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Cystathionine-β-synthase (CBS) domains are found in proteins of all living organisms and have been proposed to play a role as energy sensors regulating protein activities through their adenosyl ligand binding capacity. In plants, members of the CBSX protein family carry a stand-alone pair of CBS domains. In Arabidopsis (Arabidopsis thaliana), CBSX1 and CBSX2 are targeted to plastids where they have been proposed to regulate thioredoxins (TRXs). TRXs are ubiquitous cysteine thiol oxido-reductases involved in the redox-based regulation of numerous enzymatic activities as well as in the regeneration of thiol-dependent peroxidases. In Arabidopsis, 10 TRX isoforms have been identified in plastids and divided into five sub-types. Here, we show that CBSX2 specifically inhibits the activities of m-type TRXs toward two chloroplast TRX-related targets. By testing activation of NADP-malate dehydrogenase and reduction of 2-Cys peroxiredoxin, we found that TRXm1/2 inhibition by CBSX2 was alleviated in the presence of AMP or ATP. We also determined, by pull-down assays, a direct interaction of CBSX2 with reduced TRXm1 and m2 that was abolished in the presence of adenosyl ligands. In addition, we report that, compared with wild-type plants, the Arabidopsis T-DNA double mutant cbsx1 cbsx2 exhibits growth and chlorophyll accumulation defects in cold conditions, suggesting a function of plastidial CBSX proteins in plant stress adaptation. Together, our results show an energy-sensing regulation of plastid TRX m activities by CBSX, possibly allowing a feedback regulation of ATP homeostasis via activation of cyclic electron flow in the chloroplast, to maintain a high energy level for optimal growth.

N-terminal myristoylation, a major eukaryotic protein lipid modification, is difficult to detect in vivo and challenging to predict in silico. We developed a proteomics strategy involving subfractionation of cellular membranes, combined with separation of hydrophobic peptides by mass spectrometry-coupled liquid chromatography to identify the Arabidopsis thaliana myristoylated proteome. This approach identified a starting pool of 8837 proteins in all analyzed cellular fractions, comprising 32% of the Arabidopsis proteome. Of these, 906 proteins contain an N-terminal Gly at position 2, a prerequisite for myristoylation, and 214 belong to the predicted myristoylome (comprising 51% of the predicted myristoylome of 421 proteins). We further show direct evidence of myristoylation in 72 proteins; 18 of these myristoylated proteins were not previously predicted. We found one myristoylation site downstream of a predicted initiation codon, indicating that posttranslational myristoylation occurs in plants. Over half of the identified proteins could be quantified and assigned to a subcellular compartment. Hierarchical clustering of protein accumulation combined with myristoylation and S-acylation data revealed that N-terminal double acylation influences redirection to the plasma membrane. In a few cases, MYR function extended beyond simple membrane association. This study identified hundreds of N-acylated proteins for which lipid modifications could control protein localization and expand protein function.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) associates a chloroplast- and a nucleus-encoded subunit (LSU and SSU). It constitutes the major entry point of inorganic carbon into the biosphere as it catalyzes photosynthetic CO2 fixation. Its abundance and richness in sulfur-containing amino acids make it a prime source of N and S during nutrient starvation, when photosynthesis is downregulated and a high RuBisCO level is no longer needed. Here we show that translational attenuation of ClpP1 in the green alga Chlamydomonas reinhardtii results in retarded degradation of RuBisCO during S- and N-starvation, suggesting that the Clp protease is a major effector of RubisCO degradation in these conditions. Furthermore, we show that ClpP cannot be attenuated in the context of rbcL point mutations that prevent LSU folding. The mutant LSU remains in interaction with the chloroplast chaperonin complex. We propose that degradation of the mutant LSU by the Clp protease is necessary to prevent poisoning of the chaperonin. In the total absence of LSU, attenuation of ClpP leads to a dramatic stabilization of unassembled SSU, indicating that Clp is responsible for its degradation. In contrast, attenuation of ClpP in the absence of SSU does not lead to overaccumulation of LSU, whose translation is controlled by assembly. Altogether, these results point to RuBisCO degradation as one of the major house-keeping functions of the essential Clp protease. In addition, we show that non-assembled subunits of the ATP synthase are also stabilized when ClpP is attenuated. In the case of the atpA-FUD16 mutation, this can even allow the assembly of a small amount of CF1, which partially restores phototrophy.

