mutations or chemical inhibitors are (likely) unable to receive the anterograde signal, which can cause an accumulation of nuclear-encoded proteins in the cytosol and subsequently the formation of a negative feedback inhibiting the expression of nuclear encoded plastid proteins.

Several factors were identified to orchestrate the retrograde signaling network and the changes in the expression of photosynthetic nuclear genes have been identified [START_REF] Barajas-López | Expression of the chloroplast thioredoxins f and m is linked to short-term changes in the sugar and thiol status in leaves of Pisum sativum[END_REF]. Recently, plastidial signals were classified into two major categories depending on their function [START_REF] Pfannschmidt | Plastidial retrograde signalling -a true "plastid factor" or just metabolite signatures[END_REF]Dietzel et al., 2008;[START_REF] Pogson | Plastid signalling to the nucleus and beyond[END_REF][START_REF] Barajas-López | Expression of the chloroplast thioredoxins f and m is linked to short-term changes in the sugar and thiol status in leaves of Pisum sativum[END_REF][START_REF] Jung | Signaling between Chloroplasts and the Nucleus: Can a Systems Biology Approach Bring Clarity to a Complex and Highly Regulated Pathway?: Figure 1[END_REF]:

1-Biogenic signals control organelle biogenesis by plastid gene expression (PGE) (transcription and translation) and pigment biosynthesis (tetrapyrrole such as heme or Mg-Proto-Porphyrin-IX and carotenoid biosynthesis pathways) 2-Operational signals control modifications made by metabolic pathways in response to environmental changes or stresses including plastid redox state and redox signals by photosynthetic electron transport and reactive oxygen species (ROS) (Suzuki et al.,2011;[START_REF] Pfannschmidt | Chloroplast redox signals: how photosynthesis controls its own genes[END_REF][START_REF] Apel | Reactive oxygen species: metabolism, oxidative stress, and signal transduction[END_REF]Dietzel et al., 2008;Dietzel et al., 2008;[START_REF] Woodson | Sigma factor-mediated plastid retrograde signals control nuclear gene expression[END_REF]. The "retrograde signals" were discovered originally in plants with disturbances in the expression of the plastidial genome. [START_REF] Bradbeer | Cytoplasmic synthesis of plastid polypeptides may be controlled by plastid-synthesised RNA[END_REF] have shown that the albostrians mutant from barley presented a genetic alteration resulting in a deficiency in plastid ribosomes. This alteration blocked the synthesis of plastidial proteins. The synthesis of photosynthetic proteins encoded by the nucleus was also blocked resulting in albino striped plants [START_REF] Hess | Ribosome-deficient plastids affect transcription of lightinduced nuclear genes: genetic evidence for a plastid-derived signal[END_REF]. Very recently, the genetic defect in proplastid-to-chloroplast transition was identified as a lack of plastid-encoded RNA polymerase-associated proteins (PAPs). This lack give rise to an albino mutant phenotype in the light with a block in the PEP activities, a lack of photosynthesis genes expression and absence of the functional chloroplast [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF].

These observations led to the proposal of a missing plastid factor that activates nuclear gene expression. The authors suggested a retrograde control by the plastid affecting the nuclear gene expression [START_REF] Bradbeer | Cytoplasmic synthesis of plastid polypeptides may be controlled by plastid-synthesised RNA[END_REF][START_REF] Mandel | CLA1, a novel gene required for chloroplast development, is highly conserved in evolution[END_REF]Estevez et al., 2000). Several other mutants have been described showing similar phenotypes such as the Arabidopsis sco1 (snowy cotyledons) mutant that is mutated in the translation elongation gene encoded by plastids [START_REF] Albrecht | Characterization of the snowy cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality[END_REF][START_REF] Shimada | Arabidopsis cotyledon-specific chloroplast biogenesis factor CYO1 is a protein disulfide isomerase[END_REF] or the apg3 (albino or pale green mutant 3) mutant that is mutated in the chloroplastic ribosome 1. Both mutants show an arrested plastid development because of a defective plastidial protein synthesis and a decreased accumulation of nuclear photosynthesis genes [START_REF] Motohashi | Chloroplast ribosome release factor 1 (AtcpRF1) is essential for chloroplast development[END_REF]. Many more mutants are known in between and are publicly available in the Chloroplast Function Database II [START_REF] Myouga | The Chloroplast Function Database II: a comprehensive collection of homozygous mutants and their phenotypic/genotypic traits for nuclear-encoded chloroplast proteins[END_REF]. Furthermore, several experiments were carried out to confirm the impact of plastid protein translation on nuclear gene expression. For example, lincomycin treatment was used. Lincomycin is an antibiotic that acts specifically against the 70S prokaryotic ribosome. Its application to plants reduces the accumulation of nuclear encoded photosynthesis genes for chlorophyll a/b binding proteins (LHCB) and the small subunit of Rubisco (RBCS) (Oelmüller et al., 1986;[START_REF] Sullivan | Plastid translation is required for the expression of nuclear photosynthesis genes in the dark and in roots of the pea lip1 mutant[END_REF]. The lincomycin treatment was found to be a good indicator for the role of plastidial translation in the generation of a retrograde signal [START_REF] Barkan | Participation of nuclear genes in chloroplast gene expression[END_REF][START_REF] Ruckle | Plastids are major regulators of light signaling in Arabidopsis[END_REF]Larkin, 2016). There are also other proteins involved in the biogenic control of feedback regulation.

Disruptions in the tetrapyrrole biosynthetic pathway have been shown to result in damage of the expression of nuclear genes associated with photosynthesis (PhANG) [START_REF] Alawady | Tobacco Mg protoporphyrin IX methyltransferase is involved in inverse activation of Mg porphyrin and protoheme synthesis[END_REF][START_REF] Strand | Chloroplast to nucleus communication triggered by accumulation of Mg-protoporphyrinIX[END_REF]. Uncoupled genome (GUN) are components associated with tetrapyrrole biosynthesis [START_REF] Surpin | Signal Transduction between the Chloroplast and the Nucleus[END_REF][START_REF] Larkin | GUN4, a regulator of chlorophyll synthesis and intracellular signaling[END_REF]. gun mutants have been used to confirm the role of retrograde signaling by the tetrapyrrole pathway towards the nucleus [START_REF] Nott | Plastid-to-nucleus retrograde signaling[END_REF][START_REF] Mochizuki | The steady-state level of Mgprotoporphyrin IX is not a determinant of plastid-to-nucleus signaling in Arabidopsis[END_REF]. The two mutants gun4 and gun5 are directly involved in the chlorophyll branch of the tetrapyrrole pathway. gun5 is known as the H subunit of the Mg-chelatase (CHLH) representing the first reaction in the "Chlorophyll Branch" [START_REF] Mochizuki | Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction[END_REF]. gun4 was found to code for a Mg-chelatase activator and the GUN4 protein interacts with the Mg-chelatase complex [START_REF] Larkin | GUN4, a regulator of chlorophyll synthesis and intracellular signaling[END_REF]. These two GUNs catalyze the magnesium chelation step and the conversion of protoporphyrin IX (ProtoIX) to generate Mg-Proto-IX involving the roles of GUN4 and GUN5 in inhibiting the accumulation of phototoxic Mg-Proto-IX and in regulating chlorophyll synthesis and the plastid-to-nucleus signal [START_REF] Ankele | In vivo visualization of Mg-protoporphyrin IX, a coordinator of photosynthetic gene expression in the nucleus and the chloroplast[END_REF][START_REF] Strand | Plastid-to-nucleus signalling[END_REF]. Following norflurazon treatment, repression in the accumulation of Mg-Proto-IX, PhANGs, and LHCB1.2 has been shown in gun4-and gun5 mutants [START_REF] Zhang | Transient accumulation of Mg-protoporphyrin IX regulates expression of PhANGs -New evidence for the signaling role of tetrapyrroles in mature Arabidopsis plants[END_REF][START_REF] Strand | Chloroplast to nucleus communication triggered by accumulation of Mg-protoporphyrinIX[END_REF]. The mutants gun2 and gun3 code for components of the heme branch, which is a side pathway of tetrapyrrole biosynthesis. The genetic disruption of GUN2 and GUN3 caused an overexpression of heme [START_REF] Van Lis | Subcellular localization and light-regulated expression of protoporphyrinogen IX oxidase and ferrochelatase in Chlamydomonas reinhardtii[END_REF], which can activate a negative feedback on the accumulation of Mg-Proto-IX.

GENOMES UNCOUPLED 1 (GUN1) is a chloroplast localized pentatricopeptide-repeat protein (PPR) [START_REF] Koussevitzky | Signals from Chloroplasts Converge to Regulate Nuclear Gene Expression[END_REF]. gun1 mutants display inhibition of plastidial gene expression and translation [START_REF] Susek | Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development[END_REF][START_REF] Susek | Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development[END_REF][START_REF] Nott | Plastid-to-nucleus retrograde signaling[END_REF] which affects the expression of LHCB nuclear genes by retrograde signaling [START_REF] Jarvis | The Proteomes of Chloroplasts and other Plastids[END_REF]. Many other studies have shown the impact of tetrapyrroles and chlorophyll precursors in the expression of photosynthesis associated nuclear genes via the plastid retrograde signaling (La [START_REF] Rocca | Amitrole treatment of etiolated barley seedlings leads to deregulation of tetrapyrrole synthesis and to reduced expression of Lhc and RbcS genes[END_REF][START_REF] Kropat | Chlorophyll precursors are signals of chloroplast origin involved in light induction of nuclear heat-shock genes[END_REF].

Chlorophyll synthesis is initiated by the conversion of glutamyl-tRNA to δ-aminolevulinic acid (ALA) [START_REF] Rüdiger | Chlorophyll Metabolism, an Overview[END_REF]. Glutamyl-tRNA transcription is performed by the PEP core enzyme [START_REF] Hanaoka | Molecular genetic analysis of chloroplast gene promoters dependent on SIG2, a nucleus-encoded sigma factor for the plastid-encoded RNA polymerase, in Arabidopsis thaliana[END_REF]. The formation of tRNA-E as a precursor for tetrapyrrole biosynthesis depends on plastid transcription [START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF].

Genetic/chemical blocking of trnE expression by disrupting transcription or plastidial translation prevents the formation of Chlide a and, consequently, the reduction of LHCB import [START_REF] Bräutigam | Plastid-nucleus communication: anterograde and retrograde signalling in the development and function of plastids[END_REF][START_REF] Reinbothe | A role for chlorophyllide a oxygenase in the regulated import and stabilization of light-harvesting chlorophyll a/b proteins[END_REF]. This leads to an accumulation of LHCB precursors in the cytosol, which could activate a suppression of PhANG expression by feedback control [START_REF] Inaba | Bilateral Communication between Plastid and the Nucleus: Plastid Protein Import and Plastidto-Nucleus Retrograde Signaling[END_REF][START_REF] Inaba | Versatile Roles of Plastids in Plant Growth and Development[END_REF].

It was also shown that plants treated with norflurazon (NF) have a deficiency in carotenoid biosynthesis by blocking the enzymatic activity of phytoene desaturase (PDS) [START_REF] Chamovitz | The molecular basis of resistance to the herbicide norflurazon[END_REF][START_REF] Chamovitz | Molecular and biochemical characterization of herbicideresistant mutants of cyanobacteria reveals that phytoene desaturation is a rate-limiting step in carotenoid biosynthesis[END_REF]. This can affect the expression of the photosynthetic nuclear genes such as LHCB and RBCS genes [START_REF] Mayfield | Carotenoid-deficient maize seedlings fail to accumulate light-harvesting chlorophyll a/b binding protein (LHCP) mRNA[END_REF]Oelmüller and Mohr, 1986) and subsequently the plastidial gene expression [START_REF] Gray | Coordination of plastid and nuclear gene expression[END_REF] caused by photo-oxidative damage to the thylakoid membranes [START_REF] Susek | Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development[END_REF], a cessation of chlorophyll synthesis and chloroplast biogenesis (Dietzel et al., 2008). This carotenoid deficiency leads to the generation of a retrograde signal.

Another possibility is the environmental stress caused by the accumulation of signals related to photosynthetic redox electron transport; reactive oxygen species (ROS) such as oxygen singlet oxygen 1 O2 and H2O2, oxidative stress of the photosynthetic electron transport chain (PETC) and connected metabolite pools. This kind of oxidative stress also affects PhANGs expression by retrograde signaling (Brautigam et al., 2009). Chlorophyll molecules excited in the triplet state (P6980) can form 1 O2 in photosystem II (Krieger-Liskay et al., 2008). 1 O2 is highly reactive causing photo-oxidative damage mainly in PSII [START_REF] Gorman | New trends in photobiology: Current perspectives of singlet oxygen detection in biological environments[END_REF][START_REF] Camp | Rapid Induction of Distinct Stress Responses after the Release of Singlet Oxygen in Arabidopsis[END_REF][START_REF] Baier | Chloroplasts as source and target of cellular redox regulation: a discussion on chloroplast redox signals in the context of plant physiology[END_REF]. This induces retrograde redox signaling to the nucleus [START_REF] Apel | Reactive oxygen species: metabolism, oxidative stress, and signal transduction[END_REF][START_REF] Foyer | Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context[END_REF]. Furthermore, the transfer of electrons to O2 can cause the production of superoxide (O2 -) and hydrogen peroxide (H2O2), which disrupt the redox stability of the photosynthetic electron transport chain (PETC) [START_REF] Ivanov | Participation of Photosynthetic Electron Transport in Production and Scavenging of Reactive Oxygen Species[END_REF][START_REF] Barajas-López | Expression of the chloroplast thioredoxins f and m is linked to short-term changes in the sugar and thiol status in leaves of Pisum sativum[END_REF].

Several mutants were used to study the 1 O2 effect on operational retrograde signaling. In the dark, the fluorescent (flu) mutant of Arabidopsis thaliana accumulates free protochlorophyllide (Pchlide). FLU is a nuclear encoded plastidial protein that acts as a negative regulator of tetrapyrrole metabolism. After exposure to light, the mutant produces singlet oxygen by energy transfer from Pchlide [START_REF] Meskauskiene | FLU: A negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana[END_REF]. This leads to growth inhibition and cell death [START_REF] Wagner | The long-term response to fluctuating light quality is an important and distinct light acclimation mechanism that supports survival of Arabidopsis thaliana under low light conditions[END_REF]. In addition, the plastoquinone pool (PQ) can be also a source for redox signals. The PQ pool is an electron acceptor of PSII, localized in the thylakoid membrane and involved in plastid photo-acclimation [START_REF] Durnford | Chloroplast redox regulation of nuclear gene transcription during photoacclimation[END_REF]. It has been shown that the transcription and regulation of LHCB gene is also controlled by the redox status of the PQ pool [START_REF] Yang | The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation[END_REF][START_REF] Pfannschmidt | Chloroplast redox signals: how photosynthesis controls its own genes[END_REF].

Another approach indicates that at the end of the vegetative developmental state several cellular compounds are degraded. The best known example is the degradation of Rubisco to reduced nitrogen which causes the chloroplast to generate a retrograde signal called "degradational control" [START_REF] Krupinska | 11 -Photosynthesis and Chloroplast Breakdown[END_REF][START_REF] Pfannschmidt | Plastidial retrograde signalling -a true "plastid factor" or just metabolite signatures[END_REF]. Recently the protein Whirly 1 (Why1) is considered as a good example for the retrograde signal. When plastid are disturbed by a stress or senescence, Why1 proteins are accumulated in the cytosol (Grabowsky et al., 2008;Krause and Krupinsk, 2009).

In any case, to guarantee an optimal response of the plant to developmental or environmental constraints, it is necessary for plastids to transmit their developmental/functional state to the nucleus, which will influence PhANGs expression (as the most obvious gene group perceiving the plastid signals). In return, the nucleus controls plastids (anterograde signaling), by submitting proteins for translation and import machineries or providing post-transcriptional regulation factors for editing and splicing [START_REF] Bräutigam | Plastid-nucleus communication: anterograde and retrograde signalling in the development and function of plastids[END_REF], which are necessary to maintain plastid stability.

A coordinated response between the different genetic cell compartments (plastids and nucleus) is achieved through this mutual exchange of cytosolic and plastid signaling networks (Fernandez and Strand, 2008). Plastid signals are also known to coordinate cell cycle and chloroplast development [START_REF] Kobayashi | Regulation of Root Greening by Light and Auxin/Cytokinin Signaling in Arabidopsis[END_REF]. Thus, it has become clear that retrograde signaling plays an important role in the development of different cellular processes in the plant, such as chloroplast biogenesis.

Different types of plastid

Chloroplasts are the well-known autotrophic organelles responsible for photosynthesis, however, a plant can develop a multitude of different plastid types depending on the developmental stage or the tissue context [START_REF] Pyke | Chloroplast Division and Expansion Is Radically Altered by Nuclear Mutations in Arabidopsis thaliana[END_REF][START_REF] Dyall | Ancient invasions: from endosymbionts to organelles[END_REF]. Plastids can differ in general structure, protein composition and enzymatic function [START_REF] Pyke | Plastid biogenesis and differentiation[END_REF]Weber, 2007;Wise, 2007). Its multiplication is, however, always achieved by binary fission using a prokaryotic-type division apparatus [START_REF] Osteryoung | The Division of Endosymbiotic Organelles[END_REF][START_REF] Osteryoung | Division and Dynamic Morphology of Plastids[END_REF][START_REF] Liebers | Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development[END_REF]. All plastids are developed from the differentiation of an undifferentiated proplastid form existing in meristematic cells [START_REF] Mullet | Chloroplast Development and Gene Expression[END_REF][START_REF] Mereschkowsky | Uber Natur und Ursprung der Chromatophoren im Pflanzenreiche[END_REF][START_REF] Pyke | Chloroplast Division and Expansion Is Radically Altered by Nuclear Mutations in Arabidopsis thaliana[END_REF][START_REF] Pyke | Plastid Ontogeny during Petal Development in Arabidopsis[END_REF][START_REF] Timmis | Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes[END_REF][START_REF] Pogson | Genetic dissection of chloroplast biogenesis and development: an overview[END_REF]. Proplastids are small colorless organelles from 0.5 µm to 1 µm diameter, containing a few tubules that are connected to the inner membrane [START_REF] Pyke | Plastid biogenesis and differentiation[END_REF]. These proplastids can differentiate into different types of non-pigmented plastids called leucoplasts. These latter can develop oil bodies (Elaoiplasts; [START_REF] Pacini | Development of Plastids in Pollen and Tapetum of Ryegrass, Lolium perenne L[END_REF][START_REF] Ting | Constituents of the tapetosomes and elaioplasts in Brassica campestris tapetum and their degradation and retention during microsporogenesis[END_REF][START_REF] Pyke | Plastid biogenesis and differentiation[END_REF] or starch granules in the root, seeds and in potato tubers (Amyloplasts; [START_REF] Pyke | Plastid biogenesis and differentiation[END_REF]Wise et al., 2007). Furthermore, some plants contain chromoplasts that are differentiated directly from proplastids or most often from chloroplasts (Ljubesic, 1977;[START_REF] Bouvier | Dedicated Roles of Plastid Transketolases during the Early Onset of Isoprenoid Biogenesis in Pepper Fruits1[END_REF]. This type of plastid is rich in carotenoids [START_REF] Egea | Chromoplast Differentiation: Current Status and Perspectives[END_REF][START_REF] Liebers | Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development[END_REF]López-Juez and Pyke, 2005). In the absence of light proplastid differentiation, in dark grown plants, is blocked at a transitional state and gives rise to etioplasts [START_REF] Wellburn | Chlorophyll synthesis by isolated intact etioplasts[END_REF]. Etioplasts contain tubular membranes of paracrystalline structure called prolamellar bodies (PLB; [START_REF] Boardman | Protein synthesis by cell-free extracts of tobacco leaves: III. Comparison of the physical properties and protein synthesizing activities of 70 s chloroplast and 80 s cytoplasmic ribosomes[END_REF][START_REF] Park | Identification of the Carotenoid Isomerase Provides Insight into Carotenoid Biosynthesis, Prolamellar Body Formation, and Photomorphogenesis[END_REF]. PLB structures can facilitate greening after light exposure [START_REF] Park | Identification of the Carotenoid Isomerase Provides Insight into Carotenoid Biosynthesis, Prolamellar Body Formation, and Photomorphogenesis[END_REF][START_REF] Barrero | The ABA1 gene and carotenoid biosynthesis are required for late skotomorphogenic growth in Arabidopsis thaliana[END_REF]. PLBs contain chlorophyll precursors (protochlorophyllide; [START_REF] Reinbothe | PORA and PORB, Two Light-Dependent Protochlorophyllide-Reducing Enzymes of Angiosperm Chlorophyll Biosynthesis[END_REF][START_REF] Barthélemy | Localization of NADPH-protochlorophyllide reductase in plastids of barley at different greening stages[END_REF], NADPH, plastoglobuli or galacto-sulfo lipids [START_REF] Dehesh | The NADPH-protochlorophyllide oxidoreductase is the major protein constituent of prolamellar bodies in wheat (Triticum aestivum L.)[END_REF][START_REF] Pribil | Structure and dynamics of thylakoids in land plants[END_REF][START_REF] Bastien | Chapter One -New Insights on Thylakoid Biogenesis in Plant Cells[END_REF], ribosomes trapped within PLBs [START_REF] Bartels | Inhibition of Carotenoid Synthesis by Fluridone and Norflurazon[END_REF], a few proteins and carotenoids, lutein and violaxanthin [START_REF] Pogson | Genetic dissection of chloroplast biogenesis and development: an overview[END_REF]. Upon light exposure, etioplasts (or also proplastids) can rapidly differentiate into a chloroplast. PLBs then transform rapidly into thylakoid membranes by invaginations of the inner membrane [START_REF] Bourett | Ultrastructure of chloroplast protuberances in rice leaves preserved by high-pressure freezing[END_REF][START_REF] Pogson | Insights into chloroplast biogenesis and development[END_REF]. This process produces vesicles that can fuse together to form a thylakoid membrane system. The thylakoid membrane is characterized by cylindrical lumen structures, called grana, which are interconnected and separated from the chloroplastic inner membrane by stromal lamellae. Then this particular structure activates the protochlorophyllide oxydo reductase (POR) to convert protochlorophyllide into chlorophyllide used subsequently for chlorophyll synthesis [START_REF] Forreiter | Light-independent and light-dependent protochlorophyllide-reducing activities and two distinct NADPH-protochlorophyllide oxidoreductase polypeptides in mountain pine (Pinus mugo)[END_REF][START_REF] Ryberg | Localization of NADPH-protochlorophyllide oxidoreductase in dark-grown wheat (Triticum aestivum) by immuno-electron microscopy before and after transformation of the prolamellar bodies[END_REF]. Etioplast-to-chloroplast transition, thus, is a light-dependent process that is very rapidly appearing in the range of few hours and, therefore, is very difficult to dissect. Especially molecular regulators controlling these processes are largely unknown [START_REF] Pogson | Plastid signalling to the nucleus and beyond[END_REF] and are currently under investigation.

The role of the chloroplast

Chloroplasts are green plastids that play an important role in the plant cell development and metabolism and are already detected in the early globular stage [START_REF] Pogson | Genetic dissection of chloroplast biogenesis and development: an overview[END_REF][START_REF] Allorent | Plastid gene expression during chloroplast differentiation and dedifferentiation into non-photosynthetic plastids during seed formation[END_REF]. The chloroplast envelope controls the exchange of molecules between the stroma and the cytoplasm. The envelope has a selective and specific transport system composed of the TOC/TIC machinery (Translocon at the outer/inner membrane of the chloroplast; [START_REF] Ferro | Integral membrane proteins of the chloroplast envelope: Identification and subcellular localization of new transporters[END_REF]Jarvis and Soll, 2002). Thylakoids are lipid membranes formed mainly by mono-and digalactosyldiacylglycerol synthesized by the chloroplast itself or imported from the endoplasmatic reticulum. During the thylakoid formation, the photosynthetic apparatus is generated by the formation of protein complexes, photosystems (PS) I and II, cytochrome b6/f and ATP synthase, which all are involved in the light reaction [START_REF] Benning | Mechanisms of Lipid Transport Involved in Organelle Biogenesis in Plant Cells[END_REF]. The photosystems formation requires a coordination between chlorophyll binding proteins (light-harvesting chlorophyll-binding LHCB) and chlorophyll/carotenoid biosynthesis, which are essential for the maintenance and photo-protection of the photosynthetic complexes [START_REF] Cazzonelli | Source to sink: regulation of carotenoid biosynthesis in plants[END_REF].

Chloroplasts are the site of photosynthesis. They are responsible for detecting environmental stimuli and providing energy for the various developmental aspects of plants [START_REF] Pogson | Insights into chloroplast biogenesis and development[END_REF]. Furthermore, chloroplasts control several other metabolic reactions based on their own genetic system [START_REF] Stern | Transcription and translation in chloroplasts[END_REF], such as ribosome metabolism, amino acid biosynthesis, generation of fatty acids, lipids, vitamins, plastoquinone and phylloquinone (vitamin K), isoprenoids biosynthesis (involved in the biosynthesis of carotenoids and sterols), chlorophyll precursors and tetrapyroles (involved in chlorophyll, heme, phytochromobilin and siroheme biosynthesis), carbon oxidation via photorespiration [START_REF] Ogren | Photorespiration: Pathways, Regulation, and Modification[END_REF], plant hormones, regulation of flowering time and germination as well as leaf development and shape [START_REF] Albrecht | Characterization of the snowy cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality[END_REF].

The genetic machinery in the plastids

During evolution, a non-photosynthetic mitochondrial proto-eukaryotic cell engulfed a photosynthetic cyanobacterium giving rise to the photosynthetic eukaryotic organelle called plastid [START_REF] Dyall | Ancient invasions: from endosymbionts to organelles[END_REF]. During this endosymbiotic process, there was a loss and a massive horizontal gene transfer from the endosymbiote to the host cell nucleus [START_REF] Martin | Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus[END_REF][START_REF] Timmis | Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes[END_REF]. However, plastids have preserved parts of their ancestral prokaryotic genome that nowadays is composed of up to 100 copies of circular DNA molecules encoding about 120 genes in vascular plants. The plastome is typically divided into two single copy parts separated by two antiparallel inverse-repeated regions (IR, [START_REF] Bock | Structure, function, and inheritance of plastid genomes[END_REF]. The plastome has a circular double-stranded DNA form whose size varies from 120 to 160 kb [START_REF] Lilly | Cytogenomic Analyses Reveal the Structural Plasticity of the Chloroplast Genome in Higher Plants[END_REF] but also can appear in a linear form [START_REF] Bendich | Circular Chloroplast Chromosomes: The Grand Illusion[END_REF].

It mainly codes for photosynthesis and gene expression elements (PSI, PSII, ATP synthase, cytochrome, plastidial transcription genes (rpo), four genes encoding for ribosomal RNA for translation (rRNA; 16S, 23S, 5S and 4.5S), 30 genes encoding for transfer RNA (tRNA), chloro-respiration genes (NADH dehydrogenase complex), a single gene (clpP) encodes for a protease subunit and more than 72 genes encode for photosynthesis and ribosomal polypeptides. However, the plastid genome does not allow the synthesis of all necessary functional components in the plastid [START_REF] Sugiura | The chloroplast genome[END_REF][START_REF] Martin | Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus[END_REF][START_REF] Pfalz | Essential nucleoid proteins in early chloroplast development[END_REF]. Indeed, 95% of plastid proteomes are encoded by nuclear genes, translated in the cytosol and then imported into the plastid [START_REF] Shiina | Plastid RNA polymerases, promoters, and transcription regulators in higher plants[END_REF][START_REF] Bock | Structure, function, and inheritance of plastid genomes[END_REF][START_REF] Ferro | AT_CHLORO, a Comprehensive Chloroplast Proteome Database with Subplastidial Localization and Curated Information on Envelope Proteins[END_REF]. This indicates that the coordination between nuclear and plastidal gene expression must be tightly controlled (Lopez-Juez and Pyke, 2005;Dietzel et al., 2008). The plastid gene transcription system is composed of eukaryotic, prokaryotic and phage-type factors indicating a complex evolutionary scenario. Two types of RNA polymerases can be distinguished in plastids:

1-The Nuclear Encoded RNA Polymerase (NEP) composed of two phage-type monomeric subunits encoded by the nuclear genome called RPOTp and RPOTmp [START_REF] Shiina | Plastid RNA polymerases, promoters, and transcription regulators in higher plants[END_REF][START_REF] Liere | The transcription machineries of plant mitochondria and chloroplasts: Composition, function, and regulation[END_REF][START_REF] Börner | Chloroplast RNA polymerases: Role in chloroplast biogenesis[END_REF], Pfannschmidt et al., 2015) 2 -The Plastid Encoded RNA Polymerase (PEP), a multimeric prokaryotic-type polymerase consisting four subunits α, β, β'and β''.

Nuclear encoded polymerase (NEP)

Numerous investigations were carried out in the early 1970-1990s using the barley albostrians mutant. This mutant develops white stripes in the barley leaves. Plastids in these white sectors showed a deficiency in plastidial ribosomes, which subsequently resulted in the absence of PEP expression. However, the synthesis of plastid RNA was still detectable in these albino plants [START_REF] Siemenroth | Synthesis of ribosomal RNA in ribosomedeficient plastids of the mutant "albostrians" of Hordeum vulgare[END_REF]. This suggested the presence of a second plastidial RNA polymerase not encoded by the plastome [START_REF] Morden | Plastid translation and transcription genes in a non-photosynthetic plant: intact, missing and pseudo genes[END_REF][START_REF] Hess | Chloroplast rps15 and the rpoB/C1/C2 gene cluster are strongly transcribed in ribosome-deficient plastids: evidence for a functioning nonchloroplast-encoded RNA polymerase[END_REF]Silhavy and Maliga, 1998;Silhavy and Maliga, 1998;[START_REF] Serino | RNA Polymerase Subunits Encoded by the Plastid rpoGenes Are Not Shared with the Nucleus-Encoded Plastid Enzyme[END_REF]Allison, 2000).

Later, in 1993, biochemical studies conducted by Lerbs-Mache, former manager of our team, on chloroplasts of young spinach leaves showed the presence of a 110-kDa monomeric RNA polymerase that recognized the T7 bacteriophage promoter but not the PEP promoter. This confirmed the presence of a second phage-type transcription system encoded by the nucleus and imported into the plastid [START_REF] Lerbs-Mache | The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: singlesubunit enzyme or catalytic core of multimeric enzyme complexes?[END_REF][START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]. NEP encoding genes were firstly discovered in Arabidopsis and were named RPOT from phage T3/T7 type [START_REF] Hedtke | Mitochondrial and Chloroplast Phage-Type RNA Polymerases in Arabidopsis[END_REF][START_REF] Börner | Chloroplast RNA polymerases: Role in chloroplast biogenesis[END_REF][START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]. RPOT is considered to be the main RNA polymerase that drives the transcription of RPO genes for the PEP enzyme, ACCD (Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta), ClpP (ATP-dependent Clp protease proteolytic subunit), YCF1 and YCF2 (yeast cadmium factor proteinand 1 and 2). In monocotyledouns plants two types of NEP enzymes have been shown. RPOTp which is located only in plastids and RPOTm which is located only in mitochondria [START_REF] Hedtke | Mitochondrial and Chloroplast Phage-Type RNA Polymerases in Arabidopsis[END_REF][START_REF] Hedtke | One RNA polymerase serving two genomes[END_REF][START_REF] Chang | Functional Analysis of Two Maize cDNAs Encoding T7-like RNA Polymerases[END_REF][START_REF] Ikeda | Identification and characterization of T3/T7 bacteriophage-like RNA polymerase sequences in wheat[END_REF][START_REF] Kusumi | Characterization of a Rice Nuclear-Encoded Plastid RNA Polymerase Gene OsRpoTp[END_REF]. However, in dicotyledonous plants, such as A. thaliana, there is a third nuclear encoded polymerase type, called RPOTmp. This polymerase is imported into both plastid and mitochondria [START_REF] Hedtke | One RNA polymerase serving two genomes[END_REF]. It was suggested that RPOTmp comes from a second duplication after divergence between monocotyledonous and dicotyledonous plants. In A.thaliana RPOTmp controls the transcription of the rRNA operon in the PC promoter during seeds imbibition [START_REF] Courtois | Phage-Type RNA Polymerase RPOTmp Transcribes the rrn Operon from the PC Promoter at Early Developmental Stages in Arabidopsis[END_REF]. NEP activity is high during the first days of seed stratification and germination [START_REF] Demarsy | Building Up of the Plastid Transcriptional Machinery during Germination and Early Plant Development[END_REF]. Whereas, in mature plants, NEP activity is reduced and is replaced by the PEP activity. This suggests a predominant role of NEP in the early stages of plant development and chloroplast biogenesis.

Plastid encoded RNA polymerase (PEP)

The plastid encoded RNA polymerase originates from an ancestral cyanobacterium and has a homologous structure to the Escherichia coli RNA polymerase. It is a multimeric enzyme, whose "core enzyme" is composed of four putative subunits α, β, β´, and β" encoded respectively by the plastidial genes rpoA, rpoB, rpoC1 and rpoC2 [START_REF] Hudson | Spinach chloroplast rpoBC genes encode three subunits of the chloroplast RNA polymerase[END_REF][START_REF] Hu | Maize chloroplast RNA polymerase: the 180-, 120-, and 38-kilodalton polypeptides are encoded in chloroplast genes[END_REF][START_REF] Hu | Maize chioroplast RNA polymerase: the 78-kilodaiton polypeptide is encoded by the plastid rpoCl gene[END_REF][START_REF] Pfannschmidt | Separation of two classes of plastid DNA-dependent RNA polymerases that are differentially expressed in mustard (Sinapis alba L.) seedlings[END_REF].

