Light-driven processes: key players of the functional biodiversity in microalgae

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Ectopic expression of Rubisco subunits in maize mesophyll cells does not overcome barriers to cell type-specific accumulation

In Zea mays, ribulose bisphosphate carboxylase (Rubisco) accumulates in bundle sheath but not mesophyll chloroplasts, but the mechanisms that underlie cell-type specific expression are poorly understood. To explore the coordinated expression of the chloroplast rbcL gene, which encodes the Rubisco large subunit (LS), and the two nuclear RBCS genes which encode the small subunit (SS), RNAi was used to reduce RBCS expression. This resulted in Rubisco deficiency, and was correlated with translational repression of rbcL. Thus, as in C3 plants, LS synthesis depends on the presence of its assembly partner SS. To test the hypothesis that the previously documented transcriptional repression of RBCS in mesophyll cells is responsible for repressing LS synthesis in mesophyll chloroplasts, a ubiquitin promoter-driven RBCS gene was expressed in both bundle sheath and mesophyll cells. This did not lead to Rubisco accumulation in the mesophyll, suggesting that LS synthesis is impeded even in the presence of ectopic SS expression. To attempt to bypass this putative mechanism, a ubiquitin promoter-driven nuclear version of the rbcL gene was created, encoding an epitope-tagged LS, which was expressed in the presence or absence of the Ubi-RBCS construct. Both transgenes were robustly expressed, and the tagged LS was readily incorporated into Rubisco complexes. However, neither immunolocalization nor biochemical approaches revealed significant accumulation of Rubisco in mesophyll cells, suggesting a continuing cell type-specific impairment of its assembly or stability. We conclude that additional cell type-specific factors limit Rubisco expression to bundle sheath chloroplasts. Includes supplemental data (Table 1)

Régulation de la traduction chloroplastique chez Chlasmydomonas reinhardtii (cytochrome f et autres protéines CES...)

La biogenèse de l'appareil photo synthétique nécessite l'action concertée des génomes nucléaires et chloroplastiques pour produire en quantité stœchiométrique les sous-unités des complexes de la membrane photo synthétique, d'origine génétique double. Ce travail effectué sur l'organisme modèle Chlamydomonas reinhardtii présente un mécanisme original traduisant cette concertation: le processus CES (Contrôle par Epistasie de Synthèse), reposant sur une régulation de la traduction. En étudiant le gène chloroplastique petA codant le cytochrome f, une protéine majeure du complexe cytochrome b6f, nous montrons que sa traduction dépend de l'assemblage de la protéine au sein du complexe. En absence de ses partenaires d'assemblage, la synthèse du cytochrome f- mais pas sa stabilité est réduite. Nous présentons une caractérisation du mécanisme impliquant: I) une régulation de l'initiation de la traduction; II) un domaine régulateur porté par la protéine CES ; III) un effecteur ternaire, plutôt qu'une interaction directe entre le motif régulateur et la région 5' non-traduite du gène CES. Cet effecteur pourrait être un activateur traductionnel, qui piégé par la sous-unité non-assemblée, ne serait plus disponible pour initier la traduction. Nous avons caractérisé un activateur traductionnel, requis spécifiquement pour l'initiation de la traduction du gène petA, le gène TCA1, qui pourrait être l'effecteur du processus CES. Enfin, nous avons démontré l'existence d'autres protéines CES au sein des complexes de la membrane photosynthétique de C. reinhardtii. Le contrôle de la traduction par l'assemblage des sous-unités PsaA et PsaC du photosystème I, et des sous-unités D1 et P5 du photo système II repose sur le même mécanisme: une autorégulation négative de l'initiation de la traduction par la sous-unité CES non-assemblée. Ces observations posent la question d'une éventuelle généralisation du processus CES et de son mécanisme aux plantes supérieures, et à un autre organite, la mitochondrie.The biogenesis of the photosynthetic protein complexes requites the co-ordinate interaction of the nuclear and chloroplast genomes in order to produce in stoichiometric amounts their subunits. I contributed to the characterization of an unique mechanism of regulation: the CES process (Control by Epistasy of Synthesis) in the unicellular green alga Chlamydomonas reinhardtii. Using the chloroplast petA gene, encoding cytochrome f, a major subunit of the cytochrome b6f complex, we showed that petA translation depends on its assembly state: in the absence of its assembly partners, cytochrome f synthesis is inhibited at the level of translation initiation. Moreover, an autoregulation is exerted by a regulatory motif of the unassembled cytochrome f CES subunit. A ternary effector may "shuttle" the CES control between the regulatory motif of the protein and the 5' untranslated region of the CES (petA) transcript. I characterized the TCA1 nuclear encoded translational activator, specifically required for the initiation of the translation of the petA RNA. TCA1 may be the effector of the CES process: in our model, competitive binding of TCA1 to cytochrome f's regulatory motif would decrease petA translation efficiency. Finally, I have shown that this assembly-dependent regulation of translation also occurs for chloroplast-encoded subunits of other photosynthetic complexes of C. reinhardtii such as PsaA and PsaC subunits of Photosystem I, as well as D1 and P5 subunits of Photosystem II. In all cases, the CES mechanism is conserved. These observations raise interesting questions as to the possible generalization of the CES process to vascular plants, and to the mitochondrion.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Cytochrome f Translation in Chlamydomonas Chloroplast Is Autoregulated by its Carboxyl-Terminal Domain

The rate of synthesis of cytochrome f is decreased ∼10-fold when it does not assemble with the other subunits of the cytochrome b(6)f complex in Chlamydomonas reinhardtii chloroplasts. This assembly-mediated regulation of cytochrome f synthesis corresponds to a regulation of petA mRNA initiation of translation. Here, we demonstrate that cytochrome f translation is autoregulated by its C-terminal domain. Five cytochrome f residues conserved throughout all chloroplast genomes—residue Gln-297 in the transmembrane helix and a cluster of four amino acids, Lys-Gln-Phe-Glu, at positions 305 to 308, in the stromal extension—participate in the formation of a translation repressor motif. By contrast, positively charged residues in the stromal extension have little influence on the autoregulation process. These results do not favor a direct interaction between the repressor motif and the petA 5′ untranslated region but suggest the participation of a membrane-bound ternary effector

Role of ClpP in the Biogenesis and Degradation of RuBisCO and ATP Synthase in Chlamydomonas reinhardtii

International audienceRibulose 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 CO 2 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 Sand 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 housekeeping 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

Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastids

Plastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll-specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath-specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon-optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid-related studies of wild-type and mutant maize plants and provide material from which different plastid types may be isolated