N-terminal myristoylation, a major eukaryotic protein lipid modification, is difficult to detect in vivo and challenging to predict in silico. We developed a proteomics strategy involving subfractionation of cellular membranes, combined with separation of hydrophobic peptides by mass spectrometry-coupled liquid chromatography to identify the Arabidopsis thaliana myristoylated proteome. This approach identified a starting pool of 8837 proteins in all analyzed cellular fractions, comprising 32% of the Arabidopsis proteome. Of these, 906 proteins contain an N-terminal Gly at position 2, a prerequisite for myristoylation, and 214 belong to the predicted myristoylome (comprising 51% of the predicted myristoylome of 421 proteins). We further show direct evidence of myristoylation in 72 proteins; 18 of these myristoylated proteins were not previously predicted. We found one myristoylation site downstream of a predicted initiation codon, indicating that posttranslational myristoylation occurs in plants. Over half of the identified proteins could be quantified and assigned to a subcellular compartment. Hierarchical clustering of protein accumulation combined with myristoylation and S-acylation data revealed that N-terminal double acylation influences redirection to the plasma membrane. In a few cases, MYR function extended beyond simple membrane association. This study identified hundreds of N-acylated proteins for which lipid modifications could control protein localization and expand protein function.

N-myristoylation is a crucial irreversible eukaryotic lipid modification allowing a key subset of proteins to be targeted at the periphery of specific membrane compartments. Eukaryotes have conserved N-myristoylation enzymes, involving one or two N-myristoyltransferases (NMT1 and NMT2), among which NMT1 is the major enzyme. In the postembryonic developmental stages, defects in NMT1 lead to aberrant cell polarity, flower differentiation, fruit maturation, and innate immunity; however, no specific NMT1 target responsible for such deficiencies has hitherto been identified. Using a confocal microscopy forward genetics screen for the identification of Arabidopsis thaliana secretory mutants, we isolated STINGY, a recessive mutant with defective Golgi traffic and integrity. We mapped STINGY to a substitution at position 160 of Arabidopsis NMT1 (NMT1A160T). In vitro kinetic studies with purified NMT1A160T enzyme revealed a significant reduction in its activity due to a remarkable decrease in affinity for both myristoyl-CoA and peptide substrates. We show here that this recessive mutation is responsible for the alteration of Golgi traffic and integrity by predominantly affecting the Golgi membrane/cytosol partitioning of ADP-ribosylation factor proteins. Our results provide important functional insight into N-myristoylation in plants by ascribing postembryonic functions of Arabidopsis NMT1 that involve regulation of the functional and morphological integrity of the plant endomembranes.

In Chlamydomonas reinhardtii, the clpP1 chloroplast gene encoding one of the catalytic subunits of the ClpP protease complex contains a large in-frame insertion sequence (IS1). Based on the Escherichia coli ClpP structure, IS1 is predicted to protrude at the apical surface of the complex, likely influencing the interaction of the catalytic core with ClpC/HSP100 chaperones. Immunoblotting with an anti-ClpP1 antibody detected two immunoreactive forms of ClpP1: ClpP1(H) (59 kDa) and ClpP1(L) (25 kDa). It has been proposed that IS1 is a new type of protein intron (different from inteins). By studying transformants harboring mutations at the predicted borders of IS1 and tags at the C terminus of ClpP1 (tandem affinity purification tag, His tag, Strep.Tag) or within the IS1 sequence (3-hemagglutinin tag), we show that IS1 is not a protein intron and that ClpP1(L) results from endoproteolytic cleavage inside IS1. Processing sites have been identified in the middle of IS1 and near its C terminus. The sites can be mutated without abolishing processing.