The knock-out of these plastidial genes in tobacco resulted in an albino phenotype. This suggests an essential role of PEP in the transcription of photosynthetic genes and subsequently in the establishment and maintaining of a functional chloroplast [START_REF] Allison | Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants[END_REF][START_REF] Santis-Maciossek | Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1: molecular biology, biochemistry and ultrastructure[END_REF]. In addition, the core enzyme recognizes the prokaryotic consensus promoter sequences -35 (TTGACA) and -10 (TATAAT) in coordination with the plastidial transcription factors "Sigma Like Transcription Factors" (SLFs). These SLFs are encoded by the nucleus and imported into the chloroplast [START_REF] Liere | In vitro characterization of the tobacco rpoB promoter reveals a core sequence motif conserved between phage-type plastid and plant mitochondrial promoters[END_REF][START_REF] Hakimi | Evolutionary Conservation of C-terminal Domains of Primary Sigma70-type Transcription Factors between Plants and Bacteria[END_REF]. Then, they bind to the core enzyme in order to create an active complex called "holoenzyme" and then initiate the plastidial transcription [START_REF] Lerbs | DNA-dependent RNA polymerase of spinach chloroplasts: Characterization of α-like and σ-like polypeptides[END_REF][START_REF] Tanaka | Nuclear Encoding of a Chloroplast RNA Polymerase Sigma Subunit in a Red Alga[END_REF][START_REF] Suzuki | Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme[END_REF][START_REF] Liere | The transcription machineries of plant mitochondria and chloroplasts: Composition, function, and regulation[END_REF]. Therefore, PEP functioning is dependent on the cooperation between the two genetic systems located in the nuclear and plastid. In Arabidopsis, there are six different sigma factors [START_REF] Lerbs-Mache | Function of plastid sigma factors in higher plants: regulation of gene expression or just preservation of constitutive transcription?[END_REF].

I.5.2.1 Sigma factors

Sigma factors are a nuclear genes family encoding plastid-localized prokaryotic-type transcription factors [START_REF] Isono | Leaf-specifically expressed genes for polypeptides destined for chloroplasts with domains of σ70 factors of bacterial RNA polymerases in Arabidopsis thaliana[END_REF][START_REF] Kanamaru | An Arabidopsis Sigma Factor (SIG2)-Dependent Expression of Plastid-Encoded tRNAs in Chloroplasts[END_REF][START_REF] Fujiwara | Three new nuclear genes, sigD, sigE and sigF, encoding putative plastid RNA polymerase σ factors in Arabidopsis thaliana[END_REF][START_REF] Lysenko | Plant sigma factors and their role in plastid transcription[END_REF][START_REF] Zghidi | Nucleus-encoded plastid sigma factor SIG3 transcribes specifically the psb N gene in plastids[END_REF]. Recently, it was confirmed that sigma factors initiate promoter-specific PEP transcription [START_REF] Hakimi | Evolutionary Conservation of C-terminal Domains of Primary Sigma70-type Transcription Factors between Plants and Bacteria[END_REF][START_REF] Homann | DNA-binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression[END_REF]Privat et al., 2003). PEP is largely responsible for photosynthesis gene transcription. In Arabidopsis, PEP recognizes promoters through the association with SLFs. SLFs are expressed abundantly in photosynthetic tissues in the presence of light [START_REF] Isono | Leaf-specifically expressed genes for polypeptides destined for chloroplasts with domains of σ70 factors of bacterial RNA polymerases in Arabidopsis thaliana[END_REF][START_REF] Fujiwara | Three new nuclear genes, sigD, sigE and sigF, encoding putative plastid RNA polymerase σ factors in Arabidopsis thaliana[END_REF].

This suggested an important role of sigma factors in the transcription of the plastidial genome, the establishment of the photosynthetic apparatus and the chloroplast biogenesis.

Sequencing of the Arabidopsis nuclear genome has proven the existence of six different sigma factors (SIG1-SIG6) that perform different functions [START_REF] Fujiwara | Three new nuclear genes, sigD, sigE and sigF, encoding putative plastid RNA polymerase σ factors in Arabidopsis thaliana[END_REF][START_REF] Lerbs-Mache | Function of plastid sigma factors in higher plants: regulation of gene expression or just preservation of constitutive transcription?[END_REF]. The existence of several plastidial transcription factors for PEP may suggest that the transcription of the chloroplast genes is specifically regulated according to culture or environmental conditions [START_REF] Tozawa | Nuclear encoding of a plastid σ factor in rice and its tissue-and light-dependent expression[END_REF][START_REF] Lahiri | Complementary Expression of Two Plastid-Localized ς-Like Factors in Maize[END_REF][START_REF] Morikawa | Overexpression of Sigma Factor, ςB, Urges Staphylococcus aureus to Thicken the Cell Wall and to Resist β-Lactams[END_REF].

All these factors are orthologues of the σ70 factors in E. coli which can be divided into two groups:

 Essential sigma factors including sigma 1, 2 and 6. The corresponding gene inactivation mutants have a pale green phenotype with chlorophyll deficiency.

 Non-essential sigma factors including sigma 3, 4 and 5. The corresponding inactivation mutants do not show a phenotype deviating from wildtype.

Obtaining T-DNA insertion mutants for each of the six sigma factors allowed to characterize the specificity and the role of each of them in the regulation of PEP activity.

Essential sigma factors

 Sigma 1

Sigma 1 was shown to play an important role in transcription (Privat et al., 2003;[START_REF] Suzuki | Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme[END_REF]. Arabidopsis SIG1 factor can recognize, in vitro, the rbcL and psbA gene promoters [START_REF] Hakimi | Evolutionary Conservation of C-terminal Domains of Primary Sigma70-type Transcription Factors between Plants and Bacteria[END_REF][START_REF] Homann | DNA-binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression[END_REF].

In sig1 mutant plants, a low recognition of these promoters by SIG1 has been confirmed [START_REF] Tozawa | The plastid sigma factor SIG1 maintains photosystem I activity via regulated expression of the psaA operon in rice chloroplasts[END_REF]. In addition, transcriptomic analysis showed a strong decrease in psaA, psbB and psbE transcripts accumulation. This decrease suggested a reduction in the photosystem I and II gene expression and a deficiency in the electron transfer between the two photosystems. The conclusion from this decrease was that SIG1 regulates the expression of the PSI and II genes [START_REF] Shimizu | Sigma factor phosphorylation in the photosynthetic control of photosystem stoichiometry[END_REF][START_REF] Lerbs-Mache | Function of plastid sigma factors in higher plants: regulation of gene expression or just preservation of constitutive transcription?[END_REF].

Recently, it has been found that blue and red light leads to a high accumulation of SIG1 transcripts [START_REF] Onda | Light induction of Arabidopsis SIG1 and SIG5 transcripts in mature leaves: differential roles of cryptochrome 1 and cryptochrome 2 and dual function of SIG5 in the recognition of plastid promoters[END_REF]. This can create redox signals that regulate the phosphorylation state of SIG1 in its variable N-terminal region and then send a negative feedback to inhibit psaA transcription. Therefore, the stoichiometric ratio between PSI and PSII can be modified as a result [START_REF] Shimizu | Sigma factor phosphorylation in the photosynthetic control of photosystem stoichiometry[END_REF]. In addition, SIG1 can be regulated through interactions with other proteins such as the SIB1 protein [START_REF] Morikawa | Overexpression of Sigma Factor, ςB, Urges Staphylococcus aureus to Thicken the Cell Wall and to Resist β-Lactams[END_REF]. This may induce an alteration in the defense-related genes expression such as the response to jasmonate and salicylate [START_REF] Wang | Acetylation of Metabolic Enzymes Coordinates Carbon Source Utilization and Metabolic Flux[END_REF].

 Sigma 2

The sigma2 factor is able to interact with the PEP core enzyme and initiate plastidial transcription [START_REF] Suzuki | Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme[END_REF][START_REF] Hakimi | Evolutionary Conservation of C-terminal Domains of Primary Sigma70-type Transcription Factors between Plants and Bacteria[END_REF][START_REF] Homann | DNA-binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression[END_REF]. Therefore in Arabidopsis, SIG2 initiates the transcription of the rbcL and psbA promoter [START_REF] Hakimi | Evolutionary Conservation of C-terminal Domains of Primary Sigma70-type Transcription Factors between Plants and Bacteria[END_REF]Kanamura et al., 2001;[START_REF] Hanaoka | Molecular genetic analysis of chloroplast gene promoters dependent on SIG2, a nucleus-encoded sigma factor for the plastid-encoded RNA polymerase, in Arabidopsis thaliana[END_REF]Privat et al., 2003;[START_REF] Nagashima | DNA Microarray Analysis of Plastid Gene Expression in an <I>Arabidopsis</I> Mutant Deficient in a Plastid Transcription Factor Sigma, SIG2[END_REF]). The T-DNA insertion mutant for SIG2 showed an albino or pale green phenotype with deficiency in chlorophyll accumulation and photosynthesis proteins [START_REF] Shirano | Chloroplast development in Arabidopsis thaliana requires the nuclear-encoded transcription factor Sigma B[END_REF][START_REF] Kanamaru | An Arabidopsis Sigma Factor (SIG2)-Dependent Expression of Plastid-Encoded tRNAs in Chloroplasts[END_REF]Privat et al., 2003). Transcriptomic analysis of the sig2 mutant showed a significant accumulation of NEP transcripts [START_REF] Nagashima | DNA Microarray Analysis of Plastid Gene Expression in an <I>Arabidopsis</I> Mutant Deficient in a Plastid Transcription Factor Sigma, SIG2[END_REF]. However, the sig2 mutant has shown a reduction in the accumulation levels of tRNAs, psbD, psaJ, psbA and rbcL [START_REF] Kanamaru | An Arabidopsis Sigma Factor (SIG2)-Dependent Expression of Plastid-Encoded tRNAs in Chloroplasts[END_REF][START_REF] Hanaoka | Molecular genetic analysis of chloroplast gene promoters dependent on SIG2, a nucleus-encoded sigma factor for the plastid-encoded RNA polymerase, in Arabidopsis thaliana[END_REF][START_REF] Homann | DNA-binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression[END_REF]Privat et al., 2003;[START_REF] Nagashima | DNA Microarray Analysis of Plastid Gene Expression in an <I>Arabidopsis</I> Mutant Deficient in a Plastid Transcription Factor Sigma, SIG2[END_REF][START_REF] Tsunoyama | Blue light-induced transcription of plastid-encoded psbD gene is mediated by a nuclear-encoded transcription initiation factor[END_REF]. Thus, the SIG2 factor regulates the expression of photosynthesis genes and tRNAs.

In addition, the aligned sequences of SIG2 and SIG 6 with E. coli sigma (σ 70 ) factors revealed that SIG2 and SIG6 have a high homology (Privat et al., 2003).

The corresponding mutants showed a chlorophyll accumulation deficiency and a damaged thylakoid membrane system. This suggests a role for the two factors in the early stages of chloroplast biogenesis [START_REF] Ishizaki | A nuclear-encoded sigma factor, Arabidopsis SIG6, recognizes sigma-70 type chloroplast promoters and regulates early chloroplast development in cotyledons[END_REF][START_REF] Schweer | AtSIG6, a plastid sigma factor from Arabidopsis, reveals functional impact of cpCK2 phosphorylation[END_REF][START_REF] Malik Ghulam | Transcriptional organization of the large and the small ATP synthase operons, atpI/H/F/A and atpB/E, in Arabidopsis thaliana chloroplasts[END_REF].

 Sigma 6

The T-DNA insertion into the sig6 gene identified a mutant with pale green phenotype and chlorophyll biosynthesis deficiency [START_REF] Ishizaki | A nuclear-encoded sigma factor, Arabidopsis SIG6, recognizes sigma-70 type chloroplast promoters and regulates early chloroplast development in cotyledons[END_REF][START_REF] Loschelder | Dual Temporal Role of Plastid Sigma Factor 6 in Arabidopsis Development[END_REF].

Transcriptomic analysis of this sig6 mutant showed an increasing NEP transcript accumulation (ycf1, rpoB, rpoC1 and rpoC2). However, the mutant showed a decrease in the accumulation of PEP-dependent transcripts such as the rbcL, PSII genes (such as psbA), tRNAs (such as tRNA-E) and 16S rRNA. All these data suggest that SIG6 is a general and major transcription factor in the early stages of plastid development. In addition, the phosphorylation of SIG6 is controlled by redox-signaling mediated by the chloroplast casein kinase 2 (cpCK2; [START_REF] Schweer | AtSIG6, a plastid sigma factor from Arabidopsis, reveals functional impact of cpCK2 phosphorylation[END_REF]. This can prove the redox-induced effect in the regulation of plastid gene expression [START_REF] Tiller | Phosphorylation and dephosphorylation affect functional characteristics of chloroplast and etioplast transcription systems from mustard (Sinapis alba L.)[END_REF][START_REF] Puthiyaveetil | Transcriptional Control of Photosynthesis Genes: The Evolutionarily Conserved Regulatory Mechanism in Plastid Genome Function[END_REF][START_REF] Schweer | AtSIG6, a plastid sigma factor from Arabidopsis, reveals functional impact of cpCK2 phosphorylation[END_REF][START_REF] Türkeri | Phylogenetic and functional features of the plastid transcription kinase cpCK2 from Arabidopsis signify a role of cysteinyl SH-groups in regulatory phosphorylation of plastid sigma factors[END_REF].

Non-essential sigma factors

 Sigma 3 SIG3 is the only transcription factor expressed in Arabidopsis dry seeds (Privat et al., 2003).

This suggests its role in very early plant development steps. In addition, SIG3 gene attaches to the membrane and subsequently activates plastidial transcription [START_REF] Hakimi | Evolutionary Conservation of C-terminal Domains of Primary Sigma70-type Transcription Factors between Plants and Bacteria[END_REF]Privat et al., 2003). A T-DNA insertion sig3 mutant did not show any phenotype deviating from wildtype [START_REF] Zghidi | Nucleus-encoded plastid sigma factor SIG3 transcribes specifically the psb N gene in plastids[END_REF].

Transcriptomic analysis of this mutant showed that the corresponding gene is involved in the regulation of psbB expression by producing anti-sense mRNA [START_REF] Zghidi | Nucleus-encoded plastid sigma factor SIG3 transcribes specifically the psb N gene in plastids[END_REF]. In fact SIG3 controls the transcription of psbN which is located in the psbB operon, between psbT and psbH but on the opposite strand [START_REF] Zghidi | Nucleus-encoded plastid sigma factor SIG3 transcribes specifically the psb N gene in plastids[END_REF]Zgidi-Abouzid et al., 2011).

Therefore, the psbN gene transcription can influence the psbB operon expression.

In sig3 mutant plants, the accumulation of PSBN is greatly reduced, indicating that psbN transcription is under the specific control of SIG3 [START_REF] Zghidi | Nucleus-encoded plastid sigma factor SIG3 transcribes specifically the psb N gene in plastids[END_REF].

In addition, SIG3 is involved in atpH transcription, which allows to synthesize atpF and atpA transcripts located downstream of the same transcriptional unit [START_REF] Zghidi | Nucleus-encoded plastid sigma factor SIG3 transcribes specifically the psb N gene in plastids[END_REF]. In the absence of SIG2, accumulation of SIG3 increases. Thus, SIG3 is proposed as a candidate that can replace SIG2 thanks to their similar promoter specificity [START_REF] Hakimi | Evolutionary Conservation of C-terminal Domains of Primary Sigma70-type Transcription Factors between Plants and Bacteria[END_REF]Privat et al., 2003).

 Sigma 4

The knock-out of the SIG4 gene has generated a sig4 mutant without any recognizable deviation from wildtype appearance [START_REF] Favory | Specific function of a plastid sigma factor for ndh F gene transcription[END_REF]. However, the molecular analysis showed that the sig4 mutant is characterized by a decrease in the expression level of gene encoding the ndhF subunit. This suggests that SIG4 controls the expression of ndhF and then regulates the NDH complex assembly.

 Sigma 5

The T-DNA insertion in the SIG5 gene did not induce any phenotype deviating from wild type [START_REF] Tsunoyama | Blue light-induced transcription of plastid-encoded psbD gene is mediated by a nuclear-encoded transcription initiation factor[END_REF]. However, the sig5 mutant has been characterized as embryolethal (embryonic lethality) indicating that SIG5 can be an essential factor for plant reproduction [START_REF] Yao | AtSig5 Is an Essential Nucleus-Encoded Arabidopsis σ-Like Factor[END_REF][START_REF] Nagashima | DNA Microarray Analysis of Plastid Gene Expression in an <I>Arabidopsis</I> Mutant Deficient in a Plastid Transcription Factor Sigma, SIG2[END_REF]. In addition, it has been found that the expression of the sig5 gene is blue light dependent. This suggests a role of SIG5 in mediation of blue light signaling (Mochizuki et al., 2004;[START_REF] Tsunoyama | Blue light-induced transcription of plastid-encoded psbD gene is mediated by a nuclear-encoded transcription initiation factor[END_REF][START_REF] Onda | Light induction of Arabidopsis SIG1 and SIG5 transcripts in mature leaves: differential roles of cryptochrome 1 and cryptochrome 2 and dual function of SIG5 in the recognition of plastid promoters[END_REF].

In response to light stress (strong blue light), SIG5 factors can recognize a blue light sensitive promoter (BLRP) that participates in the repair of the photo-damaged photosystem II complex and subsequently regulate the psbD transcription [START_REF] Nagashima | DNA Microarray Analysis of Plastid Gene Expression in an <I>Arabidopsis</I> Mutant Deficient in a Plastid Transcription Factor Sigma, SIG2[END_REF][START_REF] Tsunoyama | Blue light-induced transcription of plastid-encoded psbD gene is mediated by a nuclear-encoded transcription initiation factor[END_REF].

PEP-Associated Proteins (PAPs)

Biochemical studies were carried out on mustard and Arabidopsis plants in order to characterize the structure and function of the PEP enzyme. These studies have shown that the PEP of etioplasts is composed of the core enzyme (the four subunits α, β, β, β ′, β") and sigma factors to form the holo-enzyme. After light exposition that induces an etioplast-chloroplast transition this enzyme becomes rapidly restructured and associates with additional subunits that subsequently trigger chloroplast formation [START_REF] Tiller | Phosphorylation and dephosphorylation affect functional characteristics of chloroplast and etioplast transcription systems from mustard (Sinapis alba L.)[END_REF][START_REF] Pfannschmidt | Separation of two classes of plastid DNA-dependent RNA polymerases that are differentially expressed in mustard (Sinapis alba L.) seedlings[END_REF]. Further analyses identified these subunits within the transcriptionally active chromosome (TAC) by mass spectrometry [START_REF] Yu | TAC7, an essential component of the plastid transcriptionally active chromosome complex, interacts with FLN1, TAC10, TAC12 and TAC14 to regulate chloroplast gene expression in Arabidopsis thaliana[END_REF]. The TAC is a biochemically defined fraction of the plastid, with a DNA-dependent RNA polymerase activity. It contains endogenously bound DNA and is able to express it [START_REF] Herrmann | Multiple amounts of DNA related to the size of Chloroplasts[END_REF]Igloi and Koessel, 1992;Krause et al., 2000). It is associated with the membrane in the first steps of chloroplast development.

TAC purification combined with mass spectrometry identified about 40 to 60 protein subunits in this complex. These proteins have been also identified by mass spectrometry (Pfannschmidt et al., 2000;[START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF][START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF][START_REF] Yu | TAC7, an essential component of the plastid transcriptionally active chromosome complex, interacts with FLN1, TAC10, TAC12 and TAC14 to regulate chloroplast gene expression in Arabidopsis thaliana[END_REF][START_REF] Melonek | Multifunctionality of plastid nucleoids as revealed by proteome analyses[END_REF]. 35 proteins have been identified in total in Arabidopsis, 18 of them were defined as the pTAC protein group [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF][START_REF] Lerbs-Mache | Function of plastid sigma factors in higher plants: regulation of gene expression or just preservation of constitutive transcription?[END_REF]. The PEP is a part of the TAC complex.

The smallest complex purified from mustard chloroplasts includes ten proteins of eukaryotic origin encoded by the nucleus in addition to RPO subunits [START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF]. These proteins were referred to the peak "A" enzyme [START_REF] Pfannschmidt | Separation of two classes of plastid DNA-dependent RNA polymerases that are differentially expressed in mustard (Sinapis alba L.) seedlings[END_REF]. A few years later, two other subunits were identified [START_REF] Pfalz | Essential nucleoid proteins in early chloroplast development[END_REF]. These PEP surrounding subunits have been named PEP-associated proteins (PAP; [START_REF] Pfalz | Essential nucleoid proteins in early chloroplast development[END_REF]. Therefore, in chloroplast, the core enzyme PEP is the major RNA polymerase type, which is stably associated with 12 subunits that are encoded by the nucleus [START_REF] Lerbs | Polypeptides of DNA-dependent RNA polymerase of spinach chloroplasts: characterization by antibody-linked polymerase assay and determination of sites of synthesis[END_REF][START_REF] Rajasekhar | Highly purified pea chloroplast RNA polymerase transcribes both rRNA and mRNA genes[END_REF][START_REF] Link | Redox Regulation of Chloroplast Transcription[END_REF][START_REF] Pfannschmidt | Redox Regulation and Modification of Proteins Controlling Chloroplast Gene Expression[END_REF]. The determination of the genes corresponding to each one of these twelve PAPs was carried out by homology prediction in the Arabidopsis nuclear genome. [START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF] have shown the presence of 10 PAPs (PAP1-PAP10) associated with the PEP enzyme. Two other PAPs (PAP11/MurE and PAP12/pTAC7) were added through phenotypic studies of corresponding Arabidopsis mutants [START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF][START_REF] Pfalz | Essential nucleoid proteins in early chloroplast development[END_REF]. This study has finally confirmed the existence of 12 PAPs of various functions and structures, belonging to the PEP [START_REF] Suzuki | Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme[END_REF]. These nuclear-encoded proteins are imported from the cytosol thanks to an N-terminal chloroplastidic transit peptide. Currently, the functions of the different PAPs have been mainly predicted by domain analysis of the corresponding genes in Arabidopsis and then experimentally verified by functional analysis using T-DNA insertion lines [START_REF] Pfalz | Environmental control of plant nuclear gene expression by chloroplast redox signals[END_REF]. PAPs can be divided into four major groups according to their predicted theoretical function:

1-DNA/RNA metabolism and gene expression regulation (PAP1, 2, 3, 5 and 7; [START_REF] Kindgren | Chloroplast transcription, untangling the Gordian Knot[END_REF], 2-Redox regulation and reactive oxygen species protection (PAP6 and 10; [START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF], 3-Protection against ROS, not directly related to gene expression (PAP4 and 9), 4-Unknown function (PAP8, 11 and 12;[START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF][START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF].

Interestingly, all PAP T-DNA inactivation mutants present an albino / ivory or pale green phenotype with an arrested plastid development. Transcript analyses have shown that there is typically a reduction in PEP activity (especially class 1 genes) compared to NEP activity that is not affected or even improved (class 2 and 3 genes; [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF][START_REF] Myouga | A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis[END_REF][START_REF] Arsova | Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana[END_REF][START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF][START_REF] Yagi | Eukaryotic-type plastid nucleoid protein pTAC3 is essential for transcription by the bacterial-type plastid RNA polymerase[END_REF]. These observations match perfectly with existing data about rpo-deletion mutants from tobacco [START_REF] Allison | Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants[END_REF][START_REF] Hajdukiewicz | The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids[END_REF][START_REF] Santis-Maciossek | Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1: molecular biology, biochemistry and ultrastructure[END_REF]. This suggests an important role of PAPs in the formation of the plastid encoded RNA polymerase complex for the correct regulation of plastid gene expression and chloroplast biogenesis.

However, it has been shown that albino PAP mutants can survive on a sucrose-supplemented medium where they develop a reasonable rosette and a large wild-type-like phenotype indicating that the principle processes of photomorphogenesis are functional in these mutants [START_REF] Schröter | Analysis of oligomeric protein complexes in the chloroplast sub-proteome of nucleic acid-binding proteins from mustard reveals potential redox regulators of plastid gene expression[END_REF][START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF].

The twelve sequences of the pap genes, including their predicted functional domains and three-dimensional structure, were depicted around the PEP core enzyme (Fig. 6). The phenotypes of the corresponding single T-DNA inactivation mutants including the studies describing them are summarized below (Fig. 5). All these proteins were also identified within the TAC complex or have been identified by further functional studies where they obtained other names. These are also listed. For consistency the PAP nomenclature is used throughout this thesis.

Figure 5. Phenotype presentation of pap T-DNA inactivation mutants in Arabidopsis thaliana

PAP mutants with albino/ivory phenotype (pap1, -3, -5, -6, -8, -12), albino phenotype (pap7, -10, -11), yellow / green pale phenotype (pap2), or green / albino / ivory (pap 4, -9). Thesis of Morgane Mininno thesis (2014).

The protein PAP1/pTAC3 contains a putative eukaryotic DNA-binding SAP domain. This motif is involved in the transcription, binding, repair and degradation of DNA/RNA (Aravind et al., 2000; interPRO website and Smart domain annotation). In addition, this 110-Kda protein contains two other domains [START_REF] Yagi | Eukaryotic-type plastid nucleoid protein pTAC3 is essential for transcription by the bacterial-type plastid RNA polymerase[END_REF]: A pentatricopeptide repeat (PPR) domain involved in post-transcriptional processes, such as RNA metabolism, splicing, editing, translation, RNA processing and stabilization [START_REF] Delannoy | Pentatricopeptide repeat (PPR) proteins as sequence-specificity factors in post-transcriptional processes in organelles[END_REF][START_REF] Pfalz | Site-specific binding of a PPR protein defines and stabilizes 5′ and 3′ mRNA termini in chloroplasts[END_REF], and a PNLR domain associated with the stabilization of rbcL transcripts [START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]. Inactivation of PAP1/pTAC3 resulted in a true albino phenotype. Immunoprecipitation analyses demonstrated the interaction of PAP1 with the α-subunit in the PEP complex.

Furthermore, confocal imaging confirmed the co-localization of PAP1 protein with the plastid genome. Together these analyses suggest that PAP1 is an essential plastidial transcription factor in the PEP complex, involved in plastidial transcription initiation, elongation and termination steps [START_REF] Yagi | Eukaryotic-type plastid nucleoid protein pTAC3 is essential for transcription by the bacterial-type plastid RNA polymerase[END_REF]. PAP2/pTAC2 is a protein with a predicted size of 89-kDa [START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF] containing three domains. A PPR domain, a SMR domain that is involved in DNA repair [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF] and a tetratricopeptide repeat (TPR) domain likely involved in protein-protein interactions.

Inactivation of PAP2/ pTAC2 showed plants with a slightly green phenotype and an absence of thylakoid membranes that are replaced by plastoglobuli vesicles [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF][START_REF] Myouga | The Chloroplast Function Database: a large-scale collection of Arabidopsis Ds/Spm-or T-DNA-tagged homozygous lines for nuclear-encoded chloroplast proteins, and their systematic phenotype analysis[END_REF]. Chlorophylls and carotenoids accumulated to low levels with a decrease in the chlorophyll a/b ratio due to a decrease in the photosystem I: II ratio. Transcript analyses of these mutant showed a reduction in PEP-dependent transcript accumulation (especially photosynthesis transcripts such as psbA, petB (cytochrome b6f subunit) and rbcL (for the large ribulose-1.5-bis-phosphate carboxylase / oxygenase subunit)). In contrast, NEP transcripts were expressed at normal or elevated levels such as those for the ribosomal genes, rpo, ndh, ycf, atp (for ATP synthase subunits) [START_REF] Hess | Chloroplast rps15 and the rpoB/C1/C2 gene cluster are strongly transcribed in ribosome-deficient plastids: evidence for a functioning nonchloroplast-encoded RNA polymerase[END_REF][START_REF] Allison | Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants[END_REF][START_REF] Hajdukiewicz | The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids[END_REF]Silhavy and Maliga, 1998;Krause et al., 2000). This may suggest that PAP2/pTAC2 is involved in plastid transcription and chloroplast biogenesis. PAP3/pTAC10 is a protein with a predicted size of 77-Kda containing a predicted S1-like domain involved in RNA binding. The genetic inactivation of PAP3 resulted in an albino phenotype (Steiner el al., 2011;[START_REF] Yu | TAC7, an essential component of the plastid transcriptionally active chromosome complex, interacts with FLN1, TAC10, TAC12 and TAC14 to regulate chloroplast gene expression in Arabidopsis thaliana[END_REF]. PAP3 is a part of the essential TAC proteins involved in the plastidial PEP transcription [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF]. PAP4 (FSD3; Fe superoxide dismutase 3; 26-Kda) and PAP9 (FSD2; 29-Kda) are TAC proteins most likely involved in redox regulation and ROS protection. Both contain an iron superoxide dismutase (FeSOD) domain involved in the defense against oxidative damage related to the reactive oxygen species (ROS; [START_REF] Myouga | A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis[END_REF]. The two single mutant alleles of fsd2 and fsd3 mutants have shown a pale green phenotype while the double mutant displayed a full albino phenotype in sucrose supplementation medium [START_REF] Myouga | A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis[END_REF][START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]. This suggests that they may partly replace each other in the complex if one is missing. The functionality as SOD could be demonstrated for both enzymes.

Transcript analyses further showed a significant accumulation of NEP dependent-genes and a low accumulation of PEP dependent-genes. This suggests a role of PAP4 and PAP9 in maintaining the functionality of the PEP complex and the plastidial genes expression [START_REF] Myouga | A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis[END_REF]. PAP6/FLN1 (fructokinase-like protein) is a 49-kDa protein containing a phosphorfructokinase like domain [START_REF] Arsova | Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana[END_REF][START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF]. FLN1 is a thioredoxin target protein (TRX) which is able to interact with the PAP10/TrxZ protein [START_REF] Arsova | Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana[END_REF][START_REF] Schröter | Analysis of oligomeric protein complexes in the chloroplast sub-proteome of nucleic acid-binding proteins from mustard reveals potential redox regulators of plastid gene expression[END_REF][START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF]. Indeed, PAP10 is the only redox thioredoxin domain protein. The knockout of PAP6 and PAP10 has resulted in a mutant plant with an albino phenotype [START_REF] Arsova | Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana[END_REF][START_REF] Schröter | Analysis of oligomeric protein complexes in the chloroplast sub-proteome of nucleic acid-binding proteins from mustard reveals potential redox regulators of plastid gene expression[END_REF]. These observations suggest an essential role of thioredoxin (TrxZ), PAP10, and FLN, PAP6, in PEP transcription regulation and subsequently in chloroplast transcription [START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]. PAP5 / pTAC12/ HEMERA is a 60-Kda protein that contains a glutamate-rich region (GLU-Rich) [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]. Inactivation of PAP5 resulted in mutant plants with an albino phenotype and a hypocotyl elongation under red light [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF][START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF][START_REF] Chen | Phytochrome signaling mechanisms and the control of plant development[END_REF]. Besides its participation in the plastid TAC complex it has been identified in a screen for novel phytochrome regulators and was named HEMERA in this study [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]. It has been proven that HEMERA has a dual localization both in the nucleus, thanks to an additional nuclear localization site (NLS) in the C-terminal part, and also in the chloroplast, thanks to a chloroplast transit peptide (cTP) in the N-terminal part [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF][START_REF] Pfalz | ZmpTAC12 binds singlestranded nucleic acids and is essential for accumulation of the plastid-encoded polymerase complex in maize[END_REF]. Furthermore, it was shown that the N-terminal part can interact with phytochromes B in order to form a phytochrome nuclear body (photobodies). This leads to the degradation of phytochrome-interacting-factors 1 and 3 (PIF1, PIF3) and subsequently to the photomorphogenesis initiation [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]Galvao et al., 2012).

An orthologous protein with identical properties was identified also in maize [START_REF] Pfalz | ZmpTAC12 binds singlestranded nucleic acids and is essential for accumulation of the plastid-encoded polymerase complex in maize[END_REF]. PAP7/pTAC14 is also a 49-Kda protein containing a SET domain involved in methyltransferase activity and a RubisCO-binding domain that shares similarity with histone binding motives [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF][START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]. However, a methyltransferase activity could not be shown experimentally with the following targets and no functional study of this protein has been successful to confirm this predicted function [START_REF] Alban | Uncovering the protein lysine and arginine methylation network in Arabidopsis chloroplasts[END_REF]thesis of Morgane Minnino, 2014). However, the inactivation of PAP7 causes an albino phenotype with a block in chloroplast biogenesis, accumulation of NEP dependent-genes and low accumulation of tRNAs, ribosomal proteins and PEP-dependent genes [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF][START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. The pap7-1 mutant has been used to analyze the gene expression in the albino background in detail. By comparing the gene expression profiles to light-and darkgrown Arabidopsis seedlings of the same age it was possible to define gene sets responding to light or retrograde plastid signals. In sum, the mutant exhibits a chimeric expression profile being located between the two controls with many light-induced genes at the developmental level and starvation induced genes at the metabolic level [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. PAP8/pTAC6 is a 37-Kda protein that does not have a predicted function because, until now, no homology with any other known protein was found [START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]. The knockout of PAP8/pTAC6 gene resulted in an albino phenotype with a pigment biosynthesis deficiency, a thylakoid membrane destruction and an absence of photosynthesis transcript accumulation [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF]. PAP11/MurE is an 85-Kda protein that contains the Mur ligase E domain [START_REF] Garcia | An Arabidopsis homolog of the bacterial peptidoglycan synthesis enzyme MurE has an essential role in chloroplast development[END_REF]. The MurE protein ensures the formation of uridine diphosphate-N-acetylmuramic acidtripeptide in the peptidoglycan biosynthesis in the bacteria. MurE occurs also in the purified tobacco chloroplast PEP complex [START_REF] Suzuki | Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme[END_REF]. It was also proven that PAP11 is a part of the TAC complex purified from mustard chloroplasts and A. thaliana [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF] and the corresponding gene inactivation mutant has a pale green phenotype.

Finally, the small 12-kDa PAP12/pTAC7 protein has no homology with any known functional domain. The PAP12 knockout mutants develop a true albino phenotype, show a destroyed thylakoid membrane and a decrease in PEP-dependent transcript accumulation [START_REF] Yu | TAC7, an essential component of the plastid transcriptionally active chromosome complex, interacts with FLN1, TAC10, TAC12 and TAC14 to regulate chloroplast gene expression in Arabidopsis thaliana[END_REF].

