Département de biologie moléculaire
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Research Projects

Chloroplast biogenesis and regulation of photosynthesis

A unique feature of plant and algal cells is that they contain three distinct genetic systems located in the nucleus, chloroplast and mitochondria. These systems comprise their own genome and protein synthesizing machineries. Our long-range goal is to understand the molecular cross-talk between these systems. This coordination is required for the biogenesis and function of the organelles. Using the combined approaches of molecular genetics and biochemistry, we study the interactions between the nucleo-cytosolic and chloroplast systems that are involved in the assembly of the photosynthetic apparatus. We also study the remarkable ability of photosynthetic organisms to adapt to changes in both light quality and quantity, and in particular the dynamic acclimation processes which occur in the thylakoid membranes (the photosynthetic membranes of the chloroplast).


The biogenesis of the photosynthetic apparatus and its regulation play a key role in plant development. We study this process in the green unicellular alga Chlamydomonas reinhardtii which has emerged as a powerful model system. Photosynthetic function is dispensable when this alga is grown in the presence of acetate as a carbon source, a property that greatly facilitates the isolation of mutants and their genetic analysis. Nuclear, chloroplast and mitochondrial transformation can be easily achieved. Furthermore the sequences of the nuclear, chloroplast and mitochondrial genomes of this alga have been determined (see the website of the Chlamydomonas Center: http://www.chlamy.org/ ). We also use the model plant Arabidopsis thaliana in some of our studies. It is of particular interest to compare a unicellular, motile alga like Chlamydomonas with a multicellular, sessile plant like Arabidopsis.

Chloroplast biogenesis and function depend on the concerted action of the nuclear and organellar genetic systems. While the majority of chloroplast proteins are nucleus-encoded, the chloroplast genome contains a relatively small number of genes, required mainly for photosynthesis and chloroplast gene expression. Genetic analysis of mutants deficient in photosynthetic activity has revealed a surprisingly large number of nuclear genes that are required for the expression of specific chloroplast genes. They appear to be involved in several post-transcriptional steps including RNA stability, RNA processing, splicing, translation and assembly of photosynthetic complexes.

Figure 1. Biosynthesis of the photosynthetic apparatus.
Figure 1. Biosynthesis of the photosynthetic apparatus.

Photosynthetic complexes each consist of nucleus- and chloroplast-encoded subunits. The former are synthesized as precursors on cytosolic 80S ribosomes and targeted to the chloroplast. The latter are synthesized on chloroplast 70S ribosomes. Several post-transcriptional steps in the chloroplast, such as RNA stability, processing, splicing, editing and translation, plus the assembly of the protein complexes, require the action of numerous nucleus-encoded factors. In a reciprocal manner, the state of the chloroplast is perceived by the nucleus through retrograde signaling chains.

The role of the nucleus in chloroplast biogenesis: Chloroplast RNA metabolism and translation

We are studying several nuclear mutants of Chlamydomonas that fail to accumulate specific chloroplast mRNAs. Because chloroplast RNA stability, processing and translation are closely coupled, any of these steps could be affected in the mutants. We have shown that the target site of the nucleus-encoded function affected in these mutants is often located within the mRNA 5'UTR (untranslated region). Cis-acting elements which are critical for stabilization of the RNA have been localized within their 5'UTRs. Because the nuclear transformation efficiency is high, we have used it for genomic complementation of the mutants and isolated the corresponding nuclear genes which are affected in these mutants. Some of these genes encode RNA-binding proteins derived from enzymes involved in RNA metabolism. These proteins appear to have been recruited for novel roles in chloroplast gene expression during evolution. Other genes involved in chloroplast gene expression encode proteins of the TPR (tetratricopeptide repeats), PPR (pentatrico peptide repeats) and OPR (octatrico peptide repeats) family containing degenerate repeats of 34 to 38 amino acids. Repeat-containing proteins are widespread in nature and are involved in a broad range of cellular activities. Some of these repeats appear to form RNA-binding domains.

The chloroplast psaA gene, encoding one of the apoproteins of photosystem I, has an unusual structure in Chlamydomonas reinhardtii. It consists of three exons that are scattered at widely separate loci of the plastid genome and are flanked by group II intron sequences. The mature mRNA is assembled from three separate precursors in two steps of splicing in trans. We have obtained numerous nuclear mutants that fail to assemble mature psaA RNA. They belong to three phenotypic classes: some are defective for the trans-splicing of the first split intron of psaA (class C mutants), some for the trans-splicing of the second split intron (class A), and others for both trans-splicing steps (class B). A complementation analysis has shown that in each phenotypic class there are several nuclear genes belonging to a total of at least fourteen loci. A remarkable feature is that the first psaA intron consists of at least three independently transcribed parts. The chloroplast locus, tscA, encodes a small RNA which is thought to assemble with the precursors of exon 1 and exon 2 to form the characteristic structure of group II introns. This tripartite psaA intron could represent an intermediate in the evolution of group II introns to nuclear introns. Our recent efforts to understand psaA trans-splicing have focused on the cloning and characterization of three of the nuclear genes, Raa1, Raa2 and Raa3, and the polypeptides they encode. Raa1 is a very large protein that contains five OPR repeats and is required for trans-splicing of both introns. Raa2, which is involved in splicing of the second intron, is related to pseudo-uridine synthases and is associated together with Raa1 in a 500kDa complex. The size and integrity of this complex is affected by mutations mapped in other complementation groups suggesting that other proteins are also present in this multiprotein complex. Alternatively, these other factors could be required for the assembly of the complex. Raa3, required for trans-splicing the first intron, forms a 1800 kDa complex with tscA RNA and the psaA exon1 precursor. Thus, at least two different RNA-protein complexes have been identified which could represent plastid spliceosomal complexes of group II introns.