In leaves of C4 grasses such as maize, photosynthetic activities are partitioned between bundle-sheath and mesophyll cells, leading to increased photosynthetic yield, particularly under stress conditions. As we discuss here, recent comparative chloroplast proteome analyses in maize have shown specific adaptation to C4-cell-specific differentiation of the photosynthetic apparatus, primary and secondary metabolism and metabolite transporters, as well as the chloroplast protein homeostasis machinery. Furthermore, a novel bundle-sheath-enriched 1000-kDa NADPH dehydrogenase 'supercomplex' has been identified, and we discuss its possible role in inorganic carbon concentration. These breakthroughs provide new opportunities to further unravel C4 pathways and to increase crop productivity through metabolic engineering of C4 pathways into C3 plants, such as rice.

The Plant Proteomics Database (PPDB; http://ppdb.tc.cornell.edu), launched in 2004, provides an integrated resource for experimentally identified proteins in Arabidopsis and maize (Zea mays). Internal BLAST alignments link maize and Arabidopsis information. Experimental identification is based on in-house mass spectrometry (MS) of cell type-specific proteomes (maize), or specific subcellular proteomes (e.g. chloroplasts, thylakoids, nucleoids) and total leaf proteome samples (maize and Arabidopsis). So far more than 5000 accessions both in maize and Arabidopsis have been identified. In addition, more than 80 published Arabidopsis proteome datasets from subcellular compartments or organs are stored in PPDB and linked to each locus. Using MS-derived information and literature, more than 1500 Arabidopsis proteins have a manually assigned subcellular location, with a strong emphasis on plastid proteins. Additional new features of PPDB include searchable posttranslational modifications and searchable experimental proteotypic peptides and spectral count information for each identified accession based on in-house experiments. Various search methods are provided to extract more than 40 data types for each accession and to extract accessions for different functional categories or curated subcellular localizations. Protein report pages for each accession provide comprehensive overviews, including predicted protein properties, with hyperlinks to the most relevant databases.

Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/P(i) translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are available through the Plant Proteome Database. These data are integrated with previous data, resulting in a model for C(4) photosynthesis, thereby providing new rationales for metabolic engineering of C(4) pathways and targeted analysis of genetic networks that coordinate C(4) differentiation.

During maize (Zea mays) C4 differentiation, mesophyll (M) and bundle sheath (BS) cells accumulate distinct sets of photosynthetic enzymes, with very low photosystem II (PSII) content in BS chloroplasts. Consequently, there is little linear electron transport in the BS and ATP is generated by cyclic electron flow. In contrast, M thylakoids are very similar to those of C3 plants and produce the ATP and NADPH that drive metabolic activities. Regulation of this differentiation process is poorly understood, but involves expression and coordination of nuclear and plastid genomes. Here, we identify a recessive allele of the maize high chlorophyll fluorescence (Hcf136) homolog that in Arabidopsis (Arabidopsis thaliana) functions as a PSII stability or assembly factor located in the thylakoid lumen. Proteome analysis of the thylakoids and electron microscopy reveal that Zmhcf136 lacks PSII complexes and grana thylakoids in M chloroplasts, consistent with the previously defined Arabidopsis function. Interestingly, hcf136 is also defective in processing the full-length psbB-psbT-psbH-petB-petD polycistron specifically in M chloroplasts. To determine whether the loss of PSII in M cells affects C4 differentiation, we performed cell-type-specific transcript analysis of hcf136 and wild-type seedlings. The results indicate that M and BS cells respond uniquely to the loss of PSII, with little overlap in gene expression changes between data sets. These results are discussed in the context of signals that may drive differential gene expression in C4 photosynthesis.