The inactivation of each of the PAP genes leads to the appearance of albino plants that can only survive on a sucrose-supplemented medium. Their plastids are smaller, irregular in shape, with a deficiency of the thylakoid system and a block to perform photosynthesis and chloroplast biogenesis [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF][START_REF] Myouga | A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis[END_REF][START_REF] Arsova | Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana[END_REF][START_REF] Schröter | Analysis of oligomeric protein complexes in the chloroplast sub-proteome of nucleic acid-binding proteins from mustard reveals potential redox regulators of plastid gene expression[END_REF][START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF]Garcia et al., 2011;[START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF][START_REF] Yu | TAC7, an essential component of the plastid transcriptionally active chromosome complex, interacts with FLN1, TAC10, TAC12 and TAC14 to regulate chloroplast gene expression in Arabidopsis thaliana[END_REF][START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF].

Coordination between NEP and PEP activity and chloroplast biogenesis

The plastid development during transition from the skoto-to the photomorphogenic programme requires a good functioning of plastid transcription. It is under tight control by the eukaryotic nuclear RNA polymerase II that is responsible for the transcription of gene coding NEP enzyme expression. In the early stages of plant development (or skotomorphogenesis), RPO subunits of the PEP complex. During this stage, NEP performs the transcription of PEP genes, housekeeping genes and genes encoding ribosomal proteins. This represents a critical point for the establishment of the transcription machinery of chloroplasts. Thus, NEP is more active during the early stages of plastid development, but it is already preceded by PEP activity in the green seed stage [START_REF] Hajdukiewicz | The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids[END_REF][START_REF] Demarsy | Building Up of the Plastid Transcriptional Machinery during Germination and Early Plant Development[END_REF][START_REF] Allorent | Plastid gene expression during chloroplast differentiation and dedifferentiation into non-photosynthetic plastids during seed formation[END_REF]. Prokaryotic PEP displays an E. coli-like association consisting of RPO gene products and provides the transcription of most plastid genes (PEP-B; [START_REF] Hu | Maize chloroplast RNA polymerase: the 180-, 120-, and 38-kilodalton polypeptides are encoded in chloroplast genes[END_REF][START_REF] Hu | Maize chioroplast RNA polymerase: the 78-kilodaiton polypeptide is encoded by the plastid rpoCl gene[END_REF][START_REF] Pfannschmidt | Separation of two classes of plastid DNA-dependent RNA polymerases that are differentially expressed in mustard (Sinapis alba L.) seedlings[END_REF]Pfannschmidt, 2000). After light exposure, the PEP-B core enzyme undergoes post-translational modifications to be reorganized into a eukaryotic RNA polymerase complex (PEP-A) that triggers photomorphogenesis (light induced development; Pfannschmidt and Link, 1997). At this stage PEP becomes surrounded by 12 additional nuclear encoded factors [START_REF] Schweer | AtSIG6, a plastid sigma factor from Arabidopsis, reveals functional impact of cpCK2 phosphorylation[END_REF][START_REF] Lerbs-Mache | Function of plastid sigma factors in higher plants: regulation of gene expression or just preservation of constitutive transcription?[END_REF][START_REF] Pfalz | Essential nucleoid proteins in early chloroplast development[END_REF][START_REF] Homann | DNA-binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression[END_REF][START_REF] Suzuki | Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme[END_REF][START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]Allison, 2000;[START_REF] Ishizaki | A nuclear-encoded sigma factor, Arabidopsis SIG6, recognizes sigma-70 type chloroplast promoters and regulates early chloroplast development in cotyledons[END_REF]. Afterwards, PEP takes charge of transcription, instead of NEP, and controls the photosynthesis gene transcription [START_REF] Mullet | Dynamic Regulation of Chloroplast Transcription[END_REF][START_REF] Cahoon | Analysis of developing maize plastids reveals two mRNA stability classes correlating with RNA polymerase type[END_REF].

Upon illumination, the photoreceptors phytochrome PhyA and B undergo modifications allowing their activation. Then, the active Phy are transferred to the nucleus for the regulation of phytochrome interaction factors (PIFs). These steps are preceded by the regulation of the nuclear transcription factors GLK1 and GLK2 (GOLDEN2-LIKE 1 and 2) that are required for the expression of photosynthesis and sigma factor (SIG2 and SIG6 genes).

Recently, it has been shown that one of the PAPs, HEMERA/PAP5, can interact in the nucleus with PIFs and controls the degradation of PIF1 and PIF3 [START_REF] Yang | NCP activates chloroplast transcription by controlling phytochrome-dependent dual nuclear and plastidial switches[END_REF]Yoo et al., 2019;[START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]Galvao et al., 2012). This step has been proposed to provide an anterograde signal from the nucleus to the chloroplast and stimulate, subsequently, the transition from skotomorphogenesis (etioplast/proplastid) to photomorphogenesis (chloroplast).

2015)

. At the N-terminal end, PAP7 contains a chloroplastic transit peptide (cTP) of 62 amino acids (Emanuelsson et al., 2000;[START_REF] Tanz | SUBA3: a database for integrating experimentation and prediction to define the SUBcellular location of proteins in Arabidopsis[END_REF]. Homology searches have shown that PAP7 possesses significant similarities to histone protein H3 Lys methyltransferases, enzymes known to be involved in regulating the organization and the structure of chromatin [START_REF] Trievel | Structure and Catalytic Mechanism of a SET Domain Protein Methyltransferase[END_REF][START_REF] Liu | Histone Methylation in Higher Plants[END_REF]. The PAP7 contains two predicted domains. The first one is located between positions 101 and 325 and includes a predicted SET domain characteristic of Lys methyltransferases (Tschiersch et al., 1994). There are three main amino acids that play an essential role in the putative catalytic activity of PAP7 [START_REF] Trievel | Structure and Catalytic Mechanism of a SET Domain Protein Methyltransferase[END_REF]. Asparagine at position 286 and Histidine at position 287 are involved in the binding of AdoMet (S-adénosylméthionine). Tyrosine in position 324 participates in the substrate (lysine) fixation. The second domain is located between positions 332 and 466 containing a Rubisco substrate-binding domain homologous region (putative Rubisco; [START_REF] Wang | Acetylation of Metabolic Enzymes Coordinates Carbon Source Utilization and Metabolic Flux[END_REF][START_REF] Ying | Rubisco Small and Large SubunitN-Methyltransferases BI-AND MONO-FUNCTIONAL METHYLTRANSFERASES THAT METHYLATE THE SMALL AND LARGE SUBUNITS OF RUBISCO[END_REF][START_REF] Couture | Structural and functional analysis of SET8, a histone H4 Lys-20 methyltransferase[END_REF][START_REF] Couture | Structural basis for the methylation site specificity of SET7/9[END_REF][START_REF] Couture | Structural origins for the product specificity of SET domain protein methyltransferases[END_REF]. Interestingly, the PAP7/pTAC14 amino acid sequence contains a nuclear localization signal (NLS), which is reminiscent of PAP5/HEMERA/pTAC12 and PAP8/pTAC6. Whether this signal is functional, is unknown to date. PAP7/pTAC14 is a highly conserved protein acquired during the evolution of terrestrial plants [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF][START_REF] Pfalz | Essential nucleoid proteins in early chloroplast development[END_REF]. Its predicted methylation activity is, however, not yet verified experimentally [START_REF] Alban | Uncovering the protein lysine and arginine methylation network in Arabidopsis chloroplasts[END_REF], but it was demonstrated that PAP7/pTAC14 can be associated with the thylakoid membrane [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF][START_REF] Mininno | Characterization of Chloroplastic Fructose 1,6-Bisphosphate Aldolases as Lysine-methylated Proteins in Plants[END_REF]Mininno, Thesis 2014).

In soil as well as on sucrose-supplemented medium, pap7-1/pTAC14 seeds can germinate and display green/white segregation ratios of 3:1 which is typical for a recessive mutation. One third of the green plants represent wildtype while two thirds represent heterozygous plants.

Albino seedlings grown on soil die quickly while a cultivation on sucrose-supplemented medium, under short day-light conditions (from 8 to 12 µE white light), allows for the formation of small homozygous albino rosettes that are able to reach the flowering stage (Mininno, Thesis 2014;[START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]Liebers et al., in revision).

Transcriptome analyses have been performed by quantitative real-time RT-PCR, plastid macro-arrays and whole-genome microarrays providing a precise transcript profile of the albino seedlings (see above) [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF][START_REF] Gao | Regulatory role of Arabidopsis pTAC14 in chloroplast development and plastid gene expression[END_REF][START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. Studies on protein accumulation were done so far only by western-immuno-blot analyses for a selected number of photosynthesis proteins showing an absence of PsaB, PsbD, RbcS, Lhcb3 proteins while PsaA, PsbB and OEC33 proteins continue to accumulate at very low levels compared to the wild-type plant. A systematic analysis of protein accumulation as done at the transcriptomic level is, however, lacking and our understanding of the albinism phenomenon in this mutant, therefore, is incomplete.

Ultrastructural investigation (by transmission electron microscopy) of pap7-1 mutant plastids from homozygous pap7-1 seedlings grown in the light was done by a collaborating team in the PCV laboratory. Dr. Stéphane Ravanel and his PhD student Morgane Mininno offered these data to Thomas Pfannschmidt who re-analyzed these with respect to published material of other pap mutants and albino seedlings.

Published work indicates that plastids from albino pap7-1 seedlings show irregular shapes and a smaller size when compared to WT. The plastids do not develop an organized thylakoid membrane system, but instead contain a number of unordered vesicles and potential plastoglobuli [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF].

The high quality TEM pictures obtained by Ravanel and Minnino revealed a number of additional features not yet published which are important for the understanding of the formation of the albino plastids.

1) The DNA appears to be highly de-condensed as visible from the unstained white areas (Fig. 10A).

2) The plastids contain one area with electron dense particle that form irregular rows or lines.

There exist two types, black and grey particles (Fig. 10A,a). The structure (named particle agglomeration) is not understood yet, but appears also in other albino plastids including those of other pap mutants [START_REF] Myouga | The Chloroplast Function Database II: a comprehensive collection of homozygous mutants and their phenotypic/genotypic traits for nuclear-encoded chloroplast proteins[END_REF].

3) Vesicles do bud from the inner envelope membrane, probably representing an interrupted step of thylakoid formation (Fig. 10a,B)

4) The outer envelope membrane often stains much darker than the inner one suggesting a different composition of the two membranes. 5) Plastoglobuli-like structures with a network-like arrangement can be found (Fig. 10D,E,F) that can be also seen after NF treatment [START_REF] Susek | Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development[END_REF].

These additional features provide additional hints for the understanding of the formation of the albinoic phenotype in pap7-1. However, they cannot be fully understood without more data obtained at the molecular level and here especially at the protein level. cut. D) A white plastid displaying plastoglobuli (PG)-like structures (white arrow labelled with "b") and a mitochondrion (black arrow labelled with M). A magnification of the PG-like structures is given in the bottom right corner of Fig. 1 (labelled with white "b"). E) A white plastid displaying PG-like structures and the particle agglomeration side by side (white arrows). F) A white plastid showing dispersal of PG-like structures (white arrow labelled with "c"). A magnification of this area is given in the bottom right corner (labelled with white "c"). Both magnifications demonstrate the size and staining heterogeneity of the PG-like structures and the appearance of dark-grey or black dots within them.

Aims

The thesis project focusses on the understanding of the early molecular regulation of chloroplast differentiation.

In particular, the project is concentrated on the plastid transcription machinery that is responsible for, or involved in, the specific recognition of the PEP promoters, thylakoid formation, pigment biosynthesis, plastid translation and proteins import, lipid and various metabolite biosynthesis [START_REF] Pogson | Genetic dissection of chloroplast biogenesis and development: an overview[END_REF][START_REF] Rudowska | Chloroplast biogenesis -Correlation between structure and function[END_REF]. The thesis is centered around the protein PAP7/pTAC14. The corresponding Arabidopsis inactivation mutant does not develop chloroplasts and, hence, can be used as a model for arrested plastid development. The scientific approach of my thesis is resumed in three main chapters.

The first part (Chapter 2) concerned the protein profile of the pap7-1 mutant, by LTQ-Orbitrap mass spectrometry in collaboration with the proteomic analysis platform EDyP, in order to answer the following questions:

1) Which proteins are expressed in the albino mutant and which are repressed?

2) How far does the proteome correspond the transcriptome?

3) Which biochemical properties are maintained in the mutant?

To this end, homozygous, 5-day-old light grown pap7-1 albino seedlings were used for the preparation of total protein extracts. A trilateral comparative profiling with wildtype grown in light (WTL; as model for an intact chloroplast) and wild-type grown in dark (WT; as model for an etioplast) was done to separate the photomorphogenetic effect from the light signaling effects. Then the proteomic profiles were compared to gene expression profiles from identically grown samples (from existing data done by [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. The proteomic data were further analyzed by the MapMan tool and validated by western-immuno-blot analyses of representative proteins.

The second part (Chapter 3) of the project used a molecular-genetic approach to understand and confirm, experimentally, the prediction of PAP7/pTAC14 localization in plastid and nucleus. In addition, it was sought to verify whether PAP7/pTAC14 participates, as PAP5/HEMERA/pTAC12 and PAP8/pTAC6, in the formation of photobodies and the degradation of the PIFs in the nucleus [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]Lieber et al., 2019 in revision). The following questions were in the center of the study:

1) Are the target sequences both functional and is PAP7/pTAC14 a dually localized protein present both in nucleus and plastid?

2) If PAP7/pTAC14 is found in the nucleus, does it participate in the formation of photobodies?

3) Is there a functional involvement of the nuclear version in light dependent development of the plant?

The third part (Chapter 4) of the project aims to better understand the nature of the biogenic retrograde signal pathway that is involved in chloroplast biogenesis. For this purpose two widely known inhibitors of chloroplast differentiation were used. Norflurazon, to inhibit the carotenoid biosynthesis pathway, and lincomycin, to inhibit plastid translation. Both inhibitors are known to induce albino plants and, thus, mimick the effect of the genetic defect in the pap7-1 mutant. A phenotypic, biochemical and protein accumulation analysis was performed in order to verify the role of these inhibitors in creating a negative feedback to the photosynthesis associated genes (PhANGs) and subsequently in blocking the plastid development. By comparing these data to the results from the other sections a deeper insight into chloroplast biogenesis should be gained.

Ultimatly, by combining the data from all three sections a model should be developed that explains the generation of the albinism in the pap7-1 mutant. By this it should be discussed whether PAP7/pTAC14 may be able to act as a novel mediator of the biogenic retrograde signal that controls chloroplast biogenesis.

Chapter 2 Molecular defects in the albino

Arabidopsis pap7-1 mutant

Endosymbiosis of an ancestral cyanobacterium into a eukaryotic cell gave rise to an organelle called chloroplast. The chloroplast is a defining component of the plant cell in the green lineage that carries the photosynthetic apparatus. During endosymbiosis more than 95% of the cyanobacterial genes were transferred to the host cell nucleus. The proteins encoded by these genes are translated in the cytosol and then distributed to different places in the cell. A considerable part is imported into the plastid, through the TIC-TOC translocon, where they contribute to the differentiation of the chloroplast. In multicellular organisms of the green lineage, several plastid forms have evolved, which correspond to specific cell types or developmental stages. While different plastid forms can interconvert, the most critical differentiation is that of the functional chloroplast for the initial acquisition of photoautotrophy in the young germinating seedling. The molecular regulation of this chloroplast differentiation is still largely unknown. It is, however, known that activation of plastid transcription in the light can be performed only in the presence of PAPs that assemble around the PEP complex [START_REF] Pfannschmidt | Separation of two classes of plastid DNA-dependent RNA polymerases that are differentially expressed in mustard (Sinapis alba L.) seedlings[END_REF]. This step appears to be essential to initiate chloroplast differentiation [START_REF] Pfalz | Essential nucleoid proteins in early chloroplast development[END_REF].

Inactivation of the pap7-1 gene generates homozygous mutants that are characterized by an albino phenotype and an arrested plastid development. The plastids in pap7-1 mutants display a complete absence of thylakoid membranes. They exhibit significant accumulation of NEPdependent genes and reduced accumulation of PEP-dependent transcripts (including photosynthesis genes; [START_REF] Pfalz | Essential nucleoid proteins in early chloroplast development[END_REF][START_REF] Gao | Regulatory role of Arabidopsis pTAC14 in chloroplast development and plastid gene expression[END_REF].

A recent study has analysed in detail plastid and nuclear gene expression in the pap7-1 mutant [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. Using a differential gene expression profiling, gene groups were identified, which are regulated by light, chloroplast signals or signals from the arrested albino plastids. However, until now, no study has been carried out to characterize the corresponding protein accumulation profiles in the albino plastid of pap7-1 mutants and the comparison of their accumulation levels with respect to gene expression.

This chapter presents a proteomic study using the Arabidopsis pap7-1 mutant in order to identify molecular regulators involved in the arrested plastid development. The analysis involved quantitative proteomics for total protein extracts from dark-grown and light-grown wildtype as well as light-grown homozygous pap7-1 mutants. The determinations were done in collaboration with the EDyP proteomic platform (CEA Grenoble) using an LTQ-Orbitrap mass spectrometer. Statistical analyses of the raw data were performed by Marianne Tardif (research engineer at EDyP).

homozygous pap7-1 mutant seedlings can grow slowly, show a missing greening process and develop an albino phenotype despite the typical photomorphogenesis characteristics (repression of hypocotyl elongation, opining of apical hook).

On MS medium without supplemental carbon source, pap7-1 mutant seedlings grow until the cotyledon stage. The homozygous plants become transparent and eventually die after a few weeks [START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF][START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. The albino plants, however, are able to survive on a medium with an additional carbon source without major phenotypic defects [START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF]. When pap7-1 albino seedlings are older, the leaves start to accumulate a little bit of yellow color. Apparently, homozygous plants can use sugar to survive but also to activate carotenoid biosynthesis pathway, which can protect plants from light and ROS production in the older developmental stages.

Chlorophyll content

We first measured the chlorophyll content in the total fresh weight (FW) (see Materials and Methods section). Chlorophyll a (Chl a) and chlorophyll b (Chl b) were determined for homozygous pap7-1 plants. Chlorophyll is a pigment localized in the plastid membranes (Kim et al., 2013). It is linked to the photosynthetic apparatus (PSI and PSII), exerting an important role in the harvesting of light energy which is necessary for photosynthesis [START_REF] Lin | Analysis of an Arabidopsis heat-sensitive mutant reveals that chlorophyll synthase is involved in reutilization of chlorophyllide during chlorophyll turnover[END_REF][START_REF] Lin | Identification of a Chlorophyll Dephytylase Involved in Chlorophyll Turnover in Arabidopsis[END_REF]. Therefore, the value of the Chla/b ratio is an indicator of the light harvesting binding chlorophyll protein associated with the two photosystems. Here, the WTL plants show a normal chlorophyll biosynthesis while the WTD plants show an increase in the Chl a/b ratio due to the reduction of Chl b (Fig. 2,A; [START_REF] Ashraf | Photosynthesis under stressful environments: An overview[END_REF]. In contrast, pap7-1 homozygous mutants are completely unable to produce chlorophylls.

To evaluate the maintenance of the photosystems, an absorbance spectrum was carried out after 7 days of culture of pap7-1 seedling in the light as well as WT seedling grown in light or dark, respectively. The spectrum (Fig. 2,B) showed two major peaks for WTL. The first one is at 450 nm which corresponds to Chl b. The second one is between 680 to 700 nm which corresponds to the light harvesting chlorophyll binding-protein complex (LHCB) and the photosystem II. A third small peak was detected at 480 nm which corresponds to the carotenoids. Both spectra recorded from WTD and pap7-1 are similar and do not show any peak between 400 and 700 nm. The absence of the peaks indicates that the photosystems identified and quantified (not shown). In spite of automatic validation rules (see Material and Methods section), this set still comprised low-confidence identifications and poorly quantified proteins. Indeed, proteomics data faces the challenge of missing values, which means lack of quantification of particular protein(s) in a particular replicate(s) which arises from stochasticity in sampling peptide ions in the spectrometer. As a consequence, discarding protein groups with too many missing values and implementing the remaining missing values were necessary steps before the statistical testing. Our rule (standar used at EDyP) was to keep only proteins that had at least 3 quantification values in at least one profile. Since this rule was applied independently for each pairwise comparison, the total number of protein groups eligible for differential analysis was slightly different depending on the comparison (3580 protein groups in the pap7-1 vs WTD comparison, 3613 protein groups in the pap7-1 vs WTL comparison and 3509 protein groups in the WTL-WTD comparison). In the pap7-1 vs WTD comparison, 1098 proteins (30 %) were regarded as significantly differentially abundant, 796 proteins being up-regulated in the mutant pap7-1 samples and 302 proteins being up-regulated in the WTD samples. Changes in the pap7-1 vs WTL comparison indicated just 437 proteins (12 %) as differentially abundant from which 253 proteins were up-regulated in WTL and 184 up-regulated in pap7-1 profile. In the WTL-WTD comparison, 1194 proteins (34 %) had a significant difference from which 802 proteins were up-regulated in WTL while 392 proteins were up-regulated in WTD. In total 3805 protein groups were identified and amenable to differential anlaysis from the proteomics analyses.

Integration of proteomics results with transcriptomics

The next step was devoted to compare the list of differentially accumulated proteins with an existing list of recent transcript profiling data done in our team (25444 transcripts; Grübler et al., 2017). Such a comparison is intended to determine the correlation (positive or negative) between gene expression (transcripts levels) and the corresponding protein levels. Explicitly, the integration of both transcriptomics and proteomics for further mining should help to define the pre-dominant level of regulation in the albino plastid model and to better understand the process of plastid development.

The crossing between both list were based on identity of the AGI number. Since the capacity of "proteome" coverage of the proteomics study and of the transcriptomics study was very different (3805 proteins versus 25444 transcripts; Fig. 4), AGIs from the transcriptomics list tool (Fig. 6; [START_REF] Usadel | Extension of the Visualization Tool MapMan to Allow Statistical Analysis of Arrays, Display of Coresponding Genes, and Comparison with Known Responses[END_REF][START_REF] Usadel | A guide to using MapMan to visualize and compare Omics data in plants: a case study in the crop species, Maize[END_REF]. The BINCODE summarizes proteins that are involved in a specific functional process or context. The cartography "map" corresponds to the proteins belonging to these bins. The "First BINCODE" column contains the identifier of all the main classes (1, 2, 3, 4 ..., and 35). The majority of the proteins were found to be nuclear encoded proteins. The Fig. 5 displays the distribution diagramme of the proteins over the different functional groups. The diagramme is organized from the largest towards the smallest detected protein group in the pap7-1 mutant and shows the differential accumulation between the different protein groups. The photosynthesis proteins are the most strongly detected protein group. Secondary and hormone metabolism protein groups were also strongly detected, involving the majority of carotenoid biosynthesis enzymes and the pathways for biosynthesis of abscisic acid. In addition, the group for lipid metabolism was strongly detected. This group contains the majority of glycolipids, phospholipids, acetyl COA carboxylation biosynthesis enzymes and enzymes involved in some hormone biosyntheses like jasmonic acid. In subsequent analyses of these protein groups were further investigated according to their abundance and their potential functional role in the albinism of the pap7-1 mutant.

The determined proteins are thought to be involved directly or indirectly in chloroplast biogenesis, build-up and maintenance of the photosynthetic apparatus and other linked biosynthesis pathways like pigments and lipids.

accumulation of proteins controlled by photomorphogenesis (PM). This includes all the proteins that were significantly affected by light and the plastid developmental stage.

The profile exhibits a strong accumulation of proteins involved in photosynthesis, photorespiration, energy metabolism, Calvin cycle, tetrapyrrole biosynthesis and other biosynthesis pathways. This coincides with earlier observations [START_REF] Ma | Genomic Evidence for COP1 as a Repressor of Light-Regulated Gene Expression and Development in Arabidopsis[END_REF][START_REF] Barsan | Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components[END_REF] and represents the massive restructuring necessary to establish autotrophy. The comparison between pap7-1 light vs WTL (Fig. 6,C) indicated protein accumulation changes that in literature are discussed as being caused by plastid signalling (PS) which is, however, imprecise. Distinguishing between PM and PS requires the necessity of a comparison against the dark control as a third independent control. Only this allows to distinguish between the light effect (light-signalling; LS) and the retrograde signalling impact from the albino plastid since the inclusion of it provides additional information that is essential for data interpretation.

A protein that shows up-regulation in fig. 6,A and a smaller one in Fig. 6,B would show a down-regulation in Fig. 6,C. In fact, the direct comparison of the mutant with the light-grown WT results in a dramatic over-estimation of the effect exerted by the mutation. In conclusion, studies that do not include a dark control in this context tend to interprete the impact of plastid signalling stronger than it is. However, the comparison between PM and LS situation confirmed that the light-regulation in the pap7-1 mutant works in a comparable manner as in wildtype since both proteomic profiles are highly similar. It can be concluded that the photomorphogenesis program is largely functional in the albino pap7-1 mutant. Major differences could be found only in the group of photosynthesis proteins where several proteins failed to increase upon light. Checking the identity of these proteins revealed that most of them are plastid-encoded.

LHCP, plastoquinone (PQ), cytochrome b6f complex, plastocyanine (PC) increase light absorption efficiency and the efficient transfer of electrons [START_REF] Gaffron | Zur Theorie der Assimilation[END_REF]. These are released during the oxidation of H 2 O molecules, lead to the synthesis of NADPH as well as ATP. In the presence of these two molecules CO 2 binds to ribulose-1,5-bisphosphate (in the presence of the Rubisco enzyme) and then two triose-phosphates are produced (Calvin cycle). The overall comparison of protein accumulation in the total extracts shows that photosynthesis proteins are among the largest detected protein groups. 123 total photosynthesis proteins have been detected with mass spectrometry and analyzed using MapMan (Fig. 8). The global photosynthesis protein accumulation in the pap7-1 mutant vs WTL ( Fig. 10), however, shows very low accumulation levels in the albino pap7-1 mutant which clearly corresponds to the the lack of chloroplast development. The 122 photosynthesis proteins have been grouped according to their abundance and the complex to which they belong. Five protein groups were strongly detected. The proteins of PSII and PSI, the cytochrome b6f complex, the ATP synthase complex and the subunits of RuBisCo. The photosystem II proteins are the most abundant with approximately 50 % of the photosynthesis plastidial proteins followed by PSI proteins with about 30 % (Fig. 11). The subsequent columns give relative changes in the protein accumulation in the control situation in the pap7-1 mutant grown in the light when compared to the WT-light (pap7-1 light vs. WTL, columns 1); in pap7-1 mutant grown in the light when compared to the WT-dark (pap7-1 light vs. WTD, columns 2) and in the control WTlight to WT-dark (WTL vs. WTD, (columns 3). Changes refers to log2 fold changes and protein annotation according to the MapMan software. Colour code: red (up-regulation), blue (down-regulation), NA: not attributed value.

Cross-reference of transcriptomic and proteomic data analysis

Photosynthesis proteins were analyzed separately and then their accumulation levels were compared with the relative changes in the photosynthesis gene expression in PM and LS situations. The comparison between photosynthesis transcript and protein accumulation in Fig. 12 shows that from the total 122 total detected photosynthesis proteins, present in the corresponding MapMan bin 1, 121 proteins accumulated in WTL vs WTD situation (PM).

The transketolase accumulation in Fig. 12,A The majority of proteins that have a FC.log2 ≥ 3 and ≤ 6 mainly belong to cytochrome b6, STN7, photorespiration and peroxisomal (AGT) functional groups. It is important to note that STN7 is up-regulated in the transcriptomic and proteomic levels with a FC.log2 equal respectively to 2.9 and 3.5 respectivly.

The comparison between the strongest expressed photosynthesis genes with their corresponding proteins is summarized in Fig. 12,A. Box B.

32 proteins have an FC.log2 ≥ 8, they are mainly the light harvesting and photosystem II complex are strongly light-induced.

The majority of the photosystem I and II genes are up-regulated [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. The LHCA3 was up-regulated with an FC.log2 = 1.77; LHCA4: FC.log2 = 1.17; LHCB2: FC.log2 = 3.35; LHCB5: FC log2 = 1.06. In comparison with the protein level all photosystem I and II proteins are strongly accumulated (LHCA3: FC.log2 = 12.25; LHCA4: FC.log2 = 10.47; LHCB2: FC.log2 = 10.28; LHCB5: FC.log2 = 8.63). This means that the protein accumulation behaves very much like the transcript expression. However, the observed changes were much stronger in protein accumulation suggesting a retrograde control of photosynthesis protein translation in the cytosol.

In pap7-1-light vs WTD (LS) situation 87 photosynthesis proteins were detected and only 66 of them significantly accumulated while 86 photosynthesis genes were significantly expressed in the same situation (Fig. 12,B). Some photosynthesis proteins were not detected, which led to the accumulation difference between the transcript and protein level (for example: PSBB, STN7, PETB, PSAA, LHCA2… Fig. 13,B. Box B). The comparison between photosynthesis genes and photosynthesis protein accumulation shows that the correlation between photosynthesis transcript and proteins profiles in LS situation is similar to the PM situation.

Five of these proteins have a FC.log2 ≤ -1.5 which are mainly Rubisco units Fig. 13,B.Box A.

At the transcriptomic level, 12 photosynthesis genes were down-regulated. The majority of them belongs to the PS II and cytochrome b6f complex.

61 proteins had a FC.log2 FC ≥ 1.5 where accumulation were induced by light. The top 14

proteins are also considered among the most accumulated proteins in WT (PM). Just eight of them are from PSII complex. Their accumulation level in pap7-1 was half from that in wildtype Fig. 13,A. Box C (FC.log2 in the proteomic level of LHCB4.1 = 5.12; LHCB5 = 6; LHCA3 = 7; LHCB4.2 = 4.3; PSBO1 = 7.17; PSBO2 = 8).

The results confirm that the protein levels are strongly affected in the albino plastid, in comparison with the transcript levels.

The western immuno-blot analysis in Fig. 14 using an antiserum against the cytosolic GAP DH confirmed the normalization of the protein samples since the protein accumulated to comparable amounts in all three samples. In contrast the Rubisco did not accumulate at all in the pap7-1 mutant but continued to accumulate to low level in WTD extract.

The majority of the nuclear-encoded photosynthesis proteins (LHCB1, LHCB3, STN7) exhibited strong light induction but were completely absent in pap7-1 light. Interestingly, under very long exposure time using a CCD camera for the nitrocellulose membrane very low amounts of LHCB proteins could be detected. These however exhibited a larger migration behavior than in WTL strongly suggesting that these bands represent unprocessed precursor proteins. PSI and PSII proteins, such as LHCA2 and PSAD and PSBB, accumulated to low amount in pap7-1-light mutant.

In contrast, PSBO, a lumen protein and main protein of the water-splitting complex, accumulated to normal levels in the mutant indicating that the protein import into the albino plastid is functional. The lack of nuclear encoded photosynthesis proteins is, therefore, not caused by a block of import suggesting different repression pathways.

PETB (photosynthetic electron transfer B

) is a plastid-encoded protein that encodes the cytochrome b6 (subunit of the cytochrome b6f complex). The PETB was present in nearly normal amount, and the translation appears to be functional in the pap7-1 albino.

These results indicated that the photosynthesis proteins, especially thylakoid proteins, can not accumulate in the arrested plastids, however, some plastidial proteins can accumulate. Likely, plastidial translation is still weakly functional in the pap7-1 mutant. However, cytosolic translation seems to be functional, but the import seems to be selective.

Import machinery

The plastid contains about 3000 to 4000 different proteins. Most of them are encoded by the nucleus, synthesized in the cytosolic ribosomes and then imported to the plastids [START_REF] Gray | Coordination of plastid and nuclear gene expression[END_REF][START_REF] Jung | Signaling between Chloroplasts and the Nucleus: Can a Systems Biology Approach Bring Clarity to a Complex and Highly Regulated Pathway?: Figure 1[END_REF][START_REF] Li | Protein Transport into Chloroplasts[END_REF]Soll and Schleiff, 2008). Import is a posttranslational phenomenon whose cytosolic factors can facilitate the passage of precursor proteins to the chloroplast surface (Sakamato et al., 2008). Import of nuclear encoded proteins into chloroplasts is performed in three major steps [START_REF] Olsen | The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space[END_REF]Keegstra, 1992, Young et al., 1999). First, these proteins are attached with their transit peptide to the TOC (Translocon at the outer envelope membrane of chloroplast) complex, then imported into the organelle [START_REF] Bruce | The paradox of plastid transit peptides: conservation of function despite divergence in primary structure[END_REF] through the TOC complex passed to the TIC (Translocon at the inner envelope membrane of chloroplasts) complex and finally processed for removal of the transit peptide.

The TOC/TIC machinery is localized in the outer and inner envelope membranes [START_REF] Jarvis | Targeting of nucleus-encoded proteins to chloroplasts in plants[END_REF] and the translocation occur simultaneously in a "contact sites" through the two membranes. In the stroma, the transit peptide is cleaved, the protein is completely translocated and acquired its conformation. The Table .2 shows that the majority of inner and outer proteins membranes did not display any expression change between the photomorphogenesis and the albino plastid situation.

Hence, the TOC/TIC machinery is apparently well expressed in the albino plastid.

WT-light vs WT-dark pap7-1 vs WT-dark pap7-1 vs WT-light

Locus

Table 2. Comparison of the TOC/TIC expression (transcripts and proteins) in PM, LS, and PS situations

Locus: Gene accession number. Protein annotation: From MapMan software according to biological function of their corresponding genes. The subsequent columns give relative changes in transcript and protein accumulation in the control situation in WT-light vs WT-dark (columns 1 and 2); in pap7-1 mutant grown in the light when compared to the WT-dark (pap7-1 vs WT-dark, columns 3 and 4) and in the pap7-1 mutant grown in the light when compared to the WTlight (pap7-1 vs WT-light, columns 5 and 6). Changes refers to log2 fold changes. Colour code: red (upregulation), blue (down-regulation), NA: not attributed value.

The proteomic analysis (Fig. 16,A) shows that YCF1 and TOC33 (translocase of chloroplast 33) continue to accumulate in pap7-1 albino plants. A western-immuno-blot against YCF1 was performed (antibody offer from Shikanai lab).YCF1 is the NEP-dependent hypothetical open reading frame 1 and has been recently identified as the only plastid encoded component of the TOC/TIC complex called TIC214. The western analysis shows that YCF1 accumulates to a normal amount in pap7-1 light as in a WTL at 214-KDa (Fig. 16,B). This confirms that the import machinery is still likely functional in the arrested plastid development.

with the fact that albino plastids represent, energetically speaking, a sink compartment. BXL1 and BXL2 are beta-xylosidases involved in secondary cell wall thickening, seed coat development, and formation of vascular tissues; suggesting that the albino mutants still behave like dark-grown germinating seedlings. Around half of the 51 down-regulated genes are directly or indirectly associated with photosynthesis. 26 genes corresponded to components involved in performance or regulation of light or dark reactions (11 of them being encoded in the plastome) and chlorophyll-biosynthesis (CAO, PORB and PORC). An impact on stress related genes was observed in four key genes of the jasmonic acid biosynthesis pathway that is connected to fatty acid biosynthesis; Table 3. These enzymes (AOS, AOC1, AOC2, LOX2) were much more down regulated at the level of transcript, suggesting a transcriptional regulation and, subsequently, a post-transcriptional modification.

The synthesis of these enzymes is connected to lipid metabolism. However, the disturbance of fatty acid synthesis is due to the absence of thylakoid membranes in the albino plastids [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF]. In addition, we observed a transcriptional down-regulation impact on DNAJ heat shock protein, glutathione-S-transferase or two cold-regulated genes. This may imply that the build-up of stress response pathways is coupled to the formation of a functional photosynthetic apparatus that is known to fuel the redox buffers with reduction power.

Another group of genes within this selection is associated to lipid metabolism including the plastid encoded accD gene.

The occurrence of such genes likely represents an indication for the important involvement of plastids in plant fatty acid synthesis, which apparently is disturbed as the albino plastids do not make thylakoid membranes. Apart from that, we observed an impact on the NCED gene that encodes the nine-cis-epoxycarotenoid-dehydrogenase 4, and will be discussed later.

When comparing the changes in accumulation of transcripts and proteins we observed that the proteins behave very much like the transcripts. However, in most cases the changes at the protein level were much stronger than those at the transcript level. This suggests that a strong translational up regulation does not operate in pap7-1 mutant. An impact on pigment biosynthesis pathways was also observed which is described below.

Table 3. Global comparison of transcript and protein accumulation in WT-light, WT-dark and pap7-1 light

Locus: Gene accession number, MapMan bin: functional annotation of gene products according to biological function in the MapMan software. Description: protein definition and enzyme activity, the subsequent columns give relative changes in transcript and protein accumulation in the pap7-1 mutant grown in the light when compared to the WT-light (columns 1 and 2, respectively (pap7-1-light vs WT-light fold)); in the pap7-1 mutant grown in the light when compared to the WT-dark (columns 3 and 4, respectively (pap7-1-light vs WT-dark)) and as control in WT-light to WT-dark (columns 5 and 6, respectively (WT-light vs WT-dark)), All changes are given as log2 fold changes, colour code: red (up-regulation), blue (down-regulation), ND: not detected,

Pigment biosynthesis

Tetrapyrrole biosynthesis pathway

Chlorophyll is the main photosynthetic pigment in plants and is therefore of fundamental importance for photosynthesis [START_REF] Armstrong | Identification of NADPH:protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana[END_REF]. Chlorophylls are part of the tetrapyrrole family that are considered as "pigments of life" [START_REF] Battersby | Biosynthesis of the pigments of life: formation of the macrocycle[END_REF]. The chlorophyll biosynthesis takes place in the plastid. Interestingly, tRNA-E is the precursor for the chlorophyll biosynthesis pathway (Huang and Wnag, 1986) providing a direct link between plastid transcription and chlorophyll synthesis.

Studies showed that overall levels of RNA are low in the pap7-1 mutant [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF].

More specifically it has been shown that the majority of tRNAs including tRNA-E are strongly down-regulated in the pap7-1 mutant [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF].

Since the tRNA-E is a key precursor in the tetrapyrrole pathway, its down-regulation in pap7-1 reduces tetrapyrrole biosynthesis, limits chloroplast development and chlorophyll biosynthesis (Fig. 18). The tRNA-E is involved in the glutamate activation [START_REF] Beale | The Biosynthesis of δ-Aminolevulinic Acid in Higher Plants[END_REF], to form glutamyl-tRNA (Schon et al., 1986) and ALA (5-aminolevulinic acid) biosynthesis by glutamyl-tRNA reductase enzyme [START_REF] Smith | Investigation of the subcellular location of the tetrapyrrolebiosynthesis enzyme coproporphyrinogen oxidase in higher plants[END_REF][START_REF] Mochizuki | The cell biology of tetrapyrroles: a life and death struggle[END_REF]. This enzyme is encoded by the HEMA1 gene [START_REF] Terry | A model for tetrapyrrole synthesis as the primary mechanism for plastid-tonucleus signaling during chloroplast biogenesis[END_REF]. HEMA1

accumulates at high levels in the pap7-1 mutant (FC.log2 = 1.6; Fig. 17) that appears to be in line with the development of albino phenotype. Studies have showed an increase in HEMA1

gene expression, which led to photo-bleaching for the plants developed in dark and in white light [START_REF] Mccormac | Regulation of HEMA1 expression by phytochrome and a plastid signal during de-etiolation in Arabidopsis thaliana[END_REF][START_REF] Mccormac | The nuclear genes Lhcb and HEMA1 are differentially sensitive to plastid signals and suggest distinct roles for the GUN1 and GUN5 plastid-signalling pathways during de-etiolation[END_REF]. Here, the entrance of the tetrapyrrole pathway is still, apparently, functional in the pap7-1 mutant.

FLU is a regulatory protein involved in chlorophyll biosynthesis [START_REF] Meskauskiene | FLU: A negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana[END_REF].

It is a repressor of glutamyl-tRNA reductase activity [START_REF] Terry | A model for tetrapyrrole synthesis as the primary mechanism for plastid-tonucleus signaling during chloroplast biogenesis[END_REF]. The proteomic results are completely in line with the studies that FLU enzyme showed a down-regulation in pap7-1 (FC.log2 = -1.35; Fig. 17).

ALA is converted to uroporphyrinogen III, which is the substrate of sirohemes and porphyrin pathways. Glutamate 1-semialdehyde aminotransferase (GSA) and Uroporphyrinogen III synthase (UROS), intervene in the step between ALA and uroporphyrinogen; both still accumulate in pap7-1 mutant. Furthermore, protoporphyrin IV is a precursor for the iron (heme) and the magnesium (Chl a and b) branches of the pathway.

Carotenoid biosynthesis pathway

Carotenoids are isoprenoids pigments that are synthesized in plastids (Rodriguez-Villalon et al., 2009). They are involved in several aspects of cellular development such as membrane structure, photosynthesis, regulation in growth, abscisic acid synthesis, light-harvesting and transfering the light energy to chloroplast [START_REF] Yamamoto | Carotenoids: Localization and Function[END_REF]Rodriguez-Concepción and Gruissem, 1999;Rodriguez-Concepción et al., 2001;Rodriguez-Concepción et al., 2004;[START_REF] Nambara | a. Abscisic acid biosynthesis and catabolism[END_REF]. Carotenoids improve quenching of reactive oxygen species (ROS) and thermal energy dissipation in the thylakoids, and play essential roles in photo-protection against photo-oxidative damage and photo-degradation caused by excess energy (Osmond et al., 2000;Osmond et al., 2000). Carotenoids are synthesized from isopentenyl diphosphate (IPP; [START_REF] Mcgarvey | Terpenoid metabolism[END_REF]. Two pathways synthesize IPP in plants (Rodriguez--Concepción, 2006). The first one is the mevalonate or mevalonic acid (MVA) pathway, located in the cytosol. IPP is derived from 3-hydroxy-3-methylglutaryl CoA to mevalonic acid conversion via mevalonate and then transferred to mitochondria for ubiquinone production (Rodriguez--Concepción et al., 2004).

The second one is the methylerythritol-4-phosphate (MEP) pathway, located in the plastid (Botella-Pavia et al., 2004;[START_REF] Bouvier | Biogenesis, molecular regulation and function of plant isoprenoids[END_REF] and responsible for gibberellins, abscisic acid, carotenoids and phylloquinone production (Fig. 20).

All carotenoid biosynthesis enzymes were analyzed in silico. These enzymes are all nuclear encoded proteins. From a total of 23 detected genes only a few corresponding proteins could be detected by mass spectrometry (Table . 4).

PSY (phytoene synthase) is the main enzyme in the carotenoid biosynthetic pathway and is responsible for the phytoene synthesis [START_REF] Hirschberg | Carotenoid biosynthesis in flowering plants[END_REF][START_REF] Giuliano | Metabolic engineering of carotenoid biosynthesis in plants[END_REF]. This enzyme did not show a significate fold change at the transcriptomic level (FC.log2 = -0.6). However, it was not detected by mass spectrometry.

In the light-signaling situation represented by the comparison between pap7-1 and WTD, the carotenoid biosynthesis enzymes accumulated in the same manner in mutant and WT. The comparison between the pap7-1 and WTL shows that the majority of the carotenoid biosynthesis genes did not show significant differences, except for the NCED4 (nine-cisepoxycarotenoid dioxygenase 4) that is significantly down-regulated.

In the proteomic approach, the majority of the carotenoid biosynthesis enzymes were not detected. Except ZEP (zeaxanthin epoxidase) and NCED4 enzymes that were significantly down-regulated in the pap7-1 mutant. These results were complemented by a measurement of different carotenoid biosynthesis species carotenoids did not accumulate in the pap7-1 mutant, showing a very low accumulation of the β-branch level (Fig. 21). The zeaxanthin was the most accumulating βcarotene. The low accumulation of antheraxanthin, violaxanthin, and neoxanthin was explained by the down-regulation effect of the key enzyme ZEP.

Chlorophyll and carotenoid content in WT showed that the plants are able to produce a significant pigments amount after light exposition (Fig. 24,A,B). This is due to the upregulation of MEP pathway enzymes and the availability of plastid isoprenoid precursors (Estevez et al., 2001;Botella-Pavia et al., 2004;[START_REF] Carretero-Paulet | Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase[END_REF].

MEV-treated plants showed no significant inhibition effects apart from reduced hypocotyl and root growth [START_REF] Kasahara | Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis[END_REF]Rodriguez-Concepción et al., 2004).

After light exposure, MEV-treated plants became green (Fig. 23,B). These plants can synthetize chlorophylls (0.122 μg / mg FW) and carotenoid (0.045 μg / mg FW) in a comparable amount as the wild-type control (Fig. 24,A,B). However, after light exposition, FSM-treated plants showed a yellow cotyledons with almost no chlorophyll and carotenoid production (Fig. 23,B; Fig. 24,A,B) which is likely due to the absence of IPP production in the plastid [START_REF] Rodríguez-Concepción | 1-Deoxy-d-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening[END_REF]. Carotenoid and MEP pathway availability are necessary for the efficient production of carotenoids (Rodriguez-Concepción et al., 2001).

Unlike the pap7-1 mutant whose plants are completely albino (white), MEV-treated seedlings continu to accumulate a yellow color in the leaves. In an older developmental stage, pap7-1 plants are able to accumulate a yellow coloration in the middle of the cotyledons as already shown in Fig. 1,E. These results are contradictory to what has been recently concluded (Rodriguez-Concepción et al., 2001;2004).

Here, the data showed that in the dark the treated plants are able to produce carotenoids. After light exposition, the FSM effect was observed and the FSM-treated seedling became rather yellow and not white. This shows that the effect of FSM treatment depends on the illumination condition.

By blocking both MVA and MEP pathways in the dark, plants continue to accumulate carotenoids. However, FSM-treated seedling developed directly in the light did not develop carotenoids and showed an albino phenotype (Fig. 24,C). Therefore, the pathway blocked by FSM is apparently active only in the light. These results prove that carotenoid in the dark must come from different pathways which cannot be inhibited by FSM. This might explain why pap7-1 still produce carotenoid in the dark. Immediately, when these plants are exposed in the light, the carotenoid biosynthesis is blocked.

Mitochondrial proteins accumulated rather normally in the pap7-1 mutant indicating that there is likely no negative feedback from the albino plastid on mitochondria. The ribosomal plastid-encoded proteins from "plastid translation class" (RPL2 and RPS7) accumulated to low levels in pap7-1. In addition, the nuclear-encoded ribosomal proteins (RPS1 and RPS55)

were weakly accumulated in pap7-1. RPL4 was undetectable in pap7-1 suggesting that many if not all ribosomal proteins are reduced in pap7-1 and subsequently the translation efficiency is likely reduced.

General discussion

Previous studies on retrograde signaling have focused on the transcript levels of nuclear genes. Diverse reverse genetic approaches have been used for the study of chloroplast biogenesis and plastid-nuclear communication. In several pap mutants the majority of studies have investigated gene expression and retrograde signaling only at the level of few selected transcripts and proteins [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF], Myouga et al., 2008[START_REF] Arsova | Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana[END_REF][START_REF] Egea | Chromoplast Differentiation: Current Status and Perspectives[END_REF][START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF][START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF][START_REF] Gao | Regulatory role of Arabidopsis pTAC14 in chloroplast development and plastid gene expression[END_REF][START_REF] Yu | TAC7, an essential component of the plastid transcriptionally active chromosome complex, interacts with FLN1, TAC10, TAC12 and TAC14 to regulate chloroplast gene expression in Arabidopsis thaliana[END_REF][START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. To date, no comparison between total transcript accumulation and total protein accumulation was carried out in a single pap mutant. Therefore, one of the aims in this thesis is to provide a detailed expression catalogue by addressing gene expression in relation to protein dynamics during the early stages of chloroplast biogenesis using the Arabidopsis pap7-1 mutant.

The role of PAP7 / pTAC14 in chloroplast biogenesis.

Homozygous pap7-1 mutants are indistinguishable from the wild type under darkness (Fig. 1). Moreover, it has been shown that also other ptac/pap mutant lines exhibit a wild-type-like phenotype under dark conditions [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF] suggesting that this is a general feature of pap mutants. Identification of homozygous mutants by phenotype in the dark, therefore, is not directly possible and corresponding controls could not be included in this study.

In the light, the 7-day-old pap7-1 mutant show an albino phenotype that becomes lethal in the absence of an exogenous carbon source [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF][START_REF] Gao | Regulatory role of Arabidopsis pTAC14 in chloroplast development and plastid gene expression[END_REF][START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF][START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. The albino pap7-1 mutant develops phenotypic characteristics that are highly similar to trans-plastomic mutants that are characterized by a lesion in the PEP core in tobacco plants (De Santis-MacIossek et al., 1999;[START_REF] Serino | RNA Polymerase Subunits Encoded by the Plastid rpoGenes Are Not Shared with the Nucleus-Encoded Plastid Enzyme[END_REF][START_REF] Hess | Chloroplast rps15 and the rpoB/C1/C2 gene cluster are strongly transcribed in ribosome-deficient plastids: evidence for a functioning nonchloroplast-encoded RNA polymerase[END_REF][START_REF] Allison | Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants[END_REF], Krause et al., 2000, Chi et al., 2008). This suggests that the pap7-1 mutant can be used as a general model for chloroplasts biogenesis. Furthermore, it was shown that the mutant can develop a normal wild-type like phenotype on sucrose supplemented medium suggesting that the general developmental programme is not disturbed and the phytochrome system is functional. This is comparable to studies on the albino mutant clal-1 that behaves as the wild-type in darkness and, also, upon light exposure, the light signal transduction pathway is unaffected in this mutant [START_REF] Susek | Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development[END_REF][START_REF] Mandel | CLA1, a novel gene required for chloroplast development, is highly conserved in evolution[END_REF]. These studies provide us a comparable conclusion that the signal transduction pathways and phytochromes can be functional in the absence of chloroplast development.

The lack of PAP7 protein caused remarkable damage to pap7-1 plastid morphology (Fig. 10, Chapter 1, [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF] with a total absence of thylakoid and grana membrane and the presence of globular structures, probably plastoglobuli, as in the TRXz mutants [START_REF] Arsova | Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana[END_REF], ptac-6 and ptac-12 mutants [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF]), vipp1 (Kroll et al., 2001, Arsova et al., 2007), fsd2 and fsd3 [START_REF] Myouga | A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis[END_REF]), ptac-10 (Chang et al., 2017), atmurE-1 mutant [START_REF] Garcia | An Arabidopsis homolog of the bacterial peptidoglycan synthesis enzyme MurE has an essential role in chloroplast development[END_REF], fln1 and fln2 mutants (Gilkerson et al., 2012) and ptac-7 [START_REF] Yu | TAC7, an essential component of the plastid transcriptionally active chromosome complex, interacts with FLN1, TAC10, TAC12 and TAC14 to regulate chloroplast gene expression in Arabidopsis thaliana[END_REF]. Also tapg2 albino plastid showed a damaged structure similar to the pap7-1 plastid suggesting a severe alteration of photosynthesis [START_REF] Motohashi | An essential role of a TatC homologue of a ΔpHdependent protein transporter in thylakoid membrane formation during chloroplast development in Arabidopsis thaliana[END_REF]. By analogy with these results, we can suggest an important role of pap7-1 in the formation of the thylakoid membrane. On the contrary to the pap7-1 mutant, the cla1-1 mutant presents plastids similar to proplastids with deformed prolamellar bodies and developing thylakoid membranes [START_REF] Mandel | CLA1, a novel gene required for chloroplast development, is highly conserved in evolution[END_REF] suggesting a role of CLA1 in later plastidial developmental stages.

Therefore, in the pap7-1 mutant the plastid development is arrested in early steps before thylakoid formation as in vipp1 mutants (Kroll et al., 2001, Aseeva et al., 2007). Our results share strong similarities with [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF] and allow us to confirm the implication of PAP7 in the early stages of plastid development.

PAP7 affects plastid protein homeostasis

The plastid proteome is considered as the most dominant proteome in plant cells since a single plant cell may contain many plastids that easily reach the same volume as the cytosol.

Global visualization of protein accumulation (Fig. 6) indicated that there is a minor impact of light on protein dynamics in the pap7-1 mutant (Fig. 6,A,B). However, the protein accumulation profile has shown a strong impact in the plastid status, which represents twelve percent of the total detected proteins (Fig. 6,C). The mutant showed inhibition of accumulation of proteins involved in photosynthesis, photorespiration, energy metabolism, the Calvin cycle, tetrapyrrole biosynthesis, and a number of other biosynthetic pathways. The transcriptomic profile of pap7-1 has also shown a down-regulation of photosynthesis, photorespiration and tetrapyrrole genes [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. These results prove that the over all proteomic profile follows the transcriptomic profile in pap7-1 mutant. A comparable proteomic study was performed on three RIKEN Ds-tagged albino-pale green (apg) mutant lines [START_REF] Myouga | The Chloroplast Function Database: a large-scale collection of Arabidopsis Ds/Spm-or T-DNA-tagged homozygous lines for nuclear-encoded chloroplast proteins, and their systematic phenotype analysis[END_REF]. A visual presentation of the common metabolism of three different apg mutants (each with a different degree of chloroplast damage) showed that the same proteins groups such as photosynthesis and chlorophyll synthesis are down-regulated in these mutants [START_REF] Motohashi | Common and specific protein accumulation patterns in different albino/pale-green mutants reveals regulon organization at the proteome level[END_REF]. In addition, a recent study has shown that in comparison to the proteome profile of the wildtype, the gun1 mutant (with a similar phenotype as wildtype), had a similar proteomic profile while the gun5 mutant (with pale green phenotype) exhibited a different protein accumulation profile (Moshizuki et al., 2001;[START_REF] Wu | Extensive Posttranscriptional Regulation of Nuclear Gene Expression by Plastid Retrograde Signals1[OPEN][END_REF]. The high similarities between the proteome of pap7-1, gun5, apg mutants indicate that the results obtained with the pap7-1 mutant may be regarded as typical for albino mutants.

PAP7 impact in translational and post-translational regulation of protein accumulation

The majority of the plastid proteins are encoded by nuclear genes. Two comparable proteomic analyses were done using the two albino mutants mta (from wheat) and apg2. The results

showed that their proteomes show defects mainly in groups of "protein targeting to chloroplasts", photosynthesis, response to stress and other metabolic pathways respectively [START_REF] Shi | Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflightinduced wheat albino mutant mta[END_REF][START_REF] Motohashi | Common and specific protein accumulation patterns in different albino/pale-green mutants reveals regulon organization at the proteome level[END_REF]. This corresponds to the proteome data from this study that revealed strong down-regulation photosynthesis proteins including proteins of PSI, PSII, light harvesting complex, Rubisco, cytochrome b6f complex and ATP synthase (Fig. 10, Fig. 11, Fig. 12,Table 2).

A complete analysis of the cellular proteome has shown that photosynthetic proteins constitute the mass of the whole proteome (Friso et al., 2011).

These results showed a correlation between expression levels of photosynthesis transcripts and proteins, however, a number of gene group-specific and compartment-specific effects could be observed [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. Many previous studies have concluded that all pap mutants exhibit PEP-dependent transcription defects and especially the expression of class I plastid genes were strongly suppressed (such as plastidial-encoded photosynthesis genes) (Mandal et al., 1996;[START_REF] Hajdukiewicz | The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids[END_REF][START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF][START_REF] Myouga | A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis[END_REF][START_REF] Arsova | Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana[END_REF][START_REF] Yagi | Eukaryotic-type plastid nucleoid protein pTAC3 is essential for transcription by the bacterial-type plastid RNA polymerase[END_REF][START_REF] Yu | TAC7, an essential component of the plastid transcriptionally active chromosome complex, interacts with FLN1, TAC10, TAC12 and TAC14 to regulate chloroplast gene expression in Arabidopsis thaliana[END_REF][START_REF] Chang | pTAC10, a Key Subunit of Plastid-Encoded RNA Polymerase, Promotes Chloroplast Development[END_REF]. In the pap7-1 mutant indeed a strong down-regulation of class 1 genes was observed, but a small number of transcripts remained detectable while class 2 and 3 genes accumulated normally (Grübler et al., 2017). A low number of photosynthesis proteins for class 1 and normal levels for class 2 and 3 should be expectable. Nevertheless, no photosynthesis proteins were detectable in pap7-1 suggesting a block at the translational level. Furthermore, most nuclear encoded photosynthesis genes exhibited no or only weak inhibition in pap7-1. Only the group of Lhcb genes was strongly affected. This suggests that there exists a feedback from the albino plastid towards the cytosolic translation of photosynthesis proteins indicating that retrograde signals do not only affect the transcript level but also the protein level as it was already reported in the original studies on this effect [START_REF] Bradbeer | Cytoplasmic synthesis of plastid polypeptides may be controlled by plastid-synthesised RNA[END_REF].

Consistent with these results, a recent analysis using the gun1 mutant treated with lincomycin, an inhibitor of plastid translation, has shown that the down-regulation at the protein level is much stronger than that at the transcript level in comparison with lincomycin-treated WT [START_REF] Wu | Extensive Posttranscriptional Regulation of Nuclear Gene Expression by Plastid Retrograde Signals1[OPEN][END_REF].

Validation of the proteomic assay was performed by immune-blot tests confirming that thylakoid membrane and the photosystem proteins are absent in the pap7-1 mutant (Fig. 14).

These results are in line with previous studies using the albino/yellow mutants ptac-2, ptac-6

and ptac-12 [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF] and mta [START_REF] Shi | Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflightinduced wheat albino mutant mta[END_REF] showing that the plastidial and nuclear proteins involved in photosystem and thylakoid membrane formation are absent or strongly reduced. However, interestingly, pap7-1 mutant showed a normal accumulation of the lumen protein (PSBO), cytochrome b6 (PETB), and the only plastid-encoded TIC protein (YCF1) indicating that the pap7-1 mutant is able to perform plastidial translation and import for selected proteins. These observations correspond to reports indicating that the cytochrome b6f complex is detectable in the three pap mutants ptac-2, ptac-6 and ptac-12 [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF].

The TOC /TIC proteins are among the proteins targeting to chloroplast. Their accumulation in pap7-1 did not show significant differences to the WT, both at transcript and protein level. Contrary to these observations, the albino apg2 mutant showed up-regulation of several plastid protein imports in responds to the perturbation of plastid metabolism [START_REF] Motohashi | Common and specific protein accumulation patterns in different albino/pale-green mutants reveals regulon organization at the proteome level[END_REF]. Taken together, this suggests that, likely, TOC/TIC machinery is well functional in pap7-1 mutant and the selectivity of nuclear-encoded proteins accumulation in the plastid is likely caused by retrograde translational or post-translational regulation.

To confirm this hypothesis, immuno-blot tests using longer exposure time, by CCD camera, showed low amounts of accumulation of potential LHCB protein precursors in the cytosol.

These data suggest that the initiation of Lhcb transcription and cytosolic translation might be still functional, however, LHCB import might be prevented and the accumulating proteins are subsequently send to degradation. It has been proposed that the LHCB import is possibly controlled by the availability of chlorophylls and thylakoid membranes that both are missing in the pap7-1 mutant [START_REF] Bräutigam | Dynamic Plastid Redox Signals Integrate Gene Expression and Metabolism to Induce Distinct Metabolic States in Photosynthetic Acclimation in Arabidopsis[END_REF]. This coincides with the normal accumulation of PsbO that is a chlorophyll-free soluble protein and supports the view of a negative feedback regulation from plastid import [START_REF] Kakizaki | Coordination of Plastid Protein Import and Nuclear Gene Expression by Plastid-to-Nucleus Retrograde Signaling[END_REF][START_REF] Kakizaki | New insights into the retrograde signaling pathway between the plastids and the nucleus[END_REF].

The proteome data clearly show that the translation of plastid-encoded proteins is strongly limited.

To further decipher how the selective plastid translation works, the accumulation of some ribosomal proteins has been validated by immunoblot tests (Fig. 25). The westerns showed strong repression of ribosomal proteins. However these findings are contradictory to a couple of other studies. In other pap mutants, the ribosomal RPL and RPS genes were strongly upregulated due probably to their transcription from genes containing NEP promoters [START_REF] Pfalz | pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are required for plastid gene expression[END_REF]. The lincomycin-treated gun1 mutant has shown accumulation of ribosomal proteins to high levels, which likely occurs through the post-translational regulation of protein stability/degradation [START_REF] Wu | Extensive Posttranscriptional Regulation of Nuclear Gene Expression by Plastid Retrograde Signals1[OPEN][END_REF]. In addition, a surprising accumulation of plastid ribosomal proteins and TAC subunits was shown in pale green/albino apg mutants [START_REF] Motohashi | Common and specific protein accumulation patterns in different albino/pale-green mutants reveals regulon organization at the proteome level[END_REF]. The paradox between the unexpected accumulation of plastid ribosomes and the disruption of plastid transcriptional machinery in apg mutants, also suggested post-transcriptional and translational regulation processes in order to determine plastid-encoded protein accumulation (Motahashi et al., 2012). These data suggest that in the pap7-1 mutant a differential repression of ribosomal proteins occur; this requires further investigations.

PAP7 exercise a role in retrograde signaling

Previous studies have shown that during chloroplast biogenesis, the import of nuclearencoded plastid localized proteins and retrograde signaling from plastid to nucleus is a connected process [START_REF] Pogson | Plastid signalling to the nucleus and beyond[END_REF]Waters and Langdale, 2009).

To further understand how gene expression and protein accumulation are correlated in response to the retrograde signaling, sixty two genes have been identified from the transcriptomic data set [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF] that show specific inhibition in the pap7-1 mutant and encode plastid localized proteins. The proteomic profile for this protein group is strongly down-regulated. In some cases these changes appear to be in the same order of magnitude for instance for the allene oxide synthesis pathway suggesting a predominant transcriptional control. In other cases for instance for plastid encoded photosynthesis genes the repression at protein levels exceeds strongly the repression at transcript levels suggesting a pre-dominent translational or post-translational regulation. This hypothesis is correlated with the observation of the gun1 mutant, in response to chloroplast translation inhibitor lincomycin treatment, that the protein levels have been strongly repressed suggesting translational and post-translational regulation [START_REF] Wu | Extensive Posttranscriptional Regulation of Nuclear Gene Expression by Plastid Retrograde Signals1[OPEN][END_REF]. Nevertheless, the way in which the ribosome can regulate and select proteins for translation remains largely unknown.

It should be also noted that the plastid photosynthesis transcripts show some light independent accumulation in the dark (likely through developmental control) (Pfannschmidt and Link, 1997). Thus, the light-induced increase is less strong and hence also the corresponding repression in pap7-1. Since only relative values can be given for the expression changes at transcript and protein level a direct conclusion on which level the pre-dominent regulation occurs is not easy to draw and will require more detailed studies.

In accordance with the albino cla1 mutant [START_REF] Mandel | CLA1, a novel gene required for chloroplast development, is highly conserved in evolution[END_REF], most of the plastid localized proteins in pap7-1 mutant are anyway nuclear-encoded proteins involving components for the light harvesting complex, photosystems, pigment biosynthesis (tetrapyrrols, carotenoids), stress response (such as jasmonic acid and abscisic acid biosynthesis enzymes) and lipid biosynthesis enzymes.

Several actors in retrograde signaling response such as the photosynthesis associated nuclear genes (LHCB1.4; LHCB1.3; LHCB3; RBCS2B (the small subunit of Rubisco); [START_REF] Woodson | Heme synthesis by plastid ferrochelatase I regulates nuclear gene expression in plants[END_REF]2013), have shown a correlation between repression of mRNAs and protein accumulation with strong down-regulation in the protein levels suggesting posttranscriptionally regulation by plastidial translational defects.

The lack of plastid gene expression (PGE) has been identified as a factor generating a retrograde signal that specifically inhibits expression of nuclear-encoded genes (Myouga et al., 2008). The gun1 mutant treated with lincomycin, showed the accumulation of Lhcb1 protein. This suggested a role of GUN1 in retrograde signaling [START_REF] Nott | Plastid-to-nucleus retrograde signaling[END_REF][START_REF] Wu | Extensive Posttranscriptional Regulation of Nuclear Gene Expression by Plastid Retrograde Signals1[OPEN][END_REF]. Moreover, the blocking of the plastid transcription machinery has led to a repression of seleted PhANGs in mustard (Pfannschmidt and Link, 1997) and in Arabidopsis [START_REF] Woodson | Sigma factor-mediated plastid retrograde signals control nuclear gene expression[END_REF]. These results suggest a strong relationship between the transcription of some plastid genes and PhANGs expression. However, in the current models only few photosynthesis genes were usually taken as reporter for the whole gene group, which likely is not representative as found for pap7-1 [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. Several nuclear transcription factors have been identified to affect the first stages of plastid development. Indeed, the light-induced phytochrome interaction factor 3 (PIF3) is a negative regulator of nuclear genes encoding plastid-related proteins [START_REF] Stephenson | PIF3 is a repressor of chloroplast development[END_REF]. Furthermore, Golden2-like transcription factors 1 and 2 (GLK) are required for the expression of nuclear-encoded photosynthesis genes [START_REF] Waters | GLK transcription factors regulate chloroplast development in a cell-autonomous manner[END_REF].

In rice and Arabidopsis, the knockout of GLK genes generated a pale green phenotype with a reduction in thylakoid formation [START_REF] Fitter | GLK gene pairs regulate chloroplast development in diverse plant species[END_REF][START_REF] Waters | GLK transcription factors regulate chloroplast development in a cell-autonomous manner[END_REF]. In addition, nuclearencoded photosynthesis genes were down-regulated, especially genes associated in chlorophyll biosynthesis and photosystem II. Furthermore, the wheat albino mta mutant showed very low levels of GLK gene expression and inhibition of LHCB accumulation [START_REF] Shi | Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflightinduced wheat albino mutant mta[END_REF]. Similarly, GLK1 gene is down-regulated in the pap7-1 mutant (unpublished data from Grübler) suggesting a similar mechanism for the albinism in this mutant.

PAP7 retrograde signaling involves tetrapyrrole synthesis

Furthermore, it has been shown an important role of GLKs in regulation of the tetrapyrrole pathway. GLKs control the key step in chlorophyll branch which performs the chlorophyllide a formation by protochlorphyllide oxidoreductase (PORA, PORB, and PORC) and the chlorophyllide a oxidation to chlorophyllide b (Griffiths, 1972[START_REF] Griffiths | Reconstitution of chlorophyllide formation by isolated etioplast membranes[END_REF][START_REF] Apel | The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein[END_REF]Waters et al., 2009).

Previous studies linked plastid tetrapyrrole synthesis with retrograde signaling towards the nucleus [START_REF] Mochizuki | Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction[END_REF][START_REF] Larkin | GUN4, a regulator of chlorophyll synthesis and intracellular signaling[END_REF][START_REF] Woodson | Heme synthesis by plastid ferrochelatase I regulates nuclear gene expression in plants[END_REF].

Therefore, it was investigated whether a disruption in the tetrapyrrole pathway may inhibit the development of functional plastids in pap7-1 mutant and generate a retrograde signal. The transcriptomic profile indicated that inactivation of PEP reduced the expression of tRNA-E, which is not only required for plastid translation but represents also the starting substrate of the tetrapyrrole biosynthetic pathway [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF]. Its repression therefore causes in parallel a decrease in the translation of plastid-encoded proteins, a lack of chlorophylls and leads ultimately to an ivory/albino phenotype [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF][START_REF] Williams-Carrier | A major role for the plastid-encoded RNA polymerase complex in the expression of plastid transfer RNAs[END_REF].