The composition of the chloroplast-localized protease complex, ClpP, from the green alga Chlamydomonas reinhardtii was characterized by nondenaturing electrophoresis, immunoblotting and MS. The detected ClpP complex has a native mass of approximately 540 kDa, which is approximately 200 kDa higher than ClpP complexes in higher plant chloroplasts, mitochondria or bacteria. The 540-kDa ClpP complex contains two nuclear-encoded ClpP proteins (ClpP3 and P5) and five ClpR (R1, R2, R3, R4 and R6) proteins, as well two proteins, ClpP1L and ClpP1H, both probably derived from the plastid clpP1 gene. ClpP1H is 59 kDa and contains a approximately 30-kDa insertion sequence (IS1) not found in other ClpP proteins, responsible for the high MW of the complex. Based on comparison with other sequences, IS1 protrudes as an additional domain on the apical surface of the ClpP/R complex, probably preventing interaction with the HSP100 chaperone. ClpP1L is a 25-kDa protein similar in size to other ClpP proteins and could arise by post-translational processing of ClpP1H. Chloramphenicol-chase experiments show that ClpP1L and ClpP1H have a similar half-life, indicating that both are stable components of the complex. The structure of the ClpP complex is further discussed in conjunction with a phylogenetic analysis of the ClpP/R genes. A model is proposed for the evolution of the algal and plant complex from its cyanobacterial ancestor.

Plastids are essential organelles because they contribute to primary and secondary metabolism and plant signaling networks. A high-quality inventory of the plastid proteome is therefore a critical tool in plant research. We present reference plastid proteomes for maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) with, respectively, 1564 and 1559 proteins. This was based on manual curation of published experimental information, including150 proteomics studies regarding different (sub)cellular fractions, and new quantitative proteomics experiments on plastid subfractions specifically designed to fill gaps in current knowledge. These plastid proteomes represent an estimated 40 (maize) to 50% (Arabidopsis) of all plastid proteins and can serve as a "gold standard" because of their low false-positive rate. To facilitate direct comparison of these plastid proteomes, identify "missing" proteins, and evaluate species-specific differences, we determined their orthologous relationships. The multistep strategy to best define these orthologous relationships is explained. Putative plastid locations for orthologs without known subcellular locations were inferred based on the robustness of orthology and weighing of experimental evidence, increasing both plastid proteome sizes. Examples that highlight differences and similarities between maize and Arabidopsis and underscore the quality of the orthology assignments are discussed.

The ClpP peptidase is a major constituent of the proteolytic machinery of bacteria and organelles. The chloroplast ClpP complex is unusual, in that it associates a large number of subunits, one of which (ClpP1) is encoded in the chloroplast, the others in the nucleus. The complexity of these large hetero-oligomeric complexes has been a major difficulty in their overproduction and biochemical characterization. In this paper, we describe the purification of native chloroplast ClpP complex from the green alga Chlamydomonas reinhardtii, using a strain that carries the Strep-tag II at the C-terminus of the ClpP1 subunit. Similar to land plants, the algal complex comprises active and inactive subunits (3 ClpP and 5 ClpR, respectively). Evidence is presented that a sub-complex can be produced by dissociation, comprising ClpP1 and ClpR1, 2, 3 and 4, similar to the ClpR-ring described in land plants. Our Chlamydomonas ClpP preparation also contains two ClpT subunits, ClpT3 and ClpT4, which like the land plant ClpT1 and ClpT2 show 2 Clp-N domains. ClpTs are believed to function in substrate binding and/or assembly of the two heptameric rings. Phylogenetic analysis indicates that ClpT subunits have appeared independently in Chlorophycean algae, in land plants and in dispersed cyanobacterial genomes. Negative staining electron microscopy shows that the Chlamydomonas complex retains the barrel-like shape of homo-oligomeric ClpPs, with 4 additional peripheral masses that we speculate represent either the additional IS1 domain of ClpP1 (a feature unique to algae) or ClpTs or extensions of ClpR subunits.

Plastids contain multiple copies of the plastid chromosome, folded together with proteins and RNA into nucleoids. The degree to which components of the plastid gene expression and protein biogenesis machineries are nucleoid associated, and the factors involved in plastid DNA organization, repair, and replication, are poorly understood. To provide a conceptual framework for nucleoid function, we characterized the proteomes of highly enriched nucleoid fractions of proplastids and mature chloroplasts isolated from the maize (Zea mays) leaf base and tip, respectively, using mass spectrometry. Quantitative comparisons with proteomes of unfractionated proplastids and chloroplasts facilitated the determination of nucleoid-enriched proteins. This nucleoid-enriched proteome included proteins involved in DNA replication, organization, and repair as well as transcription, mRNA processing, splicing, and editing. Many proteins of unknown function, including pentatricopeptide repeat (PPR), tetratricopeptide repeat (TPR), DnaJ, and mitochondrial transcription factor (mTERF) domain proteins, were identified. Strikingly, 70S ribosome and ribosome assembly factors were strongly overrepresented in nucleoid fractions, but protein chaperones were not. Our analysis strongly suggests that mRNA processing, splicing, and editing, as well as ribosome assembly, take place in association with the nucleoid, suggesting that these processes occur cotranscriptionally. The plastid developmental state did not dramatically change the nucleoid-enriched proteome but did quantitatively shift the predominating function from RNA metabolism in undeveloped plastids to translation and homeostasis in chloroplasts. This study extends the known maize plastid proteome by hundreds of proteins, including more than 40 PPR and mTERF domain proteins, and provides a resource for targeted studies on plastid gene expression. Details of protein identification and annotation are provided in the Plant Proteome Database.

C4 grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C4 photosynthesis is established along the developmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics of maize leaf development and C4 differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry. This was complemented by extensive qualitative and quantitative microscopy analysis of structural features (e.g., Kranz anatomy, plasmodesmata, cell wall, and organelles). More than 4300 proteins were identified and functionally annotated. Developmental protein accumulation profiles and hierarchical cluster analysis then determined the kinetics of organelle biogenesis, formation of cellular structures, metabolism, and coexpression patterns. Two main expression clusters were observed, each divided in subclusters, suggesting that a limited number of developmental regulatory networks organize concerted protein accumulation along the leaf gradient. The coexpression with BSC and MC markers provided strong candidates for further analysis of C4 specialization, in particular transporters and biogenesis factors. Based on the integrated information, we describe five developmental transitions that provide a conceptual and practical template for further analysis. An online protein expression viewer is provided through the Plant Proteome Database.

Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C4 photosynthesis. This study provides a reconstruction of how metabolic pathways, protein expression, and homeostasis functions are quantitatively distributed across BS and M chloroplasts. This yielded new insights into cellular specialization. The experimental analysis was based on high-accuracy mass spectrometry, protein quantification by spectral counting, and the first maize genome assembly. A bioinformatics workflow was developed to deal with gene models, protein families, and gene duplications related to the polyploidy of maize; this avoided overidentification of proteins and resulted in more accurate protein quantification. A total of 1,105 proteins were assigned as potential chloroplast proteins, annotated for function, and quantified. Nearly complete coverage of primary carbon, starch, and tetrapyrole metabolism, as well as excellent coverage for fatty acid synthesis, isoprenoid, sulfur, nitrogen, and amino acid metabolism, was obtained. This showed, for example, quantitative and qualitative cell type-specific specialization in starch biosynthesis, arginine synthesis, nitrogen assimilation, and initial steps in sulfur assimilation. An extensive overview of BS and M chloroplast protein expression and homeostasis machineries (more than 200 proteins) demonstrated qualitative and quantitative differences between M and BS chloroplasts and BS-enhanced levels of the specialized chaperones ClpB3 and HSP90 that suggest active remodeling of the BS proteome. The reconstructed pathways are presented as detailed flow diagrams including annotation, relative protein abundance, and cell-specific expression pattern. Protein annotation and identification data, and projection of matched peptides on the protein models, are available online through the Plant Proteome Database.

Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved in lipid biosynthesis, nitrogen import, and tetrapyrrole and isoprenoid biosynthesis are preferentially located in the M chloroplasts. By contrast, enzymes involved in starch synthesis and sulfur import preferentially accumulate in BS chloroplasts. The different soluble antioxidative systems, in particular peroxiredoxins, accumulate at higher levels in M chloroplasts. We also observed differential accumulation of proteins involved in expression of plastid-encoded proteins (e.g., EF-Tu, EF-G, and mRNA binding proteins) and thylakoid formation (VIPP1), whereas others were equally distributed. Enzymes related to the C4 shuttle, the carboxylation and regeneration phase of the Calvin cycle, and several regulators (e.g., CP12) distributed as expected. However, enzymes involved in triose phosphate reduction and triose phosphate isomerase are primarily located in the M chloroplasts, indicating that the M-localized triose phosphate shuttle should be viewed as part of the BS-localized Calvin cycle, rather than a parallel pathway.