Despite that, the tetrapyrrole biosynthesis pathway is still functional in the pap7-1 albino plastid. In fact the heme branch produces still around 30 % of the heme amount when compared to wild-type. This appears to be sufficient for the production of heme-containing mitochondrial proteins and the generation of phytochromobilin for the phytochrome system (Fig. 18; [START_REF] Thomas | Measurement of Heme Efflux and Heme Content in Isolated Developing Chloroplasts[END_REF]. The results of western blot analyses showed that pap7-1 and WTL had a similar accumulation of mitochondrial proteins. These results suggest that the arrested plastid development does not influence mitochondrial protein accumulation (Fig. 26). However, the entrance enzymes of the tetrapyrrole and its Mg-branch, HEMA1 and CHLH enzymes, show strong down-regulation (Fig. 17,18). These two nuclear-encoded plastid localized enzymes are probably connected to the down-regulation of PIF genes in the pap7-1 mutant and other systems (Grübler unpulished data; [START_REF] Leivar | Definition of early transcriptional circuitry involved in light-induced reversal of PIF-imposed repression of photomorphogenesis in young Arabidopsis seedlings[END_REF][START_REF] Shin | Phytochromes promote seedling light responses by inhibiting four negatively-acting phytochrome-interacting factors[END_REF][START_REF] Ilag | Light regulation of chlorophyll biosynthesis at the level of 5aminolevulinate formation in Arabidopsis[END_REF][START_REF] Chow | Two different genes encode ferrochelatase in Arabidopsis: mapping, expression and subcellular targeting of the precursor proteins[END_REF]. PIFs are negative regulators of chlorophyll biosynthesis in the dark to protect plants against photo-oxidation following the accumulation of chlorophyll intermediates (Toledo-Ortiz et al., 2009). Furthermore, in the TRXz (pap10) mutant, the low PEP activity resulted in repressed expression of tRNA-E, which in turn prevents NEP inhibition and likely leads to its high activity in the mutant (Hanaoka et al., 2005[START_REF] Arsova | Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana[END_REF].

In the pap7-1 mutant, the genes CHLM, PORB and PORC showed a transcriptional downregulation. The results of the protein analysis showed also a striking down-regulation effect.

These three chlorophyll key enzymes are connected to the co-expression networks with

PhANGs by the action of GLK [START_REF] Kobayashi | Regulation of Root Greening by Light and Auxin/Cytokinin Signaling in Arabidopsis[END_REF][START_REF] Kobayashi | Transcriptional Regulation of Tetrapyrrole Biosynthesis in Arabidopsis thaliana[END_REF]. The absence of protochlorophyllide oxidoreductase (POR) results in the absence of chlorophyll synthesis [START_REF] Bräutigam | Dynamic Plastid Redox Signals Integrate Gene Expression and Metabolism to Induce Distinct Metabolic States in Photosynthetic Acclimation in Arabidopsis[END_REF]. As already discussed above this may prevent the import of nuclear-encoded proteins associated with chlorophyll biosynthesis and photosystem II, such as LHCB.

Interestingly, the affected POR variants show differential responses to light. PORA accumulates only in dark grown seedlings; PORB can accumulate in darkness and after light exposition while PORC accumulates just after light exposition [START_REF] Oosawa | Identification and light-induced expression of a novel gene of NADPH-protochlorophyllide oxidoreductase isoform in Arabidopsis thaliana[END_REF][START_REF] Su | POR C of Arabidopsis thaliana: a third light-and NADPHdependent protochlorophyllide oxidoreductase that is differentially regulated by light[END_REF]. In the dark, etioplasts accumulate PORA and PORB transcripts at high levels [START_REF] Armstrong | Identification of NADPH:protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana[END_REF]. Upon light illumination, these transcripts are reduced by a negative control via phytochromes [START_REF] Mapleston | Light modulation of the activity of protochlorophyllide reductase[END_REF][START_REF] Santel | The Protochlorophyllide Holochrome of Barley (Hordeum vulgare L.)[END_REF]Forreiter et al., 1990). A microscopic ultrastructure observation of etioplasts from pap7-1 would be an important help to understand these complex regulation. However, this cannot be achieved because pap7-1 albino plants are indistinguishable from the WTD. Here, the question needs to be asked if the pap7-1 mutant has the ability to form prolamellar bodies in the dark. If truly that, it may probably suggest an increased accumulation of PORA and PORB transcripts in pap7-1 etioplasts [START_REF] Oliver | Covalent labelling of the NADPH: protochlorophyllide oxidoreductase from etioplast membranes with [3H]N-phenylmaleimide[END_REF][START_REF] Santel | The Protochlorophyllide Holochrome of Barley (Hordeum vulgare L.)[END_REF]. Therefore, to show whether the absence of chlorophyll is a limiting factor, a cop mutant was chosen since it develops a photomorphogenetic phenotype in the dark similar to the pap7-1light mutant [START_REF] Wei | Arabidopsis COP9 is a component of a novel signaling complex mediating light control of development[END_REF][START_REF] Wei | The role of the COP/DET/FUS genes in light control of arabidopsis seedling development[END_REF]. In the dark, the cop mutant showed repression of hypocotyl elongation and opening cotyledons with increased expression of light-dependent nuclear and plastid encoded genes but of course no greening [START_REF] Wei | Arabidopsis COP9 is a component of a novel signaling complex mediating light control of development[END_REF][START_REF] Chen | Phytochrome signaling mechanisms and the control of plant development[END_REF][START_REF] Ma | Genomic Evidence for COP1 as a Repressor of Light-Regulated Gene Expression and Development in Arabidopsis[END_REF][START_REF] Hills | Chloroplast Biogenesis-Associated Nuclear Genes: Control by Plastid Signals Evolved Prior to Their Regulation as Part of Photomorphogenesis[END_REF]. The morphology of cop plastid showed an absence of internal membrane system and instead accumulation of vesicles as in the pap7-1 mutant [START_REF] Wei | Arabidopsis COP9 is a component of a novel signaling complex mediating light control of development[END_REF], Kwok et al., 1996, Rohde, 2000). Thus, the formation of thylakoids most likely requires the presence of chlorophylls.

Moreover, the establishment of the thylakoid membrane requires the presence of thylakoid membrane lipids such as the galactolipid monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). Inactivation of the MGD1 enzyme, a catalyzer for MGDG synthesis from diacylglycerol, generates altered thylakoids unable to synthesize chlorophyll [START_REF] Motohashi | Chloroplast ribosome release factor 1 (AtcpRF1) is essential for chloroplast development[END_REF], 2013[START_REF] Pribil | Structure and dynamics of thylakoids in land plants[END_REF]. In conclusion of this section, a defect in GLK expression, plastidial translation of plastid localized POR enzymes, import of nuclear photosynthesis enzymes (LHCB) and lack of thylakoid, may reflect the absence of chlorophyll biosynthesis and disruption in the establishment of the photosynthesis apparatus as already shown in the pap7-1 mutant.

PAP7 is required for the stress response

The expression of the genes for the key enzymes of the jasmonic acid biosynthesis pathway (LOX, AOS, and AOC) is necessary to generate the precursors of 12-phytodienoic acid (OPDA, [START_REF] Dave | Oxylipin Signaling: A Distinct Role for the Jasmonic Acid Precursor cis-(+)-12-Oxo-Phytodienoic Acid (cis-OPDA)[END_REF][START_REF] Tamaoki | Jasmonic acid and salicylic acid activate a common defense system in rice[END_REF][START_REF] Leon-Reyes | Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway[END_REF]. OPDA biosynthesis is carried out at the inner plastid envelope membrane using mainly membrane lipids such as linolenic acid [START_REF] Douce | Biochemistry and Function of the Plastid Envelope[END_REF]). These nuclear-encoded genes have shown a considerable down-regulation at the transcriptomic level and a corresponding repression at the protein level suggesting a transcriptional regulation.

Recently, studies on maize and asian corn borer using plants grown in the presence of exogenous methyl jasmonate (MeJA), have shown an up-regulation of the expression of some genes involved in jasmonic acid synthesis and defense pathways suggesting subsequently a regulation at transcript level [START_REF] Turner | The Jasmonate Signal Pathway[END_REF][START_REF] Yamada | Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice[END_REF][START_REF] Tamaoki | Jasmonic acid and salicylic acid activate a common defense system in rice[END_REF][START_REF] Yang | Analysis of key genes of jasmonic acid mediated signal pathway for defense against insect damages by comparative transcriptome sequencing[END_REF]. A determination of the fatty acid accumulation in the pap7-1 mutant was carried out in collaboration with the team directed by Marechal (Albrieux and Jouhet, unpublished) showing that linolenic acid, as well as MGDG, are strongly reduced. This reduction is likely due to the absence of the thylakoid membrane in the albino plastid [START_REF] Miège | Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG[END_REF]Schleiffet et al., 2003;[START_REF] Aronsson | The galactolipid monogalactosyldiacylglycerol (MGDG) contributes to photosynthesisrelated processes in Arabidopsis thaliana[END_REF]. The data suggest a down-regulation of the prokaryotic fatty acid biosynthesis pathway that is plastid localized. This corresponds well to the observed down-regulation of the AccD transcript and protein (Table 3), the plastid encoded subunit of the envelope-localized acetyl-CoA carboxylase complex. The absence of thylakoid membrane, thus affects not only the chlorophyll biosynthesis and the photosystem apparatus but also hormone biosynthesis. In rice, jasmonic acid biosynthesis is induced under the control of the phytochromes (phyA and phyB, Svyatyna and Riemann, 2012). In Arabidopsis, activation of phyA by FR or a low R light can promote the induction of jasmonic acid biosynthesis (Franklin and Quail, 2010;Robson et al., 2010).

Interestingly, the biosynthesis of jasmonic acid allows the accumulation of HY5 gene in the nucleus and as a consequence the inhibition of hypocotyl elongation, regulated by phyA and subsequently by phyB [START_REF] Hsieh | Molecular interaction of jasmonate and phytochrome A signalling[END_REF][START_REF] Campos | Rewiring of jasmonate and phytochrome B signalling uncouples plant growth-defense tradeoffs[END_REF]. The jasmonic acid and active phytochromes exhibit antagonistic mutual signaling [START_REF] Hsieh | Molecular interaction of jasmonate and phytochrome A signalling[END_REF][START_REF] Wang | Phytochrome A and B Function Antagonistically to Regulate Cold Tolerance via Abscisic Acid-Dependent Jasmonate Signaling[END_REF]. It was proven that the addition of an external MeJA influences the phenotype of plants. In wild-type Arabidopsis plants, the addition of MeJA inhibited root growth by 50% due to the direct effect of MeJA. However, in the jar1 mutant (insensitive to primary root growth by MeJA) treatment sensitivity is limited and inhibition of root growth has been reduced. In the absence of MeJA, the root length of the jar1 mutant is similar to the wild-type [START_REF] Corbineau | The effects of methyl jasmonate on sunflower (Helianthus annuus L.) seed germination and seedling development[END_REF][START_REF] Staswick | Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant[END_REF]. In addition, root growth was not inhibited in the transgenic rice plants in presence of MeJA [START_REF] Yamada | Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice[END_REF]. Root growth and hypocotyl tests in the presence of exogenous MeJA seems to be an important experiment to do in the near future.

In the presence of exogenous ABA treatment, the germination rate of jar1 seeds decreased indicating a sensitivity of the jar1 mutant to ABA. This suggests that ABA and jasmonic acid can interact together in metabolic pathways [START_REF] Staswick | Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant[END_REF][START_REF] Tamaoki | Jasmonic acid and salicylic acid activate a common defense system in rice[END_REF].

PAP7 regulates carotenoid and abscisic acid biosynthesis

The two key enzymes NCED4 and ZEP of carotenoid biosynthesis pathway are nuclearencoded and plastid-localized proteins. Transcriptomic and proteomic data showed that these two enzymes are down-regulated in pap7-1 mutant especially in the transcriptomic level suggesting a transcriptional regulation (Fig. 20, Table 4).

The carotenoid biosynthesis is carried out by the plastid starting from the isoprenoid precursor IPP. The condensation of two GGPP molecules by the PSY enzyme gives phytoene and then lycopene. At this level there are two branches of α and β-carotene, which are used respectively for lutein and carotenoids synthesis (DellaPenna and Pogson, 2006). Zeaxanthin epoxidase (ZEP) enzyme catalyzes violaxanthin from an epoxidation of zeaxanthin. Then, violaxanthin is the substrate for the xanthoxin synthesis by carotenoid cleavage dioxygenase4 (CCD4). Both enzymes (ZEP and NCED4) are involved in the abscisic acid synthesis (Koornneef et al., 2002, Lefebvre et al., 2006, Frey et al., 2012). The knockout of ZEP and CCD4 genes gave alb1-7 and ccd4 nulls mutants with an ABA deficiency phenotype (aba mutant) and an increase of seed carotenoid content especially in carotene [START_REF] Gonzalez-Jorge | CAROTENOID CLEAVAGE DIOXYGENASE4 Is a Negative Regulator of β-Carotene Content in Arabidopsis Seeds[W][END_REF][START_REF] Zhang | Blue-white screening liquid can eliminate false positives in blue-white colony screening[END_REF]. These two enzymes are negative regulators of carotenoid biosynthesis and subsequently for ABA biosynthesis. This demonstrates that the inhibition of carotenoid biosynthesis pathway in pap7-1 mutant is not related to the down-regulation of ZEP and CCD4 enzymes but to another reason.

Then, an experimental strategy was performed to inhibit the isoprenoid biosynthesis by blocking the cytosolic (MVA) and plastid (MEP) IPP biosynthesis pathways (Fig. 23).

Blocking the MVA pathway by the mevinolin (MEV) showed the development of a skotomorphogenesis phenotype with yellow cotyledons due to the accumulation of low level of carotenoid. After light exposure, the MEV-treated seedlings accumulated chlorophylls and continued to develop normally. This indicates that plastid IPP pathway (MEP) can rescue the IPP biosynthesis. These data are perfectly conforming to the observations made previously [START_REF] Kasahara | Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis[END_REF], Rodríguez-Concepción et al., 2004).

Fosmidomycin (FSM) treatment blocks selectively the MEP pathway. Like after MEV traetment, FSM-treated seedlings under dark conditions generate yellow cotyledons. After light exposure, these seedlings were unable to synthetize chlorophyll pigments and showed yellow cotyledons (no bleaching). This indicates that MVA pathway could not rescue the blockage of MEP pathway in accordance with what has already proven that blocking one of the path cannot restore the activity of the other pathway [START_REF] Rodríguez-Concepción | Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development[END_REF].

Taken together, interestingly, these results suggest that there is, probably, an additional IPP precursor activated in the dark. This conclusion is contradictory to what has been shown in many other studies suggesting that in plants, all carotenoids are synthesized from GGPP produced by the MEP pathway [START_REF] Rodríguez-Concepción | 1-Deoxy-d-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening[END_REF]Rodríguez-Concepción et al., 2004;[START_REF] Gonzalez-Jorge | ZEAXANTHIN EPOXIDASE Activity Potentiates Carotenoid Degradation in Maturing Seed1[OPEN][END_REF].

Previous papers and studies have presented results from blocking MVA and MEP pathways directly in the light. Nevertheless, our study highlighted the difference between the effect of the treatment between the dark and light growth conditions.

The FSM-treated plants (500 μM) were initially grown in the dark and then de-etiolated in the light; these plants did not display a real albino phenotype (Fig. 23). Published data reported that FSM-treated seedling generate an albino phenotype and an arrest in plastid development in the light (Rodriguez-Concepción et al., 2001, 2004, Rodriguez-Villalon et al., 2009).

However, the blockage of the MEP pathway has occurred only after light exposure. This implies that FSM treatment likely blocks the MEP pathway only after light exposition (Fig. 22). Direct development in light showed that FSM-treated seedlings are completely albino suggesting that the MEP pathway is light-dependent (Fig. 24). The phenotypes of WT dark and pap7-1 grown in the dark are comparable to the FSM-treated plants (Fig. 1,C). These results show clearly that both MEP and MVA pathways are functional in the pap7-1 mutant in the dark conditions. In the dark, pap7-1 seedlings can produce carotenoids, likely from the storage lipids via glyceraldehyde 3-phosphate; GAP, which is usually an intermediate metabolite of photosynthesis. Upon light exposition, the isoprenoid production is completely blocked in pap7-1 mutant showing that carotenoid biosynthesis depends on light. Since all transcript and protein data suggest a normal activity of the pathway one can only conclude that substrate limitation hinders the production of carotenoids. In fact, one essential precursor, GAP, is produced from photosynthesis. If this is lacking also carotenoid production must stop.

The lack of carotenoids is strongly correlated with the absence of abscisic acid (ABA, [START_REF] Pizarro | Light-dependent regulation of carotenoid biosynthesis in plants[END_REF] suggesting deficiency in a number of other hormone controlled processes.

In sum, the comparison between the transcript and protein accumulation in the pap7-1 albino mutant showed that the absence of photosynthesis blocks all basic molecules for energy having a strong impact on metabolism. In conclusion, the albinism of pap7-1 is the result of several intra-plastidial and intracellular interactions (mainly anterograde and retrograde signaling) that prevents proper chloroplast differentiation without destroying the plastid. The important role of photoreceptors, especially phytochromes, has been largely noticeable in maintaining the induction and stability of several molecular aspects in chloroplast biogenesis.

In the next chapter, a detailed molecular-genetic study was established to understand the role of the PAP7 gene in light-dependent development.

Conclusion en français

L'origine de la perturbation de l'activité plastidiale du mutant pap7-1 a été partiellement étudiée par des études génétiques et transcriptomiques [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF][START_REF] Allorent | Plastid gene expression during chloroplast differentiation and dedifferentiation into non-photosynthetic plastids during seed formation[END_REF], Grübler et al., 2017). Ainsi, 3805 groupes de protéines ont été détectées et divisés en trois situations comparatives:

(1) WTL contre WTD qui correspond à la situation normale du développement du chloroplaste (photomorphogenèse; PM), (2) pap7-1 contre WTD dont les changements de l'expression sont soumis à la signalisation de la lumière (LS) et la situation (3) pap7-1 vs WTL dont les changements dépendent principalement des plastes et leurs défauts (PS).

Seulement 12 % de ces protéines ont été détectées de manière significative dans le mutant pap7-1 dont la synthèse est dépendante de l'effet de l'albinisme (PS). L'analyse globale de l'accumulation des protéines a été réalisée à l'aide de l'outil «MapMan». Les résultats ont montré un profil comparable entre le WT et pap7-1 et une régulation de la lumière chez les mutants pap7-1 comparable à celle du WT. Les plastides non fonctionnels n'ont pas eu beaucoup d'impact sur les réseaux d'accumulation de protéines dépendants de l'effet de la lumière. Le programme de photomorphogenèse est largement fonctionnel chez le mutant albinos. De plus, la comparaison entre les situations de PM et LS a montré que les protéines de photosynthèse s'accumulent de manière comparable, de sorte qu'il n'y a aucun effet de la lumière sur l'accumulation de protéines de photosynthèse.

La perturbation de la maintenance de l'appareil photosynthétique est principalement due au plastes albinos non fonctionnels.

Les protéines photosyntétiques sont les groupes de protéines les plus répendues affectées dans pap7-1. Leur profil indiquait un très faible niveau d'accumulation des protéines de PSI, la majorité des PSII spécialement les protéines des antennes de réception de la lumière.

Les résultats de la protéomique sont parfaitement en accord avec l'analyse transcriptomique. L'étude du mécanisme d'action de ces hormones sera probablement une clé imprtante des défauts moléculaires des mutants dépourvus de chloroplaste fonctionnels.

Dans le chapitre suivant, une étude génétique détaillée a été établie pour comprendre le rôle du gène PAP7 dans le développement plastidial den réponse à la lumière.

Synopsis en Français

Dans ce chapitre, nous sommes intéressés à la fonction de PAP7 sous l'angle de la génétique moléculaire afin de mieux comprendre son rôle dans la biogenèse du chloroplaste ainsi que son mode d'action au sein de la cellule.

L'étude repose principalement sur l'hypothèse initiale que des protéines du complexe PEP-PAP, dont PAP7 fait partie, seraient bi-localisées à la fois dans le chloroplaste (où les PAP ont été détectées par biochimie, [START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF] et dans le noyau grâce à la présence prédite d'un ou de plusieurs signaux de localisation nucléaire [START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF].

Cette hypothèse est soutenue par l'ensemble des publications sur le gène HEMERA/pTAC12/PAP5 [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF] ainsi que de récents travaux sur la protéine PAP8 auxquels j'ai participé (Liebers et al., 2019. soumis

Characterization of the Arabidopsis thaliana pap7-mutant

In Arabidopsis thaliana, a single nuclear gene copy located at the AT4g20130 locus encodes the PAP7/pTAC14 protein. The PAP7 gene contains a predicted chloroplast transit peptide at the N-terminal side (Fig. 1,A; cTP; covering 62 amino acids in yellow) and a potential nuclear localization signal (NLS in red). Furthermore, the PAP7 gene is characterized by the prediction of two functional domains: A SET7-like domain that is a Lys methyltransferase with a putative substrate (colored in magenta, Claude et al., 2014;[START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF] and a predicted Rubisco LSMT substrate-binding domain (RBCL-like "binding domain"; in blue).

The position L 213 indicates the last unchanged codon in the mutant allele before the T-DNA insertion point that disrupted the coding sequence introducing an early stop after two spurious codons. Therefore, the T-DNA insertion obliterates half of the protein sequence including the NLS, the putative methyl transferase domain and the putative RBCL-interacting domain (Fig. 1,A). pap7-1 is therefore a loss-of function allele.

In order to study the role of the PAP7 in chloroplast biogenesis, the SAIL_566_F06 line was used. This line was generated in a Columbia genetic background. In addition it is characterized by the insertion of a single T-DNA at the sixth exon 1438 bp downstream of the translation initiation site [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF]; Fig. 1,A). The insertion site was checked by sequencing using the insertion primer (opap7rtpF/oP7-1mR2; 503pb band), while the wildtype allele was verified by the primers opap7rtpF/opap7rtpR (835 pb band; Fig. 1,B). Seeds from heterozygous pap7-1 lines were sterilized and sown on MS agarose medium supplemented with 1% (w/v) sucrose (external carbon source), then cultivated in long days condition (16-hour photoperiod) under low intensity light (12 μE). The segregation of these plants indicated that 3/4 of germinated seeds produced green seedlings ([WT] phenotype), whereas 1/4 of these plants developed an albino phenotype (Fig. 2). The pap7-1 albino plants are characterized by a slower development compared to wild-type plants. This confirms that the knockout of the PAP7 gene produces pap7-1 homozygous albino plants. Homozygous pap7-1 mutants are unable to grow photoautotrophically and cannot survive without a supplemented external carbon source. These seedlings were subsequently used for the analysis of the loss of PAP7 gene function.

PAP7 localization using deleted cTP or NLS constructs

To further confirm the subcellular localization of the PAP7 protein, a genetic strategy decoupling the localization of this protein was performed.

Various constructions were cloned under the control of the 35S promoter; the first one containing four amino acid substitutions of conserved K and R residues in the predicted NLS was fused to RFP (pCaMV35S::PAP7𝑁𝐿𝑆 m4 -DsRed, Fig. 4,A) and the second one lacking the cTP was fused with GFP (pCAMV35S::PAP7 ΔcTP -GFP, Fig. with GFP at the C-terminal end showed fluorescence mainly in chloroplasts [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]. Therefore, in vivo data confirmed the initial prediction. PAP7 is potentially localized in both plastids and nucleus and these two compartments are essential for the function of PAP7.

In addition, disruption of the NLS or cTP compromises the respective nuclear and plastidial localization. The perturbation of the NLS function leads to a disruption in PAP7 nuclear localization. Then the cTP function depends on the protein import machinery [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]Liebers et al., in revision).

Hemi-complementation of the pap7-1 mutant

PAP7 is considered as a bi-localized protein; therefore, it possesses potential functions in nucleus and in plastids. In order to understand the role of PAP7 in both compartments, a hemi-complementation strategy was performed. This strategy aimed to uncouple the two localizations of PAP7 by mutation and to test whether in absence of the nuclear or plastidial function the complemented pap7-1 mutant develops a normal greening phenotype or not. To this end a couple of genetic constructions were generated: pPAP8::PAP7 NLSm4 ; pPAP8::PAP7 NLSm4 -GFP and pPAP8::PAP7 ΔcTP .

Search for the PAP7 nuclear function

Construction and characterization of pPAP8::PAP7 NLSm4 (pMC03a) lines

The PAP7 protein has 53.24-kDa as a calculated molecular weight and an apparent molecular weight in SDS-PAGE of about 49-kDa. It codes for a 483 amino acid protein [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF]. The sequence alignment of the PAP7 protein was generated to identify the homologous and conserved positions that are likely important for maintaining the structure and function of and homology (22 %). In addition, two residues have been identified and appear to play an essential role in the catalytic activity of the PAP7 protein. These are tyrosine Y281 and methionine M325 (Fig. 7,A,C). The amino acids in positions 332 to 465 include a homologous region to the LSMT Rubisco binding domain (Fig. 7,A,D). Interestingly, amino acids between positions 222 and 242 define the bipartite NLS. This sequence containing 19% of homology proportion and 14% of identity proportion. The two residues respectively in position 222 (lysine; k) and 242 (arginine; R) showed strong conservation (Fig. 7,A,B).

pap7-1/+ plants. Seeds from T0 plants were sown on MS sucrose supplemented medium. Fifty resistant plants were selected on hygromycin and transferred to the soil. Ten plants were identified as heterozygous and transgenic (pPAP8::PAP7 NLSm4 -GFP). Plant number 46, called pMC07a#46, has been kept for the T2 generation. Among twenty seeds were sown in the MS medium. Sixteen plants were green and four plants were homozygous pap7-1. These plants showed an albino phenotype at the beginning of the growth stage. After twenty-six days, these albino plants started to develop a greening phenotype (Fig. 11,C). Therefore, plants A, J, Y and Z were considered as transgenic in a homozygous pap7-1 background while plant C was a double heterozygous. All these plants were transplanted on soil under long day conditions.

pMC07a#46#Y and pMC07a#46#Z plants could survive under these conditions, but they grew much slower than wild-type plants. Thus, observations reflect a 1:16 Mendelian ratio, however, apparently the small quantity of sown seeds (< 100 seeds) may explain these proportions. Based on these phenotypic, genetic and statistical analyses likely the pPAP8::PAP7 NLSm4 -GFP construct can rescue the greening defect in the albino pap7-1 mutant. In addition, apparently the GFP protein does not interrupt the PAP7 function. This suggests that the GFP can be a helpful tool to follow the protein trafficking not only in the nucleus, but also in the chloroplast. Genetic analyses and a confocal microscopy observations should be done in order to verify the plastidial localization of PAP7.

photomorphogenesis the activity of several transcription factors (such as PIF1 and PIF3) is blocked. These factors are able to interact with active phytochromes by providing negative feedback on different developmental aspects (such as inhibition of seeds germination, hypocotyl growth and chlorophyll accumulation; [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]Leivar et al., 2008). Upon light exposition, PHYB converts into its active form, becomes imported from the cytosol to the nucleus and is associated with nuclear photobodies (NBs; [START_REF] Fankhauser | Transposing phytochrome into the nucleus[END_REF].

NBs are characterized by their different shapes, size, and number according to the developmental stage [START_REF] Chen | Characterization of the requirements for localization of phytochrome B to nuclear bodies[END_REF]. Interaction of PhyB with PIFs in these bodies initiate their degradation (Al-Sady et al., 2006;Lorrain et al., 2008;Shen et al., 2008).

To determine if the PAP7 protein is involved in light signaling, the pap7-1/+ heterozygous lines were crossed into a line containing a 35S::PHYB-GFP construction (called 35::PBG; [START_REF] Yamaguchi | Light-dependent Translocation of a Phytochrome B-GFP Fusion Protein to the Nucleus in Transgenic Arabidopsis[END_REF]. Consequently, the primary transformants (T1) grown under a low red light intensity (8 µE for seven days) were obtained by hygromycin selection and observed by fluorescence microscopy (Fig. 13,A). Nine PBG seedlings were transplanted into soil. These plants developed a clearly affected phenotype characterized by leaf elongation which is apparently related to the presence of PHY signaling (Fig. 13,B). Two heterozygous plants containing the transgene (35S::PBG, Fig. 13,J) were used for production of the T2 generation.

Seedlings from T2 were grown under low red light intensity (8 µE) for seven days.

Phenotypic observation showed significant inhibition of hypocotyl length in WT-PBG plants compared to the WT. However, this effect was mauch weaker in PBG pap7-1 plants (homozygous pap7-1 background) (Fig. 13,C,D).

This suggests a hyposensitivity of hypocotyl elongation of PBG pap7-1 mutant under red light conditions. Similar analyses on hypocotyl elongation showed that the dually localized albino mutants ncp-1-PBG, rcb-1-PBG as well as hmr PBG were also hyposensitive to FR and R light. This suggests a common role of all the inactivated components in phytochrome signaling [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]Chen et al., 2019;[START_REF] Yoo | Phytochrome activates the plastid-encoded RNA polymerase for chloroplast biogenesis via nucleus-to-plastid signaling[END_REF]. Therefore, PAP7 plays a role in the PhyB mediated light response. An initial observation by epi-fluorescence microscopy of the T1 generation in WT and PBG pap7-1 plants showed the appearance of nuclear photobodies (NBs) with significantly different diameters (Fig. 13,E,F). These observations were confirmed by microscopic observation in the T2 generation (Fig. 13,G,H).

To investigate this in more detail the diameter of NBs was measured. Fifty different NBs were measured in PBG WT plants exhibiting a diameter of 1.5 µm in average. In contrast, the determination of the diameter of fifty-eight NBs in PBG pap7-1 plants resulted in an average value of 0.7 µm. These results showed that NBs in the pap7-1 are much smaller than those of the PBG WT plants (Fig. 13,I). These results are in line with observations on other dually localized proteins. It was reported that hmr-2, ncp-1, rcb-1 and rcb-2 mutants are null mutants that contain a few small NBs in the nucleus [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]Chen et al., 2019;[START_REF] Yoo | Phytochrome activates the plastid-encoded RNA polymerase for chloroplast biogenesis via nucleus-to-plastid signaling[END_REF]. We can deduce explicitly that the pap7-1 mutant is disturbed in the formation of late (large) photobodies. Likely, PAP7 interacts with PHYB to activate formation of large nuclear photobodies suggesting that PAP7 is an important regulator for the action of PHYB in the NBs. From this we conclude that PAP7 is a light signaling regulator in the early developmental stages of seedling development. This can probably influence the regulation of the light-dependent gene expression, such as GLK1 expression. Recently, work carried out in our team by Liebers et al. proposed that the PBG pap8-1 showed a down-regulation of the GLK1expression in the light. Thus, it is proposed that the PAP8 protein controls GLK1 expression and indirectly the expression of PhANGs genes (Oh and Montgomery, 2014). The data presented here show significant similarities with those of the nucleo-plastidic proteins PAP8, HMR, NCP and RCB suggesting that it is also important for the initiation of photomorphogenesis and for the plastidial PEP complex [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF][START_REF] Steiner | Identification of Essential Subunits in the Plastid-Encoded RNA Polymerase Complex Reveals Building Blocks for Proper Plastid Development[END_REF]Qiu et al., 2015).

It was demonstrated that the nucleo-plastic bi-localized PAP8/pTAC6 protein can physically interact with pTAC12/PAP5/HEMERA protein in the nucleus (Liebers et al., in revision).

Furthermore, recent studies in yeast-two-hybrid assays demonstrated a potential interaction of PAP7/pTAC14 with pTAC12/PAP5/HEMERA [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF].

Here, a bimolecular fluorescence complementation (BiFC) test has been implemented to determinate the potential interaction between PAP7 protein and PAP5 and/or PAP8. BiFC is a molecular technique used to establish protein interactions networks and to detect and visualize protein-protein interactions in vivo (Costanzo et al., 2010;Miller et al., 2016). In fact, BiFC demonstrates the possibility of interaction between two proteins by generating fluorescence after physical interaction between them. To this end each protein is fused to one part of a fluorophore separated into an amino terminal (NY) and a carboxy terminal part (YC) (Kerpolla et al., 2008). When these two fragments combine by interaction of the two proteins of interest, a fluorescence is observed. Therefore, the two proteins tested likely belong to the same macromolecular complex or show at least a stable interaction. Several genetic constructions using PAP7; PAP5 and PAP8 genes without cTP, were carried out under the regulating the light signaling pathway. Here we have shown that PAP7 is essential to the mediation of light signals from the activated PHYB. This can likely regulate the expression of the light-dependent nuclear photosynthesis genes. Liebers et al. have proved that the nuclear fraction of PAP8 is essential to transduce the light signal from activated PHYB to the expression of GLK1 (one of the master regulators of nuclear photosynthesis genes). Here we have proven that PAP7 can be a new regulator candidate for the light signal from the PHYB, by interacting with PAP5 and PAP8 in the nucleus. This confirms the role of PAP7 in the initiation of photomorphogenesis in early stages of chloroplast biogenesis. Studies have shown that HMR, NCP and RCB can promote the expression of GLKs and the degradation of PIF1 and PIF3. The dual localization of these proteins has allowed for confirming their role in the regulation of the PEP assembly through an anterograde signal generating after the degradation of PIFs [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]Chen et al., 2019;[START_REF] Yoo | Phytochrome activates the plastid-encoded RNA polymerase for chloroplast biogenesis via nucleus-to-plastid signaling[END_REF]. However, biochemical and molecular experiments will be planned for future experiments in order to further confirm the role of PAP7 in the activation of PEP assembly and chloroplast biogenesis, via nucleus-to-plastid phytochrome signaling.

Conclusion en français

La protéine PAP7 a été identifiée comme une sous-unité du complexe chromosomique actif (TAC) s'associant à l'ARN polymérase codée par le plaste [START_REF] Gao | A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression[END_REF]. Le déficit en PAP7 conduit à une plante albinos, un disfonctionnement de la PEP, l'absence de l'expression des gènes de photosynthèse codés par les plastes et par la suite à un blocage de la différenciation des chloroplastes.

Dans ce chapitre, nous avons fourni des éléments expérimentaux montrant que PAP7 est bilocalisée dans les plastes et dans le noyau. Des études de complémentation fonctionnelle, utilisant le gène PAP7, ont révélé que l'insertion de l'ADN-T est responsable du défaut de la fonction de PAP7. De plus, des tests d'hémi-complémentation, par découplage des deux compartiments, nucléaire et plastidial, ont montré que PAP7 joue un rôle important dans le fonctionnement du noyau et que la localisation dans les deux compartiments semble être indispensable pour la biogenèse des chloroplastes.

Nous avons également découvert que PAP7 est essentiel pour la formation des photo-corps de phytochrome B dans le noyau au cours de la signalisation par la lumière.

Par analogie avec les travaux sur PAP8, il est possible de suspecter que PAP7 permet, de manière directe ou indirecte, la régulation de l'expression des gènes nucléaires associés à la photosynthèse (PhANGs). En effet, au sein de l'équipe nous avons apporté des évidences expérimentales sur l'importance de PAP8 pour la stabilisation de HY5 et l'expression des gènes GOLDEN LIKE (GLK), eux-mêmes impliqués dans l'expression des PhANGs au cours des étapes précoces de biogenèse des chloroplastes.

Ici, nous émettons l'hypothèse que PAP7 peut être un nouveau régulateur intervenant dans la transduction du signal lumineux par PHYB, en interagissant avec PAP5 et PAP8 dans le noyau. Des études ont montré que les protéines HMR, NCP et RCB jouent un rôle important dans l'expression des gènes GLK et dans la dégradation des gènes PIF1 et PIF3. La double localisation de ces protéines a permis de confirmer qu'elles régulent l'expression des gènes du plaste à travers un signal antérograde suite à la dégradation des PIF dans le noyau [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]Chen et al., 2019 ;[START_REF] Yoo | Phytochrome activates the plastid-encoded RNA polymerase for chloroplast biogenesis via nucleus-to-plastid signaling[END_REF]. Dans ce contexte, des expériences biochimiques et moléculaires seront planifiées afin de tester notre hypothèse sur le rôle de PAP7 dans la formation ou l'assemblage d'une part de la PEP, mais aussi d'un complexe nucléaire potentiellement impliqué dans la transduction du signal lumineux.

Chapter 4 Towards the understanding of the nature of biogenic retrograde signaling: Norflurazon and lincomycin treatment

Introduction

The study of chloroplast biogenesis has become an important research topic during the last decade. Regulation of nuclear encoded genes for plastid proteins is related to light, but also to the functional status of plastids [START_REF] Kessler | Chloroplast biogenesis: diversity and regulation of the protein import apparatus[END_REF][START_REF] Inaba | Bilateral Communication between Plastid and the Nucleus: Plastid Protein Import and Plastidto-Nucleus Retrograde Signaling[END_REF]. Since the majority of proteins involved in chloroplast differentiation are encoded by the nuclear genome, translated into the cytosol and then imported into the plastid [START_REF] Inaba | Protein trafficking to plastids: one theme, many variations[END_REF][START_REF] Jarvis | Targeting of nucleus-encoded proteins to chloroplasts in plants[END_REF][START_REF] Li | Protein Transport into Chloroplasts[END_REF], a deeper understanding of the impact of the plastid state on their expression is mandatory.

The chloroplast biogenesis is not only controlled by the coordination between the light and the plastid signaling (Larkin and Ruckle, 2008) but also by the coordination between nuclear and plastid genome (Beck, 2005[START_REF] Woodson | Coordination of gene expression between organellar and nuclear genomes[END_REF]., 2008[START_REF] Nott | Plastid-to-nucleus retrograde signaling[END_REF][START_REF] Pogson | Plastid signalling to the nucleus and beyond[END_REF][START_REF] Terry | A model for tetrapyrrole synthesis as the primary mechanism for plastid-tonucleus signaling during chloroplast biogenesis[END_REF]. [START_REF] Susek | Signal transduction mutants of Arabidopsis uncouple nuclear CAB and RBCS gene expression from chloroplast development[END_REF] performed important experiments in order to understand the retrograde communication during chloroplast biogenesis leading to the identification of the genomes uncoupled (GUN) mutants. gun2 mutants have been considered as a plastid signal regulators of tetrapyrrole biosynthesis pathway [START_REF] Moulin | Tetrapyrrole profiling in Arabidopsis seedlings reveals that retrograde plastid nuclear signaling is not due to Mg-protoporphyrin IX accumulation[END_REF][START_REF] Larkin | GUN4, a regulator of chlorophyll synthesis and intracellular signaling[END_REF][START_REF] Mochizuki | Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction[END_REF][START_REF] Woodson | Heme synthesis by plastid ferrochelatase I regulates nuclear gene expression in plants[END_REF]. The majority of gun mutants develop a yellow or undistinguishable phenotype [START_REF] Surpin | Signal Transduction between the Chloroplast and the Nucleus[END_REF]. The GUN2 gene encodes the heme oxygenase, and the GUN3 gene the phytochromobilin synthase. Lack of these two enzymes causes an over-accumulation of heme. This can lead to a negative feedback on chlorophyll synthesis [START_REF] Mochizuki | Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction[END_REF]. Thus, a positive signal can regulate the nuclear-encoded chloroplast genes. GUN4 and 5 are the Magnesium-chelatase regulators, which are involved in the chlorophyll biosynthetic branch [START_REF] Larkin | GUN4, a regulator of chlorophyll synthesis and intracellular signaling[END_REF][START_REF] Mochizuki | Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction[END_REF]. The inhibition of Mg-chelatase activity led to positive feedback on the heme biosynthesis (Cornah et al., 2003) and a down-regulation of Lhcb genes [START_REF] Strand | Chloroplast to nucleus communication triggered by accumulation of Mg-protoporphyrinIX[END_REF]. The gun mutants play also an important role in nuclear gene expression regulated by plastid signals [START_REF] Strand | Chloroplast to nucleus communication triggered by accumulation of Mg-protoporphyrinIX[END_REF]. For instance, GUN6 is considered as a positive model of plastid signal regulation. The gun6 mutant provoked the overexpression of ferrochelatase-1 (FC1) in the heme branch [START_REF] Woodson | Heme synthesis by plastid ferrochelatase I regulates nuclear gene expression in plants[END_REF]. Heme was increased and the expression of PhANGs was upregulated.

This suggested that GUN6 can be used as a retrograde signaling model to coordinate the increasing expression of photosynthesis-associated nuclear genes (PhANG) during chloroplast development after norflurazon or lincomycin treatment [START_REF] Woodson | Heme synthesis by plastid ferrochelatase I regulates nuclear gene expression in plants[END_REF].

Another group of mutants was studied for their roles in retrograde signaling. For example, the Cryptochrome 1 (CRY1) blue photoreceptor mutant and the HY5 light signaling mutant showed a chloroplast affected in proper light signaling [START_REF] Ruckle | Plastid Signals Remodel Light Signaling Networks and Are Essential for Efficient Chloroplast Biogenesis in Arabidopsis[END_REF]. In addition, the protein import mutant ppi2-2 was used to implement the effect of defective plastid protein import on nuclear gene expression [START_REF] Kakizaki | Coordination of Plastid Protein Import and Nuclear Gene Expression by Plastid-to-Nucleus Retrograde Signaling[END_REF].

Several treatments have been suggested to induce plastid signals towards the nucleus.

Fluridone and norflurazon (NF) have been widely used to block carotenoid biosynthesis.

The importance of carotenoids for chlorophyll formation becomes apparent when the synthesis of carotenoids is inhibited [START_REF] Vaisberg | Events Surrounding the Early Development of Euglena Chloroplasts: 7. Inhibition of Carotenoid Biosynthesis by the Herbicide SAN 9789 (4-Chloro-5-(methylamino)-2-(α,α,α,trifluoro-m-tolyl)-3-(2H)pyridazinone) and Its Developmental Consequences[END_REF]. Norflurazon (NF) is a potent inhibitor of phytoene desaturase, the key enzyme involved in carotenoid biosynthesis [START_REF] Hilton | Modes of Action of Pyridazinone Herbicides[END_REF][START_REF] Bartels | Chloroplast Development in 4-Chloro-5-(dimethylamino)-2-(α,α,α-trifluoro-mtolyl)-3 (2H)-pyridazinone (Sandoz 6706)-treated Wheat Seedlings[END_REF][START_REF] Breitenbach | Bleaching herbicide norflurazon inhibits phytoene desaturase by competition with the cofactors[END_REF]. This inhibition results in the accumulation of colorless carotenoid precursors in Euglena cells (Vaisberg and Schiff, 1967).

In high-light conditions, carotenoid deficiency has been shown to cause a loss of chlorophyll synthesis (Feierabend andSchubert, 1978, Frosch et al., 1979;[START_REF] Batschauer | The implication of a plastid-derived factor in the transcriptional control of nuclear genes encoding the light-harvesting chlorophyll a/b protein[END_REF].

Moreover, NF interferes with membrane lipid formation [START_REF] Hilton | Interactions of Lipoidal Materials and a Pyridazinone Inhibitor of Chloroplast Development[END_REF]. The inhibition of lipid biogenesis has been especially achieved by increasing in linolenic acid production [START_REF] Hilton | Interactions of Lipoidal Materials and a Pyridazinone Inhibitor of Chloroplast Development[END_REF]. This means a perturbation in thylakoid membrane assembly. This results in blocking of the chloroplast biogenesis.

NF-treated plants exhibit inhibition of the nuclear LHCB gene expression [START_REF] Moulin | Tetrapyrrole profiling in Arabidopsis seedlings reveals that retrograde plastid nuclear signaling is not due to Mg-protoporphyrin IX accumulation[END_REF][START_REF] Mayfield | Carotenoid-deficient maize seedlings fail to accumulate light-harvesting chlorophyll a/b binding protein (LHCP) mRNA[END_REF]Taylor, 1984, Batschauer et al., 1986), which blocks the photosynthetic membrane complex assembly [START_REF] Voigt | In-depth analysis of the distinctive effects of norflurazon implies that tetrapyrrole biosynthesis, organellar gene expression and ABA cooperate in the GUN-type of plastid signalling[END_REF][START_REF] Kim | 1O2-mediated and EXECUTER-dependent retrograde plastid-to-nucleus signaling in norflurazon-treated seedlings of Arabidopsis thaliana[END_REF]. NF treatment cause defects in the tetrapyrrole biosynthesis pathway and as a result it disrupts the accumulation of plastid proteins by activating plastid retrograde [START_REF] Saini | happy on norflurazon" (hon) mutations implicate perturbance of plastid homeostasis with activating stress acclimatization and changing nuclear gene expression in norflurazon-treated seedlings[END_REF].

It is reported that the absence of carotenoid increases the photo-sensitizing activity and thus improves the 1 O 2 production or excitation of triplet state from of chlorophyll [START_REF] Kim | 1O2-mediated and EXECUTER-dependent retrograde plastid-to-nucleus signaling in norflurazon-treated seedlings of Arabidopsis thaliana[END_REF][START_REF] Knox | Singlet oxygen and plants[END_REF]. The generation of reactive oxygen species (ROS) leads to a photo-oxidation of other plastid components [START_REF] Elstner | Activated oxygen in green plants in relation to stress situations[END_REF], bleaching of seedlings and subsequently blocking of chloroplast biogenesis [START_REF] Bartels | Chloroplast Development in 4-Chloro-5-(dimethylamino)-2-(α,α,α-trifluoro-mtolyl)-3 (2H)-pyridazinone (Sandoz 6706)-treated Wheat Seedlings[END_REF][START_REF] Jung | Effect of chlorophyll reduction in Arabidopsis thaliana by methyl jasmonate or norflurazon on antioxidant systems[END_REF][START_REF] Sagar | Effects of High Light Stress on Carotenoid-Deficient Chloroplasts in Pisum sativum1[END_REF][START_REF] Voigt | In-depth analysis of the distinctive effects of norflurazon implies that tetrapyrrole biosynthesis, organellar gene expression and ABA cooperate in the GUN-type of plastid signalling[END_REF]. Therefore, the carotenoids behave like "chemical buffers" to protect chlorophyll pigments especially during early chloroplast biogenesis [START_REF] Anderson | Role of Carotenoids in Protecting Chlorophyll From Photodestruction[END_REF]. In low red light conditions, however, NF-treated plants accumulate chlorophyll [START_REF] Sagar | Light Effects on Several Chloroplast Components in Norflurazon-Treated Pea Seedlings 1[END_REF]. Likewise, lincomycin is an antibiotic that acts specifically against the plastid translation by binding to the plastid 70S ribosomes while the cytosolic 80S ribosomes remain unaffected [START_REF] Gray | Coordination of plastid and nuclear gene expression[END_REF][START_REF] Cottage | The Arabidopsis plastid-signalling mutant gun1 (genomes uncoupled1) shows altered sensitivity to sucrose and abscisic acid and alterations in early seedling development[END_REF]Bradbeer et al., 1979, Rapp andMullett, 1991). Both inhibitors induce the same phenotype in wild-type seedlings as the pap7-1 mutant (an albino phenotype), but they act on different molecular targets (plastid carotenoid synthesis and plastid translation versus plastid transcription). In order to understand how these different molecular defects can cause the same phenotypic effects we used the classical treatments norflurazon (NF) and lincomycin (Linco) and tested protein accumulation in treated plants by western-immuno-analyses.

Phenotypic description of NF and Linco-treated Arabidopsis plants

NF and Linco treatments are well-described in literature. In order to test whether the effect of these a number of initial phenotypic tests were done. An increasing concentration gradient of NF was used (2 µM, 5 µM and 10 µM) to determine the concentration required to block carotenoid biosynthesis. A 0.5 mM of lincomycin was used as mentioned in the literature [START_REF] Cottage | The Arabidopsis plastid-signalling mutant gun1 (genomes uncoupled1) shows altered sensitivity to sucrose and abscisic acid and alterations in early seedling development[END_REF][START_REF] Ruckle | Plastid Signals Remodel Light Signaling Networks and Are Essential for Efficient Chloroplast Biogenesis in Arabidopsis[END_REF]Saini et al., 2010;Kakizaki et al., 2011). After filtration in a 0.2 µm filter, the necessary volume was added directly to the MS 1% sucrose medium to reach the indicated final concentration. After 48 h of stratification, treated

Arabidopsis WT-Col0 seedlings were exposed for 6 hours to continuous light in order to initiate the germination process. Seedlings were grown for 3 days in dark condition. Under dark condition, the plants treated with NF and Linco showed an etiolated phenotype similar to the control (WT-Col0 without treatment; Fig. 1). They developed an apical hook with closed unexpanded cotyledons (Rodriguez et al., 2009). Linco-treated seedling showed a slightly weaker yellow coloring in the leaves compared to the control leaves. In contrast, NF-treated plants showed a fully albino phenotype caused by the blocking of carotenoid biosynthesis.

Taken together these results indicate that the two treatments do not affect WT seedlings development, but both interfere fully or in part with carotenoid biosynthesis which is in contrast to the pap7-1 mutant that exhibited a normal yellow color in the dark.

control conditions. In contrast, hypocotyls of NF-treated plants were significantly shorter (approx. 10 mm). This effect was enhanced by the presence of exogenous sucrose.

Similarly, also with Linco treatment seedlings had a shorter hypocotyl length than WT and the effect was more pronounced in the presence of sucrose.

It has been shown before that an exogenous carbon source delays germination rate [START_REF] Xu | Effects of Sucrose on Germination and Seedling Development of Brassica Napus[END_REF] and inhibits seedling development (Jang, 1997;John, 2003), which explains the inhibition of hypocotyls length in our conditions. Several studies have shown that sucrose supplementation can promote root growth but also can inhibit the elongation of hypocotyl by negative feedback from sucrose signaling. Here, we show that this interaction is enhanced by the block of plastid biogenesis confirming an interaction with retrograde signaling.

In sum, the phenotypes induced by NF and Linco treatments were found to be reproducible in our hands and hence respective conditions were used for the subsequent western-immunoanalyses. Hypocotyl length (cm) 0 Sucrose 1 % sucrose ribosomal subunit. The second proteinwas RPL2, a protein of the large subunit of the plastid ribosome. In both treatments, we observed a complete inhibition of RPL2. In contrast, for RPS7 trace amounts remained detetable after NF treatment suggesting that the direct inhibition of plastid translation by Linco is more effective than the indirect photooxidative destruction after NF treatment. We also tested two NEP-dependent gene products, RPOB as subunit of the PEP complex and ACCD, a subunit of the ACC complex. Interestingly ACCD accumulated to almost WT-levels in NF and Linco-extracts while RPOB was not detectable in both treatments. This suggests that maybe translation of selected proteins might be possible even after inhibitor treatment. This interesting finding will require further analyses.

Nuclear encoded protein accumulation

NF and Linco completely repressed the nuclear-encoded photosynthesis proteins (LHCB1;

LHCB3; STN7). LHCA2, however, accumulated weakly in the NF extract. Even more accumulated PSBO that was found in both extracts. These results suggest that nuclearencoded proteins can be imported into the plastids of NF and Linco treated plants and that they can even accumulate to significant amounts suggesting that plastids in these treated plants are functional to some degree (Fig. 7,A). RPS1; RPS5; RPL4 are nuclear encoded proteins of plastid ribosomes. Their accumulation was strongly inhibited in both treatments, but trace amounts appeared to be detectable. In contrast, FTSZ1 was present in all extracts in a nearly comparable manner (Fig. 7,B). This may suggest that the inhibitor treatments induce differential effects within the plastid or that plastid protein complexes are maybe differentially susceptible to degradation induced by the inhibitors. It anyway provides further indication that some protein complexes and activities might be retained in the albino plastids of the treated plants.

those from plastid tetrapyrrole biosynthesis (TPB) and plastid gene expression (PGE) that can be found is almost all reviews, therefore, is largely artificial. Since also the target genes such as Lhcb genes are identical in all cases one can postulate the working hypothesis that they all act through a common mechanism, e.g. through the action of PAP7 and the other dually localized PAPs. Since RPOB (Fig. 6) is not detectable in both NF and Linco treated plants one can assume that the PEP complex is inactive as in pap7-1 mutants and thus mimic the phenotype of the mutant. Smaller molecular differences are likely due to the chemical effects of the inhibitors and their side effects.

General conclusion

The development of functional chloroplasts starts from undifferentiated proplastids. This process is complex and depends (among others) on the proper functioning and coordination of the plastid-encoded polymerase (PEP) and the nuclear encoded polymerase (NEP) that both are required for the expression of the plastid genome. The PEP complex consists of four plastid encoded core subunits that are surrounded by 12 PEP-associated proteins (PAPs)

encoded by the nucleus and imported from the cytosol. Accumulation of PAPs in plastids is light-dependent and occurs in parallel with chloroplast biogenesis [START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF].

Interestingly, the genetic inactivation of each one of these PAPs blocks chloroplast differentiation by disrupting plastid PSII gene expression resulting in an albino phenotype of homozygous mutant plants.

Using the Arabidopsis pap7-1 albino mutant as a model for arrested plastid development this study provides new information on molecular processes initiating chloroplast biogenesis. It investigates at a systems level the protein accumulation in the mutant background and characterizes the double localization of the PAP7 protein and its functional implications in plastid and nucleus that eventually lead to the albino phenotype of the mutant.

First, the total protein accumulation profile of the mutant has been analyzed in comparison to dark-and light-grown wild-type plants. The specific effects of the mutation on the protein accumulation then were aligned to the corresponding changes observed at the transcriptomic level obtained in earlier studies [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF] providing a detailed view on the gene expression processes in the mutant background. In general, the protein accumulation followed the observed gene expression changes, however, a number of gene-specific and compartmentspecific features could be observed. For instance accumulation of plastid-encoded photosynthesis proteins showed a strong down-regulation while the effect at the transcript level was less prominent suggesting predominant translational activation of them upon illumination. In contrast, cytosolic translation of photosynthesis genes remained functional and the lumen protein PsbO even accumulated to normal level within the albino plastids indicating that the import machinery is functional in the albino plastids. Lhcb proteins, however, did not accumulate and only traces of precursor proteins were detectable suggesting that thylakoid membrane proteins are likely not imported and, thus, degraded pointing to potential selectivity of the import machinery. Subunits of plastid ribosomes showed downregulation indicating that plastid translation is at least partially compromised in the albino plastids. However, some plastid proteins such as YCF1 were found to accumulate normally indicating that selected proteins may be translated without restriction pointing to a potential developmentally determined translation preference. In sum, neither plastid transcription nor plastid translation is completely inactive in the albino plastids. Rather it appears that both processes are developmentally arrested in a restricted functional state that likely corresponds to a state before the PEP is decorated with PAPs.

The protein data together with pigment determinations indicated that the tetrapyrrole biosynthesis pathway remained functional in the mutant, but the chlorophyll biosynthesis branch was specifically blocked. This branch is completely encoded in the nucleus and under the control of the nuclear GLK transcription factors. Recent transcriptomic analyses have shown that GLK1 is down-regulated in the pap7-1 mutant [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF] providing a potential explanation for the lack of chlorophyll. Essential carotenoids were also lacking in the mutant leading to a down-regulation of xanthoxin, the precursor for the important hormone abscisic acid. Neither genes nor proteins revealed a down-regulation suggesting that the pathway, in principle, should be functional. However, one initial precursor (glyceraldehyde-3-phosphate) cannot be produced due to the lack of photosynthesis suggesting that the biosynthetic pathway is likely not functional because of substrate lack. In contrast, transcripts and proteins of the plastid-localized and nuclear-encoded allene oxide synthesis pathway were all found to be down-regulated. Since the albino plastid does not build thylakoid membranes the starting substrate MGDG is not available. This is likely caused by the down-regulation of transcripts and protein for the plastid-encoded acetylCoA carboxylase subunit D (ACCD). It can be assumed that the albino plastids are uncapable to produce OPDA, the precursor of jasmonic acid, another important hormone in early plant development. It appears that the albino plastids are highly defective in pigment and metabolite syntheses caused by repression of selected genes in plastid AND nuclear genome suggesting strongly the involvement (or the missing) of a retrograde signaling compound.

Based on in silico analyses, PAP7 contains a predicted chloroplast transit peptide (cTP) and a nuclear localization site (NLS, [START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]. Here, in vivo analyses confirmed these predictions. PAP7 is a bi-localized protein with the ability to locate in parallel in the nucleus and plastids. PAP7/pTAC14 can be first imported in the chloroplast and then return to the nucleus as already shown for pTAC12 [START_REF] Pfalz | ZmpTAC12 binds singlestranded nucleic acids and is essential for accumulation of the plastid-encoded polymerase complex in maize[END_REF]. Functional complementation of the pap7-1 mutant in heterozygous Arabidopsis pap7-1/+ plants by the genetic construction pPAP8::PAP7 was performed. This construction could rescue the pap7-1 mutant and the transgenic plants could develop a wild-type phenotype in the mutant background. These results are significant not only because of the identification of the PAP7 open reading frame being sufficient to fully complement the pap7-1 mutant, but also because these data confirm the importance of PAP7 in chloroplast biogenesis during the early developmental stages.

Furthermore, the results confirmed in vivo that the PAP7 can be expressed under the control of the PAP8 promoter. Therefore it does prove the co-expression between PAPs (Liebers et al., 2017). The hemi-complementation tests showed that a separation between nuclear and plastid functions seems to be difficult. Without one of the two predicted plastidial or nuclear sites, pap7-1 mutants cannot restore the wild-type phenotype. The two functional nuclear and plastidial versions, thus, are both essential and complementary for chloroplast biogenesis.

While the consequences of a lack of PAP7 in the plastid are well understood through its compromising effect on PEP activity, the corresponding consequences in the nucleus are unknown. To shed light on this question pap7-1 was crossed into a line with GFP-labelled phytochrome B (PBG). The resulting plants revealed that the lack of PAP7 deteriorates the formation of nuclear late photobodies. Therefore, the nuclear fraction of PAP7 protein can offer a new (retrograde) regulator candidate for the light signaling by activated phytochrome B. This can probably regulate the expression of GLK genes. PAP7 interacts in the nucleus with two other regulators of the PhyB mediated light response; PAP5 and PAP8 [START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF]Liebers et al., 2019, unpublished). In turn, phytochromes can be involved in the control of jasmonic acid biosynthesis and the different developmental aspects such as hypocotyl length.

This study shows that PAP7 is essential for the build-up of photosynthesis apparatus as a key step for chloroplast biogenesis which is responsible for the biosynthesis of almost all molecules necessary for plant function (carotenoid, chlorophyll, membrane lipids). The results suggest that in the absence of PAP7 plastids accumulate less mRNA and less corresponding proteins, but also less pigments or hormones. This leads to an arrest of the plastid development in the absence of an exogenous carbon source with a final albino appearance. In future, a detailed study of the interactions between hormones and light signals during chloroplast differentiation seems to be a very important area of scientific investigations. In addition, most of the proteins present in the chloroplast are encoded by nuclear genes. Therefore, it will be important to understand the selectivities observed for the plastid import and translation machineries.

In conclusion, our study shows that PAP7 represents a new regulator of chloroplast biogenesis and its implementation into photomorphogenesis (Fig. 1).

In the chloroplast, PAP7 is required for the establishment of a functional PEP complex. This complex is mainly responsible for the proper expression of plastidial PSII and tRNA genes providing essential components for the build-up of the photosynthesis apparatus and for plastid translation. PAP7 exhibits a dual localization and performs a second function in the nucleus. The data strongly suggest that the nuclear fraction of PAP7 interacts with PAP5 and PAP8 to activate the expression of GLK1 through the establishment of a correct light signal transduction from activated PHYB. GLK1, then, most likely activates the transcription of a number of nuclear-encoded photosynthesis genes (e.g. LHCBs), tetrapyrrole biosynthesis genes and, possibly, allenoxide biosynthesis genes. Corresponding proteins are imported into the plastid where they contribute to the establishment of the photosynthesis apparatus and protein complexes of connected functional processes such as chlorophyll biosynthesis.

Carotenoid biosynthesis, however, appears to be not a direct target of PAP7, but depends on precursors provided by functional photosynthesis. Another indirect target appears to be the synthesis of lipids for the thylakoid membrane through the action of the plastid-localized ACCD subunit that is part of the plastid fatty acid synthesis complex. Nuclear PAP7 might be also involved in the expression of the key enzymes of the plastid-localized allene oxide synthesis pathway that leads to the production of the jasmonic acid (JA) precursor OPDA starting from thylakoid lipids. JA is known to interact with phytochrome-mediated photomorphogenesis. OPDA, thus, likely represents an additional yet unrecognized retrograde signal that links proper chloroplast biogenesis to phytochrome-mediated plant development and hypocotyl elongation. In sum, PAP7 most likely acts, together with other dually localized PAPs, as integrator of light-induced gene expression events in plastid and nucleus which are required to provide the essential components for chloroplast biogenesis.

Conclusion générale

Le développement de chloroplastes fonctionnels commence à partir de proplastes [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF], ce qui pourrait expliquer le manque de chlorophylle.

L'absence des caroténoïdes essentiels dans le mutant a entraîné, également, une régulation à la baisse de la xanthoxine (le précurseur de l'importante hormone acide abscissique). Cela permet de proposer que la voie de synthèse des caroténoïdes doit être fonctionnelle.

Néanmoins, en absence de photosynthèse, le précurseur initial (glycéraldéhyde-3-phosphate) ne peut pas être produit. Cela conduit à l'inhibition de la voie de biosynthèse des caroténoïdes.

Comme les plastes albinos ne forment pas les membranes de thylakoïdes, les lipides membranaires tels que les MGDG ne sont pas synthétisés. Ceci est probablement causé par la régulation à la baisse de l'expression des transcrits et des protéines de la sous-unité D de l'acétyl-CoA carboxylase (ACCD). De plus, les transcrits et les protéines de la voie de la synthèse de l'allène oxyde synthase (AOS) ont montré un profil de la régulation négatif.

On peut supposer que les plastes albinos sont incapables de produire l'OPDA qui est le précurseur de l'acide jasmonique (une autre hormone importante dans le développement précoce des plantes). Ici il semble que les plastes albinos sont très défectueux dans les synthèses des pigments et des métabolites, suite à la répression de gènes sélectionnés dans le génome plastidiale et nucléaire. Ces données suggèrent fortement l'implication (ou l'absence)

de la signalisation rétrograde.

Basé sur les analyses in silico, PAP7 contient un peptide de transit chloroplastique (cTP)

prédit et un site de la localisation nucléaire (NLS, [START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]. Ici, les analyses in vivo ont confirmé ces prédictions. PAP7 est une protéine bi-localisée capable de se localiser en parallèle dans le noyau et les plastes. D'abord, PAP7 peut être importé dans le chloroplaste puis elle peut retourner dans le noyau, comme indiqué précédemment pour la protéine pTAC12 [START_REF] Pfalz | ZmpTAC12 binds singlestranded nucleic acids and is essential for accumulation of the plastid-encoded polymerase complex in maize[END_REF]. La complémentation fonctionnelle du mutant pap7-1 a été réalisée chez les plantes hétérozygotes pap7-1/+, par la construction génétique pPAP8::PAP7.

Perspectives

The results of this thesis provided several answers to the questions that were asked at the beginning of the project. However, the conclusions from these data also suggested new questions that will require further analysis. In perspective, this work opens several new scientific avenues that are detailed below.

1) The pap7-1 protein profile. Indeed, the protein profile study of the pap7-1 mutant showed many gaps in relation to the complete understanding of the distribution of certain proteins within the albino plastid as well as the rest of the cell. For example, the analysis showed that the plastid import machinery is still functional in the pap7-1 mutant since the nuclear encoded PSBO subunit of the watersplitting apparatus is accumulating to normal levels. However, a couple of other nuclear encoded photosynthesis proteins were not imported into the plastid, notably thylakoid membrane proteins. Interestingly, the analysis by western immuno-blotting showed that the nuclear encoded LHCB1 and LHCB3 precursor proteins accumulated in the cytosol while the corresponding matured proteins were completely absent in the albino plastids. At the same time the corresponding transcript accumulation was strongly repressed suggesting that the defect in LHCB import may have a negative feedback on the Lhcb gene expression. It is tempting to speculate that this might be part of a cytosolic unfolded protein response (UPR) that could act as potential retrograde signal towards the nucleus. It would be interesting to see whether over-expression of LHCB constructs with non-functional transit sequences do induce also a Lhcb repression. Furthermore, it will be important to understand the specificity of the import machinery in the albino plastid. It will be important to verify how the TOC-TIC machinery exerts a selective import into the mutant plastid of these protein groups or, as another possibility, whether these proteins were imported but then quickly degraded in the plastid. For this one could perform additional analyses using a genetic cloning strategy in which a translational fusion of LHCB with GFP is transformed into Arabidopsis plants in order to follow the trafficking of LHCB and the mechanism of their import/ accumulation in the plastid.

2) Connections to the jasmonic acid biosynthesis pathway

Additionally, the proteomic data of the pap7-1 albino mutant showed a significant downregulation of the nuclear-encoded plastid-localized enzymes lypoxygenase, allene oxide synthase and allene oxide cyclase (LOX, AOS and AOC) that represent the key enzymes of the jasmonic acid precursor biosynthesis (OPDA).

It was shown that jasmonic acid biosynthesis is induced under the control of phytochromes.

Activation of phytochrome by low red light may promote the induction of jasmonic acid biosynthesis and its interaction with the hypocotyls growth of the mutant. A detailed study of the OPDA synthesis can be a point to understand the hormonal activity in the pap7-1 mutant.

Furthermore, using an exogenous supplemented methyl jasmonate (MeJA) treatment could be another important experiment to be performed. Ideally, the addition of MeJA can improve a couple of phenotypic characteristics in pap7-1 mutant such as inhibition of hypocotyl and root elongation. Functional interaction between these phenotypic characteristics and phytochromes can be a way to study whether PAP7 can control the interactions between OPDA, jasmonic acid biosynthesis and late nuclear photobodies accumulation in pap7-1 mutant.

3) Molecular studies of PAP7 functions in the nucleus

In this work it has been confirmed by BiFC tests, that PAP7 can interact with a presumptive nuclear protein complex (including PAP8 and PAP5) that likely mediates light signaling by activated phytochrome B. A confocal observation seems to be a way in order to get a good detection of photobodies localization.

To further confirm this interesting discovery, additional experiments using PHYB-PAP7, seems to be interesting. In this context, it can be proposed to quantify the expression of some nuclear transcription factors such as GLK1, GLK2, PIF1, PIF3, HY5, PSY, HMR/PAP5 that may be crucial for the initialization of photomorphogenic responses.

In addition, the hemi-complementation test have shown the importance of both plastidial and nuclear PAP7 for full complementation of the pap7-1 mutant. However, silencing of the constructs due to position effects cannot be fully ruled out at the current stage of investigation and require further investigation. To further study this, it will be necessary to clone the two different constructs (PAP7 ΔcTP and PAP7 NLSm4 ) in fusion with GFP in order to monitor PAP7 trafficking and to prove if the GFP disrupts the expression of PAP7.

Another highly appreciated point that can be approached experimentally is to define the function of PAP7. A genetic strategy involving the deletion of some conserved amino acid in the SET domain, like the highly conserved trypsin, can be a tool to confirm the methyltransferase activity of PAP7 and to define the precise function of this protein.

All these combined data will allow understanding the role of PAP7 in the nucleus and in chloroplast biogenesis. In addition, these data will present a real scientific tool in order to set up PAP7 as the first protein of the PEP-PAP complex covering a detailed genetic, transcriptomic and proteomic study.

I Biological materials 1 Plant materials

Arabidopsis thaliana ecotype Columbia (Col-0) and the mutant line pap7-1/pTAC14-1 (SAIL_566_F_06) were used in this study.

Surface sterilization of Arabidopsis seeds

Seeds were surface-sterilized in a sterilization solution (1 ml of solution for a volume equivalent to 100 µl of seeds). The sterilization solution was prepared by dissolving 1.5 g of chlorine INOV'CHLORE (INOV'CHEM) in 40 ml of sterile distilled water, and then absolute ethanol (50 % v/v) was added to dilute the solution. After gentle shaking for 5 to 8 minutes, the seeds were washed three times with absolute ethanol and then air-dried in a laminar flow hood overnight.