Chlamydomonas reinhardtii mutants defective in the chloroplast ATP synthase are highly sensitive to light. The ac46 mutant is affected in the MDH1 gene, required for production or stability of the monocistronic atpH mRNA encoding CFO-III. In this and other ATP synthase mutants, we show that short-term exposure to moderate light intensities—a few minutes—induces an inhibition of electron transfer after the primary quinone acceptor of photosystem II (PSII), whereas longer exposure—several hours—leads to a progressive loss of PSII cores. An extensive swelling of thylakoids accompanies the initial inhibition of electron flow. Thylakoids deflate as PSII cores are lost. The slow process of PSII degradation involves the participation of ClpP, a chloroplast-encoded peptidase that is part of a major stromal protease Clp. In the light of the above findings, we discuss the photosensitivity of ATP synthase mutants with respect to the regular photoinhibition process that affects photosynthetic competent strains at much higher light intensities.

In the green alga Chlamydomonas reinhardtii, the ClpP protease is encoded by an essential chloroplast gene. Mutating its AUG translation initiation codon to AUU reduced ClpP accumulation to 25 to 45% of that of the wild type. Both the mature protein and the putative precursor containing its insertion sequence were present in reduced amounts. Attenuation of ClpP did not affect growth rates under normal conditions but restricted the ability of the cells to adapt to elevated CO(2) levels. It also affected the rate of degradation of the cytochrome b(6)f complex of the thylakoid membrane in two experimental situations: (1) during nitrogen starvation, and (2) in mutants deficient in the Rieske iron-sulfur protein. The ClpP level also controls the steady state accumulation of a mutated version of the Rieske protein. In contrast, attenuation of ClpP did not rescue the fully unassembled subunits in other cytochrome b(6)f mutants. We conclude that proteolytic disposal of fully or partially assembled cytochrome b(6)f is controlled by the Clp protease.