Surface sterilization of Arabidopsis seeds for in vitro culture

Seeds were surface sterilized in 1 ml of 70 % ethanol, 5 min in a sterilization solution (30 % bleach and 0.02 % Triton X100), washed three times with 1 ml of sterile water with shaking in order to suspend the seeds.

In vitro culture of Arabidopsis

Sterilized seeds were grown on MS plates (1x Murashige and Skoog; MS). One liter of agar MS medium contained 4.31 g of MS base salt mixture (SIGMA powder); 5 g of sucrose; 0.5 g of 2-N-Morpholino-ethanesulfonic acid (MES); 5 g of plant agar granulated and eventually charcoal (0.2 g for 250 ml MS). Then, the pH (HANNA instruments pH 213 Microprocessor pH Meter) was adjusted to 5.7 using 1M KOH. The plates were stratified for 2 days at 4°C.

Then, the plates were transferred to a growth chamber. The growth of wild-type light (WTL)

and the mutant pap7-1 was performed directly in long-day conditions (16 h light /8 h darkness 130 to150 μE of illumination), at 23°C for 5-7 days. The seeds of wildtype dark (WTD) were first exposed during 6 hours to light to enhance germination. Then, the plates were placed under continuous darkness for 5 to 7 days. WTD seedlings were harvested under green light to avoid de-etiolation. For chemical treatment with inhibitors such as Norflurazon (NF), Lincomycin (Linco), mevalonic acid (MEV), and fosmidomycin (FSM), the necessary volume was added directly to the MS medium to reach the indicated final concentration (Chapter 2 and 4). The WT and pap7-1/+ heterozygous seeds were grown on MS medium containing different inhibitors, and then the dishes were stratified for 2 days at 4°C. The dishes were exposed for 6 hours to the light at 23°C followed by three days in the dark and de-etiolated for three days in the light before harvesting. The used concentrations for different treatments such as NF [START_REF] Cottage | The Arabidopsis plastid-signalling mutant gun1 (genomes uncoupled1) shows altered sensitivity to sucrose and abscisic acid and alterations in early seedling development[END_REF][START_REF] Kim | 1O2-mediated and EXECUTER-dependent retrograde plastid-to-nucleus signaling in norflurazon-treated seedlings of Arabidopsis thaliana[END_REF][START_REF] Sagar | Light Effects on Several Chloroplast Components in Norflurazon-Treated Pea Seedlings 1[END_REF], Linco (Cottage et al., 2010), FSM andMEV (Rodríguez-Concepción et al., 2004), were taken from literature.

In vivo culture of Arabidopsis

Plants already grown on MS sucrose (1 %) medium were transplanted, or seeds were directly sown on the soil (Nutrilor or Hydro) mixed with vermiculite (4:1). The pots were then placed in the culture room and covered with a plastic film, during the first week, to maintain a humid atmosphere. The plants were cultivated under long day conditions (16 hours of light and 8 hours of darkness) with a light intensity of 80 µE and a constant temperature of 23°C in standard conditions. The plants were watered twice a week.

Harvesting and storage of seeds

Plants cultivated in vitro were harvested after 5 to 7 days of growth for subsequent extractions (depending on the presence or absence of chemical treatment). The plants were harvested using a pincer under a magnifying glass and the samples were placed in a 50 ml falcon tube, pre-cooled in liquid nitrogen (N2). Then the tubes were stored at -80°C. For plants cultivated in soil, after 4-5 weeks of growth in soil the plants were ready to be harvested. The harvested seeds were stored at 16°C until use.

Seed germination assay

Seeds of the genotypes used were placed in small Petri dishes (diameter 5 cm) containing one to two Whatman papers. Seeds were imbibed with 1 ml of sterile water and then the Petri dishes were exposed to light at 23°C for one week. This method facilitates the counting of germinated seeds and determined their germination rate (Zhong et al., 2016).

Bacterial strains

Escherichia coli DH5α strain was used for bacterial transformation, plasmid multiplication, and Agrobacterium tumefaciens (strain GV3101/pMP90) was used for plant transformation.

These two bacterial strains were grown on Luria Bertani (LB, 10 g/L of Bactotryptone, 10 g/L NaCl and 5 g/L of yeast extract),) or LB agar using appropriate antibiotics (carbenicillin (100 mg/ml), spectinomycin (100 mg/ml) for selection of resistance conferred by plasmid integration. Then, E. coli and Agrobacterium tumefaciens were grown at 37°C or 28°C, respectively.

II Molecular biology

PAP7 has a predicted nuclear localization signal (NLS) and a predicted chloroplastic transit peptide (cTP). Therefore, PAP7 is considered as bi-localized protein. In this study, a molecular approach was used in order to enhance the understanding of the role of PAP7 in chloroplast biogenesis. A strategy was developed to uncouple the localization of PAP7 so that only one protein version could accumulate either in nucleus or plastid testing its functional importance by hemi-complementation.

Plasmid vectors

The following table represents the plasmids used in this study: Contains a CAMV35S promoter and a PAP10 fused to GFP (35S:: PAP10-GFP). This plasmid was used as an expression vector for the BiFC tests. pBB301a

Contains the CAMV35S promoter and PAP10 fused to DsRed (35S:: PAP10-DsRed). This plasmid was used as an expression vector for the BiFC and biolistic tests. pRB1637

Contains the CAMV35S promoter and a PHYB gene fused to GFP at the C-terminus (35S::PHYB-GFP). This plasmid was used to clone the PHYB-GFP in pap7-1/+ heterozygous plants.

DNA amplification by the polymerase chain reaction "PCR"

Before cloning the gene of interest, the DNA fragment has been modified by site directed mutagenesis. The recombinant plasmid pAG09a was used as the template, containing the coding sequence of PAP7orf merged with the GFPorf. The primers used to introduce the mutation were shown in Table 2. into the PAP7 NLS . For that, two PCR reactions were performed. The first one was carried out in three steps using specific primers containing the desired mutation. The pair of primers opap7_Fxho/op7m3R amplified the fragment "A 682 bp "; the primers op7m3F/op7m4R amplified the fragment "B 99 bp " and finally the fragment "C 747 bp " was amplified by the primers op7m4F/opap7_Rxba. Subsequently, the two mutagenesis reactions were performed and fused. The three PCR products, generating overlapping gene segments, were used as a matrix DNA for the second PCR, and amplified by the distal primers opap7_Fxho (1) / opap7_Rxba in order to obtain the complete amplify PAP7 NLS mutant fragment (PAP7 NLSm4 ) of 1528 bp. The open reading frames of the PAP7 (pAG09a) and for PHYB::GFP (pRB1637) constructs were amplified from cDNA. For the generation of PAP7 NLSm4 , the open reading frame of the PAP7 was amplified by a PCR directed mutagenesis in order to insert four amino acids in the predicted bi-partite NLS. The deletion version of PAP7 was carried out by PCR using opap7_DcFXho/opap7_Rxbal to remove the cTP gene (ΔcTP).

Each PCR reaction was made in a final volume of 31 μl. The PCR ensured to obtain sufficient amounts of DNA. The tubes were then placed in the thermocycler for 35 cycles of PCR amplification. The used program is resumed in the Table 4.

Agarose gel electrophoresis

In order to add a single-base extension (adenosine) to the 3' extremity, the PCR was followed by a quick A-tailing using the Taq polymerase for 5 min at 75°C (Promega). Then, PCR products were analyzed after separation by agarose gel electrophoresis. The agarose concentration varies according to the DNA fragment size (Sambrook et al., 1989). Agarose gel was prepared in TAE buffer (40 mM Tris-Acetate, 1 mM EDTA; pH 8.0). The SYBR ® Safe stain was added to visualize the DNA fragments on the gel. DNA samples were loaded onto an agarose gel and separated at 120 V in TAE buffer for 30 min. The size of the fragments was determined according to the size marker (1kb Plus, Invitrogen TM ).

Purification of DNA fragments from Agarose gel

The gel band containing the interesting fragment was cut out from the gel under UV light.

The existing impurities in the nucleic acids (salt, protein, agarose…) were removed after successive washing, using the GeneClean ® III Kit (according to the manufacturer's instructions). The pure DNA fragment was deposited on an agarose gel in order to check the precise size of the fragment and the quality of purification.

Cloning of PCR products into pGEM ® -T Easy vector

PCR fragments, with A-Tailing in the 3' end, were cloned into pGEM®-T Easy vector (Promega). The ligation was carried out in a total volume of 12 µL at 4°C for overnight. The reaction mixture is constituted: 1μl pGEM®-T Easy; 3 μl PCR product (molecular ratio vector:insert from 1 to 3 to facilitate vector-insert contact); 1.2 μl buffer ligase 10X; 1.2 µl T4 ligase and water (Smith, 2007). The insert, which already has been cloned into pGEM®-T Easy vector, has been digested with restriction enzymes and then cloned into:

 An expression vector comprising the GFP or RFP gene under control of a CAMV35S

promoter. It allows for observing directly the transformed seeds (T1 or T2) and the transformed cells under a magnifying glass or light microscope.

 A binary vector pBB389 (pPAP8::PAP8) which allows the expression of the gene of interest.

The PAPs are likely controlled by a common regulator and display similar expression patterns [START_REF] Liebers | PAP genes are tissue-and cell-specific markers of chloroplast development[END_REF]. The PAP7 and PAP8 promotors are likely interchangeable. Therefore, the PAP8 promoter has been used in the following cloning reactions in addition to some technical reasons. This vector has been cloned by Dr. Robert Blanvillain. It has the general characteristics of transformation plasmids such as the right (RB) and the left (LB) borders, the hygromycin phosphotransferase resistance gene (hptII) for the selection in plants and the spectinomycin resistance gene for E.coli or Agrobacterium tumefacian selection.

Bacterial transformation by heat shock

The E. coli strain DH5α cells was thawed on ice in presence of 8 μl of ligation product for 5 minutes. Then, a heat shock was performed at 42°C for 45 s and cells were immediately incubated on ice for 2 min before the addition of 180 µl of SOC medium. Cells were grown for 20 min at 37°C. After this time, cells were spread on LB plates using the appropriate antibiotics for selection of resistance (carbenicillin (0.2 mg/ml) and in the presence of X-Gal (20 mg/mL) and IPTG (0.2 g/mL) to allow the blue-white screening. The plates were incubated at 37°C overnight. In the next day, only white colonies were checked by colony PCR.

Analysis of recombinant bacteria and plasmid DNA mini-prep isolation

Positive clones were then cultivated in 6 ml of liquid LB medium with antibiotics at 37°C overnight with agitation (180 rpm). On the next day, bacteria cultures were harvested and were centrifuged at 4000 G for 10 min. Furthermore, plasmid DNA was extracted from bacteria culture using QIAprep Spin Miniprep kit according to the supplier's instructions [START_REF] Stephen | A rapid method for isolating high quality plasmid DNA suitable for DNA sequencing[END_REF]Simoen et al., 2003).

Nucleic acid quantification

The nucleic acid concentration assay was performed by spectrophotometric methods (Thermo Scientific™ NanoDrop™) at 260 nm and their quality was controlled on agarose gel.

9 Plasmid DNA sequencing 1 μg of DNA was prepared in a 7 μl of final volume. Sequencing of plasmids containing the coding sequences was done by "Eurofins industry" (https://www.eurofins.fr/).

The DNA was amplified by PCR and the used primers are located in the two sides of the cloning cassette. There are universal primers located in T3 and SP6 promoters. The check of sequence compatibility was done by ApE and SnapGene ® Viewer 4.1.4 software.

Digestion of DNA by restriction enzymes

The gene of interest (from the recombinant plasmid) was cloned into a binary vector between two restriction sites (Xho, XbaI and BamH1). The reaction mixture consisted of 1 µg of DNA, 1 unit per μg of restriction enzymes, CutSmart ® buffer 10X and adjusted to a final volume of 40 µl with sterile water. Then the digestion was performed at 37°C during two hours. The reaction mixture is loaded on agarose gel (1% TAE buffer). The fragment of interest was cut out of the gel and then purified. After purification, the ligation was carried out in the presence of T4 DNA ligase. Finally, a bacterial transformation was performed using E. coli.

11 Transformation of Agrobacterium tumefaciens using electroporation 100 ng of DNA was added to 50 μl of electro-competent A. tumefaciens bacteria. The culture was incubated for 5 min on ice. Then, an electroporation (Bio-Rad Gene Pulser Electroporation System) was performed at 25 kV/cm. The penetration of DNA in electrocompetent bacteria is achieved by the weakening of the bacterial wall which increases their permeability (creation of pores) following the application of the electric shock.

400 μl of SOC nutritive medium was immediately added to the bacteria. The culture was then placed at 28°C for 3 h in order to initiate the expression of the selection gene. The bacteria were spread on LB agar medium containing the antibiotics rifampicin (50 mg/l), gentamicin (20 mg/l) and the transgene selection antibiotic (spectinomycin 50 mg/l). The culture was incubated at 28°C for 2 days.

Transformation of Arabidopsis using the floral dip method

The floral dip method is the simplest method used for the production of transgenic Arabidopsis plants transformed by A. tumefaciens [START_REF] Zhang | Blue-white screening liquid can eliminate false positives in blue-white colony screening[END_REF]. The method is taken from an adapted protocol (Bechtold et al., 2000). Plant transformation was carried out when the plants initiated the flowering stalk. Several colonies were cultured in 5 ml of liquid LB medium, containing antibiotic for selection (gentamycin 20 mg/l, rifampicin 50 mg/l, spectinomycin 50 mg/l), and then incubated at 28°C with agitation at 225 rpm overnight. On the next day, the cultivated bacteria were used to prepare 300 ml of total culture. When the DO600nm exceeded 1, the cells were harvested by centrifugation at 6000 rpm for 20 min. Then, the bacteria were suspended in 5 % sucrose solution containing 2.2 g MS, 300 μl Silwet L-77, 20 µ l BAP, 1 ml of Gamborg B5 vitamins enables the genes to be transferred. The suspension was diluted in 200ml of this medium until obtaining a DO600nm = 2.4. The transformation is carried out by putting the plants in the Agrobacterium solution for 10 to 15 minutes. After transformation, the plants were placed in the culture room. After about 3 months, we obtained transformed transgenic lines containing the gene of interest.

% pre-cooled ethanol. Then, the pellet was dried and suspended in 50 μl of sterile distilled water. The DNA was stored at -20°C.

Screening of T-DNA insertion mutants by PCR

Two to three µl of the DNA were used to perform a PCR screening. The Arabidopsis T-DNA insertion mutants contain a T-DNA with a kanamycin resistance gene (http://signal.salk.edu/tdna_protocols.html). We have already verified that pap7-1 mutant (At4g20130 / SAIL 566_F06) is a homozygous pap7-1/pap7-1 by PCR. Analysis showed that pap7-1 carried one T-DNA inserted in exon 6. Transformed plants were selected on an agar medium supplemented with antibiotic. The screening of mutants was also carried out by PCR (40 cycles) using three primers; it amplified specifically the wild-type allele or the mutant allele of the gene of interest and the transgene allele. Oligonucleotides were designated by Robert Blanvillain (Table 2). Genotyping of pap7-1 and complementation lines was performed using the same primers used in cloning. The PCR products were separated on 1% agarose gel (Chapter 3).

Transient transformation of onion epidermis: biolistic test

Genetic transformation of plant cells by bombardment (here micro-projectile-mediated DNA transfer to onion cells) produces stably transformed transgenic plant cells [START_REF] Hamada | An in planta biolistic method for stable wheat transformation[END_REF]. Micro-projectile bombardment involves the precipitation of DNA on gold microparticles that were used to generate transgenic genes [START_REF] Ismagul | A biolistic method for high-throughput production of transgenic wheat plants with single gene insertions[END_REF]. The biolistic canon system (Bio-Rad PDS-1000/He) was used as a gene delivery system. 30 µl of gold micro-particles (Seashell technology; 0.6 μm; 30 mg/ml) were combined with 20 μl of plasmid DNA and 20 μl of binding buffer. The mixture was suspended in 70 µl of precipitation buffer (Seashell technology TM ). After agitation, the coated DNA is sedimented by incubation for 5 minutes. The supernatant was removed and the granulated gold particles were washed with 500 μl of absolute ethanol already cooled and then taken up in 30μl of ethanol. An onion piece was placed in the PDS1000 chamber. An aliquot (ten microliter) of gold particles was deposited on the "Macrocarrier" (a plastic cylinder) for bombardment. This device is alimented by a helium gas that accelerates the macrocarrier, on which gold particles coated with DNA have been dried (Kikkert, 1983). Gold particles were delivered to the onion cells using this particle gun (BioRad). The bombarded onions were placed in the dark for 16

h. The next day, the transformed cells were allowed to express the construct before fluorescence microscopy was done.

Total proteins extraction

This protocol was developed in the laboratory. The plant material was crushed in a mortar with liquid nitrogen. 100 mg of powder was added to an equal volume of 4X SDS loading dye buffer (200 mM Tris, pH8; 0.5% β-mercaptoethanol; 20 % glycerol; 4 % SDS; 0.2 % blue bromophenol) in order to get a soluble protein fraction and to keep the other constituents in suspension. SDS is an ionic detergent that denatures the proteins. This denaturing agent has a long hydrophobic tail with a negative charge (the sulfate groups). SDS has been used as a surfactant, covering the intrinsic charge of proteins by negative charges [START_REF] Shapiro | Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels[END_REF].

The reason why β-mercaptoethanol has been used in the buffer is to reduce the disulfide bonds within or between the proteins and allowing subsequently the protein denaturation. The proteins were denatured at 95°C for 10 min (Benchmark Digital Dry Bath Heat Block). Then, the cell membrane debris, as well as the insoluble particles, were removed by centrifugation at 4°C for 15 min at full speed. The total soluble proteins, in the supernatant, were quantified and stored at -20°C.

Membrane protein extraction

The extraction method was used to make gel electrophoresis for membrane protein extracts of Arabidopsis plants. The methods, as well as the choice of buffers, have been provided by Dr.

Silva Lerbs-Mache, LPCV Grenoble, to optimize the structural state of the membrane proteins. 600 mg of plant tissues were ground in the mortar in the presence of liquid nitrogen.

3 ml of TPC was added to the fine powder (100 mM HEPES pH 7.3; 50X protease inhibitor; 100 mM DTT; H2O). DTT denatures and precipitates proteins by preventing the formation of disulfide bridges, which created an insoluble protein complex. The protease inhibitor prevents the degradation of protein and promotes their preservation for a long time. Centrifugation for 30 min at 11200 rpm at 4°C allowed separating the insoluble and membrane proteins (pellet) from the soluble protein (supernatant). The supernatant was stored at -20°C, while the pellet was crushed again by a blue pistil in a 1.5 ml Eppendorf tube with 300 μl of TPA (0.5 M Tris pH 6.8%; 20 % SDS; 100 mM DTT; H2O). Cell debris was removed by centrifugation at 11,000 rpm for 5 min at 30°C. After a freeze in liquid nitrogen, vortex mixing was performed followed by centrifugation at 11,000 rpm at 4°C for 30 min. The supernatant was taken up in the 4X loading buffer and then denatured at 95°C for 10 min in order to perform the SDS-PAGE analysis.

Then, migration was carried out in pH 8.3 migration buffer (3.03 g Tris base; 14.4 g glycine;

1 % SDS) at 50 V for 15 minutes to concentrate the proteins and then at 100V for about 1 hour for a good separation between the protein bands (Bio-Rad Mini-Protean ® Tetra System, China).

5 Protein staining techniques for mini-gels

Protein staining with Coomassie blue

Protein staining was done by incubating the gel for a few hours in a Coomassie blue solution (ethanol 25 %; acetic acid 10 %; 0.5 % Coomassie blue R250 (Bio-Rad). First, the Coomassie blue was removed by washing with a strong discoloration buffer (40 % ethanol; 10 % acetic acid) during one hour, then several washing cycles were performed in a weak discoloration buffer (10 % ethanol; 7 % acetic acid) until optimal wash.

Protein staining with silver nitrate

The silver staining method is characterized by its high resolution, transparent background and sensitivity [START_REF] Swain | A silver stain protocol for proteins yielding high resolution and transparent background in sodium dodecyl sulfate-polyacrylamide gels[END_REF]. Each step was carried out at room temperature with agitation. The fixation of the proteins on the gel was performed by washing for 10 min with a solution composed of 40 % ethanol; 10 % acetic acid. Then the gel was rinsed with distilled water for 10 minutes. Once the proteins are fixed, the staining was sensitized by incubating the gel for 5 min in 40 % ethanol; 0.025 % glutaraldehyde and 0.01 % formaldehyde. The gel was rinsed at first with 40% of ethanol and 60% of distilled water for 20 min, and second by distilled water. The gel was incubated for one minute in a sodium thiosulfate solution (0.02 %) and then rinsed twice for one minute in distilled water. A silver nitrate solution (0.1 %) generated the proteins staining for 20 minutes and the gel was rinsed for one minute with water. Staining was developed by adding a solution of 2.5 % sodium carbonate and 0.04 % formaldehyde until solution turns yellow. The addition of 5 % acetic acid for 5 min stops the reaction. The gel was stored in distilled water.

Protein transfer from gel to nitrocellulose membrane in western blot

After the electrophoresis separation, the proteins were transferred from the gel onto a nitrocellulose membrane with a 0.2 μm of porosity (Privat et al., 2003). The transfer was made in a "sandwich" system (Bio-Rad) consisting of a sponge/3 Whatman papers/gel/ nitrocellulose membrane/3 Whatman papers / sponge. These elements were imbibed in the transfer buffer (3.03 g Tris; 14.4 g glycine; 4 ml SDS 10 %; 200 ml ethanol). The transfer was carried out at 100 V for 80 min with constant agitation in a Biorad migration system in the presence of an ice block. The transfer quality was controlled by Ponceau S staining (1 % acetic acid; 0.5 % Ponceau S) for the nitrocellulose membrane for 15 min at room temperature. This is a rapid and reversible staining method for locating protein bands on western blots. The discoloration was carried out with distilled water. Staining was completely eliminated by continuous washing with TBS-Tween solution (20 mM Tris; 150 mM NaCl; 0.1 % Tween 20).

Immunodetection

After the transfer, the membrane was blocked in a TBS-Tween saturation solution with dry milk (5%) for at least 1 hour, with agitation at room temperature, in order to block the free membrane sites (without protein fixation). Then, the membrane was washed for 5 min with TBS solution. The membrane was incubated with agitation in a TBS-Tween (0.1%); dry milk (5%) solution containing the primary polyclonal antibody (obtained from Agrisera and diluted according to the antibody) for at least one hour at room temperature. Afterwards, the membrane was washed 3 times, 10 min, with TBS-Tween to eliminate non-specific interactions. The membrane was incubated in the secondary antibody for one hour. The membrane was washed again three successive times in T-TBS and finally washed for 10 minutes in TBS alone. Proteins were revealed using the ECL western blotting detection system (Bio-Rad) for different exposure times. The ECL is a chemiluminescent substrate for the detection of the enzyme activity of horseradish peroxidase (HRP).

Detecting protein bands

In a first step, the membrane was incubated in the chemiluminescent detection reagents. Next, the membrane was placed in the Bio-Rad ChemiDoc MP with an Image Lab software system,

NanoLC-MS/MS approaches

In order to obtain a global picture of the protein accumulation in the pap7-1 mutant, a nanoLC MS approach in collaboration with the proteomics platform from team EDyP, BIG was performed. The goal of this approach was to obtain a protein profile of sufficient detail which can be compared to the existing transcriptomic data.

Experimental strategy

Total protein extracts were isolated and prepared in triplicate from six to seven day-old mutant and wild-type seedlings. Therefore, the plants were grown under the same conditions.

Protein extraction was performed from three independent Arabidopsis plant samples. For pap7-1 homozygous sample preparation, triplicates were treated as technical replicates. A technical draw-back of working with an albino mutant is the problem that no homozygous mutant seeds can be generated. For this reason, it was necessary to cultivate larger amounts of heterozygous mutants by collecting corresponding seed material and spreading enough of that on MS medium in a way that even after 3:1 segregation sufficient albino mutants could be harvested. In total, triplicates of WTL, WTD and pap7-1 light were harvested and immediately used for total protein extract preparations. Aliquots of each extracts were tested by SDS-PAGE for equal loading and quality control.

Proteins digestion

The samples were subjected to a standardized protocol of analysis typically used by EDyP.

Each protein sample (20 μg) was stacked by a 1 cm-migration on the top of a NuPAGE 4-12 % gel (Invitrogen) before Coomassie blue staining (R250, Bio-Rad). Stacking bands containing the proteins were manually excised for in-gel digestion with trypsin using a variable), and oxidation (M, variable). Minimum peptide length was set to seven amino acids.

Minimum number of peptides, razor + unique peptides and unique peptides were set to 1.

Maximum false discovery rates, calculated by employing a reverse database strategy, were set to 0.01 at peptide and protein levels. For quantification, the "Match between runs" option (with matching time set to 0.7 min) was allowed between all samples. The protein iBAQ was used for quantification (Schwanhausser et al., 2011).

Statistical processing of proteomics results

Statistical analysis was performed using ProStaR (Wiezoreck et al., 2017). At first step, proteins identified in the reverse and contaminant databases were discarded from the list.

After log2 transformation, iBAQ values were normalised by median centering over the whole experiment. Next steps were applied by only considering replicates from pairwise comparisons that is, pap7-1 vs WTD, or pap7-1 vs WTL, or WTL vs WTD. Thus, for each pairwise comparison, proteins exhibiting less than 3 quantification values in one condition were discarded. Missing value imputation was performed within each replicate using the 2.5% quantile value. Statistical testing was conducted using Limma. The Limma statistical test provided abundance fold changes (FC) with associated p-values. The final set of proteins whose abundance was significantly different between two samples were sorted out using a log2 (FC) cut-off of 1.5 and a p-value cut-off leading to a FDR <1% according to the Benjamini-Hochberg estimator.

Bioanalysis

The comparison between the differential analysis results from the proteomics study (this study, unpublished) and a recent transcriptomics study [START_REF] Grübler | Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1[END_REF] were carried out by the MapMan tool version 3.6.0RC which is free software. Only proteins that exhibited significantly differential expression in at least one comparison in either proteomics or transcriptomics study were considered. A prior organization of an excel board was necessary.

The main columns (FC.Log2) were recognized by MapMan. The p-value columns were prefixed with "d_". The statistics result columns indicated that a significant fold change comparison in the protein (or transcript) is indicated by 1, otherwise the 0 corresponds to a no significant result. This step was important to filter the significant proteins in MapMan. The "NA" (originaly present in the excel files to indicate that a protein is absent from a comparison set) were replaced by "X". Figures of different metabolic

Light microscopy

Before the fluorescence microscopy observation, transient expression was followed by a light microscope (Nikon eclipse E6000), equipped with emission and excitation filters. Also Nikon was used to observe plant growth through its image processing and analysis software.

Furthermore, a "Keyence VHX-5000" was used as a digital microscope to focus on plant details with zoom range from 20X to 1000X.

Photography tools

Photographs of the plants on agar medium were taken by a Nikon camera. In addition, the digital mobile phone camera "Wiko PULP 4" was used for their features performing in taking some photos of developed plants.

Figure 2 .

 2 Figure 2. The major steps of the endosymbiote integration into its host (Dyall et al., 2004) -Principal steps of endosymbiotic uptake, gene transfer and development of targeting signals for proper localization of proteins is indicated. The scheme is valid for plastids and mitochondria.

Figure 3 .

 3 Figure 3. Different types of plastids and their potential interconversions (Lopes-Juez and Pyke, 2005) -The figure summarizes the plastid types that can arise from the undifferentiated proplastid as well as the interconversions after a first differentiation.

Figure 4 .

 4 Figure 4. Representation of the functions of the six sigma factors in the plastid transcription machinery in Arabidopsis -This figure was taken from the Ruhr-Universität Bochum website (http://www.ruhr-uni bochum.de/pflaphy/Seiten_dt /J_Ortelt_d.html ).

Figure 7 .

 7 Figure 7. Global overview of the components of the plastid transcription machinery (gene localization, protein trafficking and transcriptional activity of all components) Pfannschmidt et al., 2015. -The three genetic compartments of a plant cell are represented (nucleus, chloroplast and mitochondria). The boxes correspond to the genes and their corresponding proteins are circled and colored with the same color as the gene. The nomenclature of genes and proteins was already used in the main text. The black arrows indicate the flow of information from genes through transcriptions and translations (white boxes). The yellow color corresponds to the plastidial sigma factors encoded by the nucleus. These plastidial transcription factors ensure specific recognition of promoters by the PEP complex (blue). Blue coloration corresponds to the NEP genes.Green coloration corresponds to the PAP genes in the nucleus. These genes probably require a master regulator (colored orange). The PAP subunits surround the PEP core enzyme. The relative positions of PAPs are determined through the literature. PAPs limited by broken red lines displays a second nuclear location besides their plastid localization. Unknown nuclear or plastid trafficking routes are represented by broken red arrows[START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]

Figure 7 .

 7 Figure 7. Principle of photosynthesis and different actors of light-dependent reaction at the thylakoid membrane in the chloroplast (Queen Mary, University of London, Jon Nield; http://macromol.sbcs.qmul.ac.uk/). Photosystem II (PSII), Cytochrome b6f complex (Cyt b6f), Photosystem I (PSI). These three complexes and the ATP synthase work together to produce ATP and NADPH.

Figure 9 .

 9 Figure 9. Visualization of the photosynthesis protein accumulation in pap7-1 light vs WT-darkEach spot corresponds to a protein (refers to log2 fold change value). Protein accumulation level is colour-coded (Red squares = up-regulation; blue squares = down-regulation); A colour code is indicated in the top left corner of the figure. MapMan software[START_REF] Thimm | MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes[END_REF]; https://mapman.gabipd.org/)

Figure 4 .

 4 Figure 4. Visualization of the photosynthesis protein accumulation in pap7-1 vs WT-light Each spot corresponds to a protein (refers to log2 fold change value). Protein accumulation level is colour-coded (Red squares = up-regulation; blue squares = down-regulation); A colour code is indicated in the top left corner of the figure. MapMan software (Thimm et al., 2004; https://mapman.gabipd.org/)

  . Box A is special as it represents the only downregulated component in response to light. It is a photosynthesis key enzyme involved in glycolysis and regeneration of ribulose in the Calvin cycle. This enzyme is down-regulated at the transcript level (FC.log2 = -0.61) and it is more repressed at the proteomic level (FC.log2 = -1.58).

Figure 9 .

 9 Figure 9. Overview of chloropast import (Jarvis, 2008) An overview of different steps of protein import into chloroplasts via the TOC/TIC import machinery (shown in yellow; center of figure) and subsequent distribution of proteins within the plastid.

  Dans ce chapitre, différentes études ont été présentées afin de mieux comprendre les étapes précoces de la différenciation des chloroplastes. Une stratégie protéomique permet d'identifier les différents groupes de régulateurs moléculaires impliqués dans la biogenèse des chloroplastes. L'inactivation de gène PAP7 entraîne un blocage génétique dans la transition étioplaste-chloroplaste ainsi que le développement des plantes pap7-1 présentant un phénotype albino à la lumière. Les mutants d'Arabidopsis pap7-1 sont affectés dans la formation des thylakoïdes. Ils présentent une activité NEP élevée et un faible niveau d'expression des gènes PEP. La stratégie d'analyse protéomique a été réalisée par une méthode de spectrométrie de masse quantitative (Orbital-LTQ) en collaboratoin avec notre partenaire EDYP. Ensuite, le profile protéique du mutant pap7-1 a été déterminé. Les résultats ont été validés par des tests d'immuno-détection. En parallèle, la corrélation entre le profil protéique et l'expression de gènes (analyse macroarray) a été menée afin de comprendre les différents aspects de régulations transcriptionnelles et post-transcriptionnelles. Le profil comparatif d'accumulation protéique a été réalisé en utilisant des plantes de type sauvage qui ont été cultivées à la lumière et à l'obscurité comme un contrôle relatif du profil d'expression du mutant pap7-1.

L

  photosynthèse doit être expliqué par diverses raisons telle que l'absence des lipides membranaire, la perturbation dans les voies de biosynthèse des pigments et des hormones.Les analyses biochimiques ont montré que la voie du tétrapyrrol est toujours fonctionnelle dans le plaste albino. La branche de biosynthèse de l'hème est également fonctionnelle tandis que la branche de biosynthèse des chlorophylles est bloquée par la forte inhibition des enzymes CHLM et POR.La biosynthèse des caroténoïdes est fortement régulée dans pap7-1. Tous les caroténoïdes détectées dans les plantules pap7-1 (lutéine et bêta-carotène) sont déplétés.Les précurseurs de la production d'isoprénoide par la voie cytosolique et plastidiale ont été analysé en utilisant des inhibiteurs chimiques. Les analyses ont montré que ces deux voies sont fonctionnelles dans le mutant pap7-1. Néanmoins, la perturbation de la biosynthèse des caroténoide est originaire d'un blocage au niveau d'une troisième voie, probablement, de la biosynthèse d'IPP. La régulation de la production des caroténoïdes peut permettre aux plantes de réaliser la photosynthèse(Rodreguez et al., 2001). Une analyse détaillée de la corrélation entre le blocage du développement des chloroplastes, la photosynthèse et la synthèse des isoprénoïdes plastidiques semble être importante.De plus, plusieurs protéines plastidiales sont à l'origine de la biosynthèse de certaines hormones comme l'acide jasmonique. Les enzymes clés de la biosynthèse de l'acide

  4,B). The DsRed and the GFP open reading frames were introduced between XhoI and BamHI restriction sites at the Cterminal end of the PAP7 gene. After transient expression in onion epidermal cells by bombardment with gold particles, a microscopic observation was performed. NLS disruptionshowed exclusively localized red fluorescence in plastids (Fig.4,C) and no signal was observed in the nucleus. However, a cTP suppression showed a dispersed green fluorescence just in the nucleus and the cytosol (Fig.4,D). The merged figure showed a red fluorescence corresponding to the plastid and a green fluorescence corresponding typically to the nucleus (Fig.4,E). These results are in line with the study done by[START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF] Fusing HMR/PAP5 gene with GFP at the N-terminal position showed that the fluorescence was located in the nucleus due to the disruption of the cTP function, whereas the fusion of PAP5

  the protein. The local alignment was performed by Elena Nicolin during her L1 training of the restricted PAP7 protein sequence on both nuclear localization site and SET domain. The amino acids between positions 281 to 325 define the SET domain that is characteristic for Lys methyltransferase proteins. The local alignment showed significant levels of identity (31 %)

Figure 4 .

 4 Figure 4. The effect of NF and Linco on Arabidopsis hypocotyl elongation Hypocotyl length was determined in six days-old Arabidopsis WT plants treated with 5 µM NF or 0.5 mM Linco. WTC indicates the control seedlings grown on MS medium without inhibitors, NF indicates norflurazon and Linco indicates lincomycin treatments

  including a camera system to document fluorescence. A CCD camera, in a darkroom with an UV trans-illumination lighting, was used for prolonged exposure time which can lead to an increase in fluorescence intensity and subsequently to the detection of small samples and accurate quantification of image data. The machine is connected to a computer connected to Image Lab ™ Software version 5.1. Once the membrane (or gel) is in place the software results an exposure time optimization for the band intensity. The separated protein bands were visualized by fluorescence of proteins in the visible range, with excitation by UV light. The colored gels were scanned with the scanners connected to the same computer.

Freedom

  EVO150 robotic platform (Tecan Traging AG, Switzerland) as follows. Gel bands manually excised were washed six times by successive incubations in NH4HCO3 25 mM and then in CH3CN 50 % v/v, NH4HCO3 25 mM. After dehydration in pure CH3CN, reduction was carried out with DTT 10 mM in NH4HCO3 25 mM (45 min at 53°C) and alkylation with iodoacetamide 55 mM in NH4HCO3 25 mM (35 min in the dark). Alkylation was stopped by the addition of DTT 10 mM in NH4HCO3 25 mM (10-min incubation). Gel pieces were then washed again in NH4HCO3 25 mM and dehydrated with pure acetonitrile. Modified trypsin (sequencing grade, Promega) in NH4HCO3 25 mM was added to the dehydrated gel pieces for incubation at 37 °C overnight. Peptides were extracted from gel pieces in three sequential embedded in MaxQuant. Trypsin/P was chosen as the enzyme and 2 missed cleavages were allowed. Precursor mass error tolerances were set respectively at 20 ppm and 4.5 ppm for first and main searches. Fragment mass error tolerance was set to 20 ppm. Peptide modifications allowed during the search were: carbamidomethylation (C, fixed), acetylation (Protein N-ter,

  pathways were generated by MapMan analysis.Also several databases were used to study the different proteins groups in detail (At_chloro http://at-chloro.prabi.fr/at_chloro/ ; NCBI https://www.ncbi.nlm.nih.gov/; ChloroKB http://chlorokb.fr/ ; tair https://www.arabidopsis.org/).IV Microscopy and imaging techniques 1 Fluorescence microscopyFluorescence microscopy was used to determine the protein localization. The strategy has been developed to test the transformation efficiency and to study the localization of green fluorescent protein in the plastid and nucleus (GFP; 477 nm in the blue light), the cytosolic and nuclear localization (NLS) of red fluorescent protein (DsRED; 558 nm), and the interaction by a split YFP (yellow fluorescence protein; 514 nm). The observation was carried out on a part of the onion epidermis where onion cells were transiently transformed. The transformed cells were deposited between slide and cover slip in the presence of a few drops of water. Then, the samples were observed under a fluorescence microscope by different light sources using excitation filters (AxioScope with AxioCam MRc5 camera; Zen software).

Table 1 . The list of different biochemical characteristic of PEP-associated proteins (PAPs)* Subunit Name AGI number Size (kDa) (Theor/Exper) Predicted protein Domain

 1

	Function

Table 1 . Summarizing table of the most detected plastidial photosynthesis proteins

 1

	Symbols	pap7-1 light vs. WTL	pap7-1 light vs. WTD	WTL vs. WTD
	PSBA	-10.63	NA	10.71
	PSBB	-10.96	NA	11.40
	PSBC	-11.32	NA	10.90
	PSBD	-11.72	NA	11.98
	PSBE	-10.24	NA	9.92
	PSBF	-9.18	NA	9.26
	PSBG	-3.62	NA	NA
	PSBH	-11.50	NA	11.57
	PSBQ-2	-6.96	3.48	10.42
	PSBL	-10.62	NA	9.43
	PSBO-1	NA	7.17	8.45
	PSBO2	NA	7.95	9.28
	PSBP	-2.19	5.02	7.21
	PSBR	-6.78	NA	6.86
	PSBS	NA	2.35	3.54
	PSB27	-4.26	NA	7.80
	LHCA2	-7.07	NA	8.11
	LHCB1.3	-5.14	6.62	11.73
	LHCB1.4	-8.59	NA	8.67
	LHCB2	-5.51	4.80	10.28
	LHCB3	-7.30	NA	9.56
	LHCB4.1	-5.44	5.13	10.56
	LHCB4.2	-6.62	4.29	10.91
	LHCB4.3	-5.49	NA	5.57
	LHCB5	-2.70	5.94	8.63
	LHCB6	-5.65	5.28	10.92
	STN7	-3.41	NA	3.49
	PETA	-9.46	-4.39	5.05
	PETB	-9.23	NA	6.74
	PETC	-3.33	1.84	5.17
	PETD	-7.72	NA	7.79
	PSAA	-10.01	NA	9.56
	PSAB	-9.30	NA	9.72
	PSAC	-8.66	NA	8.16
	PSAD	-5.47	3.61	9.08
	PSAE	-3.91	3.31	7.22
	PSAE-2	-2.87	6.41	9.25
	PSAF	-6.64	3.75	10.37
	PSAG	-7.16	NA	8.43
	PSAH-1	-6.36	2.87	9.21
	PSAK	-6.58	NA	9.15
	PSAL	-8.69	NA	5.87
	PSAN	-3.96	NA	NA
	PSAP	NA	2.27	3.56
	PSBQ-1	-3.70	NA	4.59

pap7-1 vs WT-light pap7-1 vs WT-dark WT-light vs WT-dark

					stess proteins
	Locus at5g20630	Transcripts Proteins 1.25 0.00	Transcripts Proteins -0.10 1.44	Transcripts Proteins -1.35 1.44	Protein annotation GER3
	at2g42750	-1.20	ND	1.36	Photosynthesis ND	2.56	ND	DNAJ heat shock protein
	atcg00430 at2g42540	-1.03 -1.24	-3.62 ND	-0.38 0.91	ND ND	0.65 2.14	2.78 ND	PSBG COR15A
	at1g76100 at3g25760	-1.04 -1.63	ND ND	1.21 -0.21	ND ND	2.25 1.42	ND ND	PETE1 AOC1
	at3g08940 at3g02730	-1.06 -1.12	-6.62 -1.44	0.60 1.64	4.29 2.55	1.65 2.77	10.91 3.99	LHCB4.2 TRXF1
	at4g02770 at1g52400	-1.12 -1.37	-5.47 -1.13	0.83 1.82	3.61 1.69	1.95 3.19	9.08 2.82	PSAD-1 BGLU18
	at5g38420	-1.37	-7.36	-0.62	-4.20 Ribosomal proteins	0.75	3.17	RBCS-2B
	atcg00020 atcg00330	-1.41 -1.04	-10.63 -6.87	-1.37 -0.59	ND -4.23	0.04 0.45	10.71 2.57	PSBA RPS14
	at1g08380 atcg00650	-1.49 -1.41	ND ND	1.44 -0.23	ND ND	2.93 1.19	ND ND	PSAO RPS18
	at4g05180 atcg00640	-1.56 -1.96	-6.96 ND	1.22 -1.73	3.48 ND	2.77 0.23	10.42 ND	PSBQ-2 RPL33
	at2g05070	-1.62	-5.51	2.55	4.80 Divers	4.16	10.28	LHCB2.2
	atcg00720 atcg00560 at5g20250 at1g15500	-1.63 -1.76 2.23 1.15	-9.23 -10.62 2.07 2.74	-1.64 -1.08 2.24 0.35	ND ND 3.71 4.00	-0.02 0.69 0.01 -0.80	6.74 9.43 1.65 1.26	PETB DIN10 (Dark inducible 10) PSBL ATNTT2
	at3g54890 at5g04810	-1.80 1.17	-6.65 ND	0.48 1.05	3.50 ND	2.28 -0.12	10.12 ND	LHCA1 PPR
	at3g27690 at1g29395	-1.87 -1.24	-5.51 ND	0.76 -2.47	4.80 ND	2.63 -1.23	10.28 ND	LHCB2.3 COR414-TM1
	atcg01010 at5g49360	-2.06 1.92	-2.43 1.83	-0.75 1.06	ND 0.22	1.30 -0.86	2.51 -1.60	NDHF BXL1
	atcg00730 at1g02640	-2.13 1.10	-7.72 1.47	-2.00 -1.22	ND -0.80	0.13 -2.32	7.79 -2.22	PETD BXL2
	at4g20130 at1g32900	-2.26 -1.55	ND -2.73	-1.83 -0.12	ND ND	0.43 1.43	ND 2.81	PTAC14 Starch synthase
	at1g29910 at1g78370	-2.44 -1.92	ND -0.71	3.42 1.39	ND 1.55	5.85 3.30	ND 2.26	CAB3 ATGSTU20
	at2g34430 at2g34290	-2.44 2.34	-8.59 ND	4.61 0.08	ND ND	7.05 -2.26	8.67 ND	LHCB1 Protein kinase
	atcg00490 at5g60250	-2.83 1.14	-6.59 ND	-2.84 1.90	-3.98 ND	0.00 0.76	2.61 ND	RBCL Zinc finger
	atcg00710 at1g02280	-3.42 1.07	-11.50 0.95	-3.16 0.50	ND 3.86	0.26 -0.58	11.57 2.91	PSBH TOC33
	atcg00680 at5g35066	-3.56 -1.02	-10.96 ND	-3.39 -0.62	ND ND	0.17 0.40	11.40 ND	PSBB unknown protein
	at1g21500	-1.02	ND	1.38	Lipids metabolism ND	2.41	ND	unknown protein
	at3g02620 at5g37360	1.33 -1.12	0.35 -3.37	-0.52 1.17	2.94 ND	-1.85 2.28	2.59 5.26	Acyl desaturase unknown protein
	at4g33030 at5g57345	-1.00 -1.28	-0.08 ND	-0.22 1.49	2.41 ND	0.79 2.77	2.48 ND	SQD1 unknown protein
	atcg00500 at4g17970	-1.30 -1.32	-1.45 ND	-0.59 0.66	0.59 ND	0.71 1.98	2.04 ND	ACCD unknown protein
	at4g16155 at1g52220	-1.31 -1.67	-0.92 0.12	0.08 0.91	-0.31 4.72	1.39 2.57	0.61 4.57	Dihydrolipoyl dehydrogenase unknown protein
	at2g31810	1.28	0.73	1.13	-0.43	-0.15	1.61	Acetolactate synthase
	at5g14060	-1.75	-0.40	0.27	1.41	2.02	1.78	CARAB-AK-LYS
				Hormones and pigments metabolism
	at2g14750	-1.40	ND	0.03	ND	1.43	ND	APK (APS KINASE)
	at5g23010	-1.79	-2.70	4.09	ND	5.88	4.11	MAM1
	at3g25770	-1.56	-2.25	2.44	1.61	4.00	3.86	AOC2
	at4g19170	-1.66	-3.31	2.37	ND	4.02	2.89	NCED4
	at3g45140	-2.01	-2.78	4.10	4.17	6.11	6.95	LOX2
	at5g42650	-2.20	-1.99	-0.76	1.84	1.44	3.83	AOS
	at1g44446	-1.08	ND	0.97	ND	2.05	ND	CH1
	at1g03630	-1.19	-2.41	0.17	-0.77	1.36	1.64	POR C
	at4g27440	-1.25	-2.79	-3.11	-5.99	-1.87	-3.21	POR B

WT-light vs WT dark pap7-1 light vs WT-dark pap7-1 light vs WT-light

	Locus	Protein annotation	Transcripts Proteins Transcripts	Proteins	Transcripts	Proteins
	AT3G14440 NCED3	-1.93	NA	-2.19	NA	-0.26	NA
	AT4G32810 CCD8	-1.05	NA	-1.27	NA	-0.22	NA
	AT1G78390 NCED9	-0.48	NA	-1.04	NA	-0.56	NA
	AT1G30100 NCED5	-0.25	NA	-0.58	NA	-0.33	NA
	AT3G24220 NCED 6	-0.1	NA	-0.29	NA	-0.2	NA
	AT4G18350 NCED2	0.13	NA	0.03	NA	-0.11	NA
	AT2G44990 CCD7	0.14	NA	0.11	NA	-0.03	NA
	AT4G25700	BETA-OHASE 1	0.31	NA	1.17	NA	0.86	NA
	at1g67080	ABA4	0.38	NA	0.14	NA	-0.24	NA
	at1g10830	sodium symporter	0.41	NA	0.47	NA	0.06	NA
	AT5G57030 LUT2	0.54	NA	0.31	NA	-0.23	NA
	AT5G52570	BETA-OHASE 2	0.65	NA	-0.75	NA	-1.4	NA
	AT5G17230 PSY1	1.3	NA	0.7	NA	-0.6	NA
	AT1G31800 LUT5	NA	3.07	NA	3.51	NA	0.41
	AT3G63520 CCD1	0.64	3.42	1.07	3.68	0.43	0.27
	AT1G06820 CRTISO	0.76	2.66	0.76	2.67	0.01	-0.01
	AT4G15110 CYP97B3	0.43	1.88	0.46	1.67	0.03	-0.21
	AT1G08550 VDE	0.75	4.16	1.49	3.75	0.74	-0.43
	AT3G04870 ZDS	NA	3.06	NA	2.58	NA	-0.48
	AT4G14210 PDS3	0.66	4.26	0.77	3.78	0.11	-0.49
	AT1G57770 FAD/NAD	2.17	3.59	2.54	3.03	0.37	-0.59
	AT5G67030 ZEP	2.39	3.43	1.88	1.93	-0.51	-1.51
	AT4G19170 NCED4	4.02	2.89	2.37	NA	-1.66	-3.31

Table 4 . Carotenoid biosynthesis enzyme accumulation in WTL, WTD and pap7-1

 4 Comparison between the light-induced expression changes of carotenoid biosynthesis genes and proteins. The heatmap displays the expression changes of the indicated genes as log2 fold change according to a decreasing gradient. The colour code corresponds to ≤-1 and ≥1. Left margin indicates gene identity, right margin indicates the encoded protein according to current nomenclature in literature. The respective sample comparison is indicated on the top table.

  Le découplage de la localisation de PAP7 nous a permis d'établir une étude d'hémicomplémentation. La stratégie repose sur la complémentation séparée du mutant pap7-1 par le variant nucléaire (PAP7 ΔcTP ) ou par le variant chloroplastique (PAP7 NLSmuté ). Les résultats de ces analyses montrent que le variant nucléaire est incapable de restaurer le verdissement de la plantule mais que le variant chloroplastique permet au mutant pap7-1 de verdir. Cependant, la temporalité de ce verdissement est altérée de telle sorte que nous suspectons un rôle régulateur de la protéine PAP7 dans le noyau en complément de la fonction essentielle de la forme plastidiale pour la biogenèse du chloroplaste. Au sein de l'équipe nous avons fait usage des différentes constructions moléculaires pour tester, expérimentalement, les possibilités d'interactions de quelques protéines PAP dans le noyau.Les études précitées des mutants pap5 et pap8 ont révélé que la voie de signalisation de la lumière par le phytochrome B est altérée dans ces 2 mutants. Ceci, suscite l'hypothèse que le mutant pap7-1 pourrait présenter le même défaut de signalisation. Je montre dans ce chapitre d'une part que les PAP possédant un NLS peuvent «intéragir» par complémentation de fluorescence (BIFC) en dehors du chloroplaste et notamment dans le noyau. Et d'autre part que la formation des photocorps de phytochrome B, induits par la lumière est profondément altérer dans le mutant pap7-1. L'ensemble des résultats présenté dans ce chapitre fait l'objet d'une discussion sur le rôle de PAP7, élargi au complexe nucléaire des PAP, dans la coordination de l'expression des génomes nucléaire et chloroplastique et notamment en ce qui concerne les gènes associés à la photosynthèse PHANGs (photosynthesis-associated nuclear genes) et PhAPGs (photosynthesis-associated plastid genes).

pour publication). Une conséquence implicite de cette hypothèse de bi-localisation nucléo-chloroplastique serait que les protéines PAP localisées dans le noyau pourraient également former un complexe fonctionnel multi-protéique.

Dans un premier temps, je présente la validation expérimentale in vivo que la protéine PAP7 est à la fois détectée dans le noyau et les chloroplastes avec la possibilité de découpler cette localisation par mutagenèse dirigée de la séquence de PAP7. A cet égard, des constructions moléculaires ont été générées pour tester la fonctionnalité de PAP7, avec ou sans étiquette fluorescente, par des expériences de complémentation fonctionnelle du mutant pap7-1. Une partie de ces analyses n'est pas terminée et suit son cours.

A l'occasion de ces expériences de génétique moléculaire, nous avons caractérisé de façon précise l'altération génétique de l'allèle pap7-1, ainsi que certaines altérations phénotypiques du mutant homozygote albino associé.

Table 1 . Numbers of each phenotype population in T2 generation

 1

	Genotype/phenotype	Plants
	pMC07a#46	Y	Z
	Albino plants	5	2
	Green plants	70	68
	Albino/Total (%)	3	6,66
	Segregation of albino plants	<1/16	1/16

  It will be an interesting field of future research to test this in detail. The data presented here indicate that it is worth to follow this experimental line est très similaire à celui des plantules traitées avec NF et Linco, de plus, au niveau moléculaire, il existe de nombreuses similitudes. Bien que les plates soient albinos dans ces trois cas, ils ne sont pas complètement inactifs. Ils conservent certaines activités ainsi qu'une certaine accumulation de protéines encodées à la fois par le Comme la RPOB n'est pas détectée dans les plantes traitées par NF et Linco, on peut supposer que le complexe PEP est inactif comme dans les mutants pap7-1 et imite ainsi le phénotype du mutant. De plus, des petites différences moléculaires sont probablement dues aux effets chimiques des inhibiteurs et à leurs effets secondaires. Cela sera un domaine de recherche intéressant pour le futur afin de les tester en détail. Les données qui ont été présentées ici indiquent qu'il est intéressant de suivre des expériences de manière plus approfondie.

	more thoroughly.
	5 Conclusion en français
	L'aspect phénotypique de pap7-1 noyau et par les plates.
	L'idée générale selon laquelle le traitement NF conduit à la photo-destruction de toutes les
	parties internes du plaste, en ne laissant qu'une enveloppe vide, semble être une simplification
	excessive. Néanmoins, les plastes et leurs protéines sont clairement endommagés. La
	différence entre l'effet des inhibiteurs et du mutant pap7-1 est expliquée par le fait que les
	inhibiteurs ont besoin d'un certain temps pour pénétrer dans la plante et atteindre tous les
	plastes dans toutes les cellules. Toutefois, le défaut génétique du pap7-1 est déjà actif pendant
	la maturation de la formation des graines. Le défaut génétique est donc plus immédiat et plus
	direct. Néanmoins, les trois traitements agissent de la même façon sur l'inactivation de
	l'expression des gènes plastidiales. La distinction entre les signaux biogéniques de la
	biosynthèse du tétrapyrrole plastidiale (TPB) et l'expression génique plastidiale (PGE) est
	largement artificielle. Comme les gènes cibles tels que les gènes de Lhcb sont identiques dans
	tous les cas, on peut proposer que ces trois inhibiteurs agissent par un mécanisme commun.

  indifférenciés. Ce processus complexe dépend (entre autres) du bon fonctionnement et de la coordination entre la polymérase codée par le plaste (PEP) et la polymérase codée par le noyau (NEP) qui sont toutes les deux nécessaires à l'expression du génome plastidiale. Le complexe PEP se compose de quatre sous-unités centrales codées par le plaste. Elles sont entourées par douze protéines associées à la PEP (PAP) qui sont codées par le noyau et importées du cytosol. L'accumulation des PAP dans les plastes dépend de la lumière et elle se produit parallèlement à la biogenèse des chloroplastes[START_REF] Pfannschmidt | Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle[END_REF]. Il est intéressant de noter que l'inactivation génétique de chacune de ces PAP bloque la différenciation des chloroplastes en perturbant l'expression des gènes du PSII des plastes, ce qui donne un phénotype albinos de plantes mutantes homozygotes. par le plaste a montré une forte régulation négative alors que l'effet au niveau de la transcription était moins important. Ces données ont fourni une régulation prédominante de la traduction de ce type des protéines, lors de l'illumination.En revanche, la traduction cytosolique des gènes de la photosynthèse est restée fonctionnelle. La protéine de lumen PsbO est accumulée à un niveau normal, indiquant que la machine d'import est fonctionnelle dans les plastes albinos. Cependant, les protéines Lhcb ne se sont pas accumulées et seules des traces de précurseurs ont être détectées, ce qui suggère que les protéines de la membrane du thylakoïde ne sont pas importées et sont dégradées. Ces données indiquent une sélectivité potentielle de la machine d'import dans les plastes albinos. L'expression des gènes sous-unités de ribosomes de plastes ont montré une régulation négative indiquant que la traduction plastidiale est partiellement inhibée dans les plastes albinos. Toutefois, nous avons constaté que certaines protéines plastidiales comme YCF1 s'accumulent normalement, ce qui indique que certaines protéines sélectionnées peuvent être traduites sans restriction. Il s'agit d'une préférence potentielle de la traduction qui est déterminée par l'état du développement plastidiale. En résumé, ni la transcription plastidiale et ni la traduction plastidiale ne sont pas complètement inactives dans les plastes albinos. Il semble plutôt que les deux processus sont arrêtés dans un état fonctionnel restreint qui correspond probablement avant la décoration du PEP par les PAP.

	En utilisant le mutant albinos d'Arabidopsis pap7-1 comme un modèle d'arrêt du
	développement de plastes, cette étude fournit de nouvelles informations sur les processus
	moléculaires initiant la biogenèse des chloroplastes. Le mutant pap7-1 a été utilisé pour
	l'étude de l'accumulation de protéines, la caractérisation de la double localisation de la PAP7 et ses implications fonctionnelles dans les plastes et les noyaux qui conduisent finalement au phénotype albinos du mutant. Tout d'abord, le profil d'accumulation de protéines totales du mutant a été analysé en comparaison avec celui des plantes de type sauvages qui ont été cultivées à l'obscurité ou directement à la lumière. Ensuite, les effets spécifiques de la mutation sur l'accumulation des protéines ont été alignés selon les changements correspondants observés au niveau transcriptomique obtenus lors des études précédentes (Grübler et al., 2017). Ceci a montré une vue détaillée sur les processus d'expression génétique dans les mutants. En général, l'accumulation des protéines a suivi les changements observés dans l'expression des gènes. Cependant, nous avons pu observer un certain nombre de caractéristiques spécifiques de quelques gènes et protéines. Par exemple, l'accumulation des protéines de la photosynthèse biosynthèse du tétrapyrrole demeurait fonctionnelle chez le mutant. Toutefois, la branche de la biosynthèse de la chlorophylle est spécifiquement bloquée. Cette branche est complètement codée dans le noyau et sous le contrôle des facteurs de transcription nucléaire GLK. Des analyses transcriptomiques récentes ont montré que GLK1 est régulé à la baisse dans le qui sont codées Les données protéomiques ainsi que les dosages des pigments ont indiqué que la voie de mutant pap7-1

  Cette construction pouvait sauver le mutant pap7-1 et les plantes transgéniques pouvaient développer un phénotype de type sauvage dans le fond mutant. Ces résultats sont significatifs non seulement parce que la séquence codante de PAP7 est suffisant pour complémenter entièrement le mutant pap7-1, mais aussi parce que ces données confirment l'importance de PAP7 dans la biogenèse des chloroplastes durant les premiers stades du développement. De plus, les résultats ont confirmé in vivo que PAP7 peut être exprimé sous le contrôle du promoteur PAP8. Par conséquent, ces données corrobore la coexpression entre les PAP[START_REF] Liebers | Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development[END_REF]. L'analyse des tests d'hémi-complémentation montrent qu'une séparation entre les fonctions nucléaires et plastidiques semble être difficile. de la signalisation lumineuse par le phytochrome B activé. Ceci peut probablement réguler l'expression des gènes GLK. PAP7 interagit dans le noyau avec deux autres régulateurs (PAP5 et PAP8) de la réponse lumineuse médiée par PhyB ; PAP5 et PAP8[START_REF] Chen | Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes[END_REF] Liebers et al., 2019, non publié). A leur tour, les phytochromes peuvent être impliqués dans le contrôle de la biosynthèse de l'acide jasmonique et dans des autres différents aspects du développement tels que la longueur de l'hypocotyle.Cette étude montre que PAP7 est essentielle à la biogenèse du chloroplaste. Le chloroplaste est indisponsable à la vie oxygénique sur terre tel qu'il est le responsable de la biosynthèse de presque toutes les molécules nécessaires au fonctionnement des plantes (caroténoïdes, chlorophylles, lipides membranaires). Les résultats suggèrent qu'en absence de PAP7, les plastes accumulent moins d'ARNm et moins de protéines correspondantes, mais aussi moins de pigments ou d'hormones. Cela conduit à l'arrêt du développement plastidiale et un développement de phénotype albinos en absence d'une source de carbone exogène.

	Sans l'un des deux sites plastidiaux ou nucléaires prévus, les mutants pap7-1 ne peuvent pas
	restaurer le phénotype du type sauvage. Les deux versions nucléaires et plastidiales sont donc
	à la fois essentielles et complémentaires pour la biogénèse des chloroplastes. Bien que les
	conséquences d'un manque de PAP7 dans le plastide soient bien comprises, les conséquences
	correspondantes dans le noyau sont inconnues. Pour répondre à cette question biologique, le

mutant pap7-1 a été croisé avec une ligné exprimant le phytochrome B étiquetée par la GFP (PBG). L'absence de PAP7 détériore la formation des photo-corps nucléaires tardifs. Par conséquent, la fraction nucléaire du PAP7 peut offrir un nouveau candidat régulateur (rétrograde) Dans l'avenir, une étude détaillée des interactions entre les hormones et les signaux lumineux au cours de la différenciation des chloroplastes semble être un domaine de recherche scientifique très important. De plus, la plupart des protéines présentes dans le chloroplaste sont codées par des gènes nucléaires. Par conséquent, il sera important de comprendre les modalités de sélectivités observées pour les mécanismes d'import et de traduction plastidiale.

Table 1 . Description of the different plasmids used Plasmid Transgene description pAG09a

 1 Contains the CAMV35S promoter, the coding sequence of the PAP7orf gene with a cTP and NLS and flanking XhoI and BamH1 restriction sites. The PAP7 is translationally fused to GFP pBB511Contains the CAMV35S promoter, a PAP7 without cTP, fused to GFP at the C-terminus (35S::PAP7 ∆cTP -GFP). pBB389Binary vector that contains PAP8 full-length with its own promoter (pPAP8::PAP8). The plasmid has a cTP and a NLS flanked by XhoI and Xbal sites. pBB300a

Table 2 . Primers used for site-directed mutagenesis List of primers Sequence

 2

	5'-3'

in position 222 and 241, and arginine (R), in position 223 and 242, by a neutral glycine (G)

Table 3

 3

		. PCR composition
	PCR composition	Added volume (µl)
	Matrix	0.5
	Primer 1	1
	Primer 2	1
	dNTP	1
	Buffer 5X	6
	Phusion TM	0.3
	Distilled Water	21

Table 4

 4

		. PCR program
	PCR program	Temperature (°C)	Time
	Initial denaturation	98	1 min
	Denaturation	98	20s
	Hybridization	52	15s
	Elongation	72	45s
	Final elongation	72	2min

Table 5 . Antibodie summary table Antiserum Antibody clonality

 5

			Theoretical	Description
			molecular weight
			( kDa)
				Nuclear encoded non-photosynthesis proteins
	RPS1	Rabbit polyclonal	20	Is localized in mitochondria and involved in protein translation
	RPS5	Rabbit polyclonal	27.5-27.8	Plastid ribosomal protein
	RPL4	Rabbit polyclonal	26-32	Plastid ribosomal protein
	FTSZ1	Rabbit polyclonal	40	Protein of the plastid cell division apparatus
	PAP7	Rabbit polyclonal	49-56	Anti-plastid transcriptionally active 14
	PAP8	Rabbit polyclonal	37	Anti-plastid transcriptionally active 6
				Nuclear encoded photosynthesis proteins
	LHCB1	Rabbit polyclonal	25-28	The most abundant chlorophyll a/b-binding protein located in the thylakoid membrane of the chloroplast
	LHCB3	Rabbit polyclonal	26-28.7	A chlorophyll a/b-binding protein located in the thylakoid membrane of the chloroplast
	LHCA2	Rabbit polyclonal	24-27.7	Chlorophyll a/b-binding protein of PSI.
	STN7	Rabbit polyclonal	55	Main kinase required for LHCII phosphorylation.
	PSBO	Rabbit polyclonal	33	Subunit of the water splitting complex of PSII located in the lumen.
				Plastid encoded proteins
				Photosynthesis proteins
	RBCL	Rabbit polyclonal	52.7	Large subunit of Rubisco.
	PSAA	Rabbit polyclonal	55-60	A core protein of PSI.
	PSBB	Rabbit polyclonal	56	A chlorophyll-binding protein located in the membrane, where it serves as the core antenna of
				PSII
	PETB	Rabbit polyclonal	23-25	A crucial component of the Cytb6f complex.
				Ribosomal proteins
	RPS7	Rabbit polyclonal	17.4	A part of the small subunit of the ribosomal complex.
	RPL2	Rabbit polyclonal	29.9	A constituent of the large subunit of the ribosome.
				NEP dependent proteins
	RPOB	Rabbit polyclonal	121	RNA polymerase subunit β
	ACCD YCF1	Rabbit polyclonal Rabbit polyclonal	55	ACCase subunit beta (acetyl-coenzyme A, carboxylase (subunit β). TIC214, component of the import machinery.
				Mitochondrial proteins
	ACO1	Rabbit polyclonal	98	An enzyme involved of the Krebs cycle in the
				mitochondria.
	S4	Rabbit polyclonal	40-43	A constituent of the mitochondrial ribosome.
	Cyt C	Rabbit polyclonal	12.5-14	Located in inner mitochondrial membrane. It is an essential component of the electron transport chain
	VDAC1	Rabbit polyclonal	29	Voltage-dependent anion channels: class of porin ion channel located on the outer mitochondrial
				membrane.

Remerciements

Remerciements 2

Ces travaux de thèse ont été réalisés régulières d'entrée au CEA

Chapter 3 Functional analysis of PAP7 in Arabidopsis thaliana

Chapter 5 Materials and Methods

Proteins Assay

The extracted proteins are assayed according to the calorimetric method of bicinchoninic acid (Smith test): MicroBC Assay Protein Quantification kit (Pierce™ BCA protein, Sigma Aldrich, Poole, UK). The proteins reduce Cu 2+ to Cu + in an alkaline medium. Bicinchoninic acid is a specific reagent for Cu + , which forms a complex with maximum optical absorption at 562 nm (Smith et al., 1985). The reaction showed a color change of the protein solution to purple and the absorbance is directly proportional to the protein concentration. Then, the protein concentration was calculated according to a reference curve obtained for a standard protein (BSA) in a concentration range from 1 ng /µl to 200 ng /µl.

The reaction was carried out at room temperature including 150 μl of standard and samples were piped into 96 micro-plate wells. 150 μl of BCA reagent (mix A + B + C 25:25:1) was added in each well and followed by a quick vortex [START_REF] Hussain | Comparative Analysis of Protein Quantification Methods for the Rapid Determination of Protein Loading in Liposomal Formulations[END_REF].

Incubation at 37°C for 60 min was performed. Afterwards, the absorbance (OD) was performed at 562 nm against the blank (MicroBC assay reagent). A table with the absorbance results obtained during the assay and a standard curve was generated. The protein quantity was obtained from the standard amount of added BSA protein.

Proteins separation by SDS polyacrylamide gel electrophoresis (SDS-PAGE)

Laemmli showed in 1970 that proteins could be separated by SDS-PAGE and the mobility of proteins correlated very well with their molecular weights (Weber and Osborn, 1969).

Proteins corresponding to 30 µg were separated on a mini-SDS polyacrylamide gel which makes the possibility to observe the protein separation profile and to detect the proteins after western-immuno-blotting.

The acrylamide gel consisted of a separation gel (10 % of acrylamide; 1.5 M Tris/HCl pH 8.8, 0.4 % SDS) and a stacking gel (4 % of acrylamide; 0.5 M Tris/HCl pH 6.8, 0.4 % SDS), which was poured on top of the solid separation gel. The spacers had a thickness of 0.75 mm to 1.5 mm, which determines the capacity of the gel.

APS creates free radical SO4 2-ions that reacts with the acrylamide and initiates their polymerization. The added TEMED is a catalyst that accelerates the gel polymerization.

1 ml of isopropanol was added on the gel which prevented oxygen from entering and inhibiting the polymerization. After the polymerization, the alcohol was removed with filter paper. Afterwards, a suitable comb was inserted between the glass plates without creating bubbles. The comb was carefully pulled out after polymerization of the stacking gel leaving pockets for the sample application.

Contributions scientifiques

Publications:

1) Chieb M, Liebers M, Chevalier F, Lerbs-Mache S, Blanvillain R, Pfannschmidt T. "Determination of the DNA/RNA associated sub-proteome from chloroplasts and other plastid types" Methods Mol Biol. 2018;1829:253-271. doi: 10.1007/978-1-4939-8654-5_17.

2) Monique Liebers