Photosynthesis is a process that converts light energy into organic energy in the chloroplasts of embryophytes. This process requires a protein apparatus encoded by both the nuclear and the plastidial genomes of the plant cells. Among the nuclear encoded proteins addressed to the chloroplast are many factors involved in the different maturation steps of chloroplast transcripts (cleavage, splicing, editing, stabilization and translation). In particular, pentatricopeptide repeat proteins (PPR) are involved in all these steps, including editing, through their sequence specific RNA binding PPR motifs. Organellar RNA editing is performed by a protein complex called the editosome. Within this complex, a PPR protein is the specificity factor and probably also the editing enzyme through its DYW domain. Many other editing factors within this complex have been discovered and characterized. However, many questions concerning the function of editing and the functioning of editosome are remaining. During my PhD thesis, I tried to answer three of these questions.First, I investigated whether new editosome proteins could be identified using new independent approaches. Indeed, the discovery of new factors could allow the identification of editing regulators, paving the way for a better understanding of the molecular editing mechanism and its biological function. Protein complex purification (realized before my PhD) and a yeast two hybrid screening were performed. The first approach only identified PPR proteins, while the second did not yield any specific interaction. However, the search for editosome components allowed the identification and characterization of CBSX2, a potential regulator of thioredoxin activity. CBSX2 was shown to specifically inhibit the in vitro reductase activity of some thioredoxins. This inhibition is mediated by protein-protein interaction and reverted by CBSX2 AMP binding.I also investigated the requirement of the DYW domain for editing. According to the main stream hypothesis, DYW domains (of PPR proteins) carry the editing activity. Consequently, they must be an essential component of the editosome. However, the characterization of PPR proteins that do not carry a DYW domain but are required for editing challenges this hypothesis. To test the DYW requirement for editing, complementations of editing mutants with several truncated PPR proteins have been performed. So far, these experiments should allow us to validate (or not) a model in which the DYW domain is necessary and provided in trans to PPR proteins by a small sub-family of particular PPR-DYW proteins.Finally, I investigated whether plastid differentiation was linked to differential editing of some transcripts. In particular, C4 photosynthetic plastid differentiation served as a model. In particular, regulations that drive C4 differentiation could rely on organellar gene expression. In this context, editing could participate to differential transcript maturation. Data analysis of C4 plastid transcriptomes required the development of a specific bioinformatic analysis pipeline. This study revealed differentially expressed plastidial genes without editing and splicing modification.; La photosynthèse est un processus permettant la conversion de l'énergie lumineuse en énergie organique dans les chloroplastes des embryophytes. Les complexes photosynthétiques sont d'une origine génétique double. Leurs sous-unités sont codées à la fois par le génome nucléaire et par le génome chloroplastique. Parmi les protéines codées par le génome nucléaire et adressées au chloroplaste, se trouvent de nombreux facteurs impliqués dans les différentes étapes de la maturation des transcrits des gènes chloroplastiques (clivage, épissage, édition, stabilisation et traduction). En particulier, les protéines à motifs pentatricopeptide repeat (PPR) sont impliquées dans toutes ces étapes, notamment l'édition, via leur capacité à lier des séquences spécifiques d'ARN par leurs domaines PPR. L'édition des ARN des organites est réalisée par un complexe protéique nommé l'éditosome. Au sein de ce complexe, les protéines PPR sont les facteurs de spécificité mais également très probablement les enzymes catalysant la réaction grâce à leur domaine DYW. De nombreux facteurs d'édition composant ce complexe ont été découverts et caractérisés. Toutefois, de nombreuses questions portant sur le rôle de l'édition et le fonctionnement de l'éditosome restent encore ouvertes. Au cours de cette thèse, j'ai cherché à répondre à trois questions.En premier lieu, j'ai cherché à identifier de nouveaux facteurs associés à l'éditosome par des approches indépendantes de celles déjà utilisées. En effet, la découverte de nouveaux facteurs pourrait permettre l'identification de régulateurs de l'édition, ouvrant la voie à une meilleure compréhension à la fois du fonctionnement moléculaire de l'édition mais aussi de sa fonction biologique. Une approche par purification de complexes (réalisée avant la thèse) et un crible double hybride chez la levure ont été entrepris. La première approche a permis d'identifier de nouvelles protéines PPR associées à l'éditosome. La seconde approche a permis d'identifier la protéine CBSX2 dont l'interaction avec un membre de l'éditosome n'a pas semblé spécifique. Toutefois, elle a conduit à la caractérisation fonctionnelle de CBSX2 en tant que régulateur potentiel de l'activité des thiorédoxines. Mes résultats ont montré qu'in vitro CBSX2 inhibe spécifiquement l'activité réductrice d'un sous-groupe de thiorédoxines. Cette inhibition implique des interactions entre protéines et est levée par la liaison d'AMP à CBSX2.J'ai également cherché à savoir si le domaine DYW des PPR est indispensable dans l'éditosome. En effet, selon l'hypothèse la plus couramment admise, le domaine DYW serait l'enzyme de la réaction d'édition, il serait donc nécessaire dans le complexe. Néanmoins, la caractérisation de protéines PPR ne possèdent pas de domaine DYW mais requises pour l'édition remet en question cette hypothèse. Pour cela, la complémentation de mutants d'édition avec une combinaison de formes tronquées de protéines PPR a été entreprise et est toujours en cours. Elle devrait permettre prochainement de valider (ou non) un modèle selon lequel le domaine DYW est nécessaire et apporté en trans aux protéines PPR par une petite sous-famille de protéines PPR-DYW particulières.Enfin, j'ai cherché à savoir si la différenciation des chloroplastes s'accompagnait d'une édition différentielle de certains ARN. En particulier, le cas de la différenciation des chloroplastes des plantes « en C4 » a été étudié. Dans ce cas, une régulation de l'expression des gènes des organites pourrait être impliquée dans la mise en place de la différenciation chloroplastique. Dans ce contexte, l'édition pourrait jouer un rôle dans la régulation de la maturation des transcrits. L'analyse comparative des données produites a nécessité le développement d'un pipeline d'analyse bio-informatique spécifique. Cette étude a permis de montrer une expression différentielle des gènes chloroplastiques sans modification de l'édition et de l'épissage.