Figure 2. Assembly of PSI in Chlamydomonas.
Figure 2. Assembly of PSI in Chlamydomonas.

The core PSI subunits are encoded by the chloroplast genes psaA, psaB, and psaC, indicated by colored boxes. The corresponding RNAs are shown as wavy lines. The psaA gene is fragmented into three exons whose transcripts are assembled into the mature psaA mRNA through two trans-splicingreactions mediated in part by the nucleus-encoded factors Raa1, Raa2, Raa3, Rat1, and Rat2. Only those factors whose genes have been identified are shown. 70S represents chloroplast ribosomes. Mab1 is specifically required for the processing/stability of the psaB mRNA and Tab2 and Tab3 are necessary for its translation. PsaB is the first PSI subunit to be inserted into the membrane. In its absence the PsaA subunit cannot assemble and inhibits directly or indirectly its own synthesis through the CES process (control by epistasy of synthesis) characterized by Wollman and coworkers. Once CPI consisting of PsaA and PsaB has been formed, the next subunit PsaC can assemble to form RCI. In the absence of CPI, PsaC inhibits its own synthesis. The other PSI subunits are integrated subsequently into the complex. Ycf3, Ycf4, and Ycf37 are specific PSI assembly factors that act at various stages of assembly.

Tab2 and Tab3 are two nuclear genes that control translation of the chloroplast psaB mRNA via cis-acting sequences in its 5' UTR. Mab1 is required for the processing/stability of the same chloroplast mRNA. Tab3 belongs to the family of OPR proteins. These 38 amino acid repeats are also found in a family of chloroplast proteins such as the trans-splicing factor Raa1 and the translation factors Tbc2 and Tda1. Thus the OPR proteins of Chlamydomonas may be the functional counterparts of the large family of PPR proteins which have similar functions in RNA metabolism in higher plants.

Adaptation to a changing light environment through state transitions

Plants and algae have the remarkable ability to adapt to changes in light quality and quantity. They balance energy input and consumption in the short term through non-photochemical dissipation of excess energy and through state transitions. This adjustment occurs at the level of the primary reactions of photosynthesis catalyzed by photosystem II (PSII) and photosystem I (PSI) which are linked in series through the plastoquinone pool, the cytochrome b6f complex (Cytb6f ) and plastocyanin. Because the antenna systems of PSII and PSI have different light absorption properties, changes in light conditions lead to unequal excitation of the photosystems and to changes in the redox state of the plastoquinone pool. Reduction of the plastoquinone pool by overexcitation of PSII relative to PSI leads to the activation of a kinase through Cytb6f and to phosphorylation of the light-harvesting system of PSII (LHCII) (state 2), a fraction of which is then displaced from PSII to PSI. Overexcitation of PSI relative to PSII leads to the oxidation of the plastoquinone pool, inactivation of the kinase and dephosphorylation of the mobile LHCII and its return to PSII (state 1).This reversible redistribution of excitation energy between the two photosystems leads to an overall increase in photosynthetic quantum yield. It also triggers a change from linear to cyclic electron flow (or vice-versa) and plays a key role in ATP homeostasis.

Figure 3. State transitions.
Figure 3. State transitions.

Transition from state 1 to state 2 occurs when the redox state of the plastoquinone pool (PQ) is reduced, for example, as a result of preferential excitation of PSII relative to PSI. Docking of plastoquinol (PQH2) to the Qo site of Cytb6f leads to the activation of the protein kinase Stt7/STN7, which phosphorylates LHCII. Phosphorylated LHCII dissociates from PSII and binds to PSI. In C. reinhardtii, this state 1 to state 2 transition leads to a switch from linear electron flow to cyclic electron flow. Upon preferential excitation of PSI relative to PSII, the kinase is inactivated and the PPHI/TAP38 phosphatase dephosphorylates LHCII, which moves back to PSII.

Using a fluorescence video-imaging screen based on the differences in fluorescence yield in state 1 and state 2, we have isolated a dozen of mutants deficient in state transitions. Amongst these mutants, stt7 was found to be deficient in the thylakoid serine-threonine protein kinase Stt7. The Stt7 kinase is required for LHCII phosphorylation and state transitions. It is conserved in Arabidopsis and belongs to a small family of thylakoid protein kinases including Stt7 and Stl1 in Chlamydomonas and STN7 and STN8, respectively, in Arabidopsis. We have found that Stt7 is associated with LHCII, Cytb6f and PSI and that its level changes during state transitions. Although we have been able to identify several of its substrates (collaboration with Alex Vener, Linkoping University), the mechanism of regulation of the kinase remains unknown and is under investigation. Using bioinformatics and reverse genetics approaches we have identified the PPH1 phosphatase in Arabidopsis which is able to dephosphorylate the LHCII proteins under state 1 conditions. The function of other chloroplast phosphatases is under investigation. The ultimate aim is to understand the regulation of this chloroplast-phosphatase thylakoid network.

Figure 4. Thylakoid kinase and phosphatase signaling network.
Figure 4. Thylakoid kinase and phosphatase signaling network.

The redox state of the plastoquinone pool is influenced by light quality and quantity, cellular ATP and CO2 level, and modulates the activity of the Stt7/ STN7 and Stl1/STN8 kinases required for the phosphorylation of LHCII and PSII, respectively. It may also act on CSK. Activation of the Stt7/STN7 kinase depends on its close interaction with the Cytb6f complex. After it is phosphorylated, LHCII moves from PSII to PSI. The dephopshoryaltion of LHCII is mediated by the phosphatase PPH1/TAP38. In addition, the redox state of Trx also influences the activity of Stt7/STN7. The proposed action of CK2 on Stt7/STN7 and of CSK on chloroplast gene expression is still tentative and will need further experimental support. Besides its role in short-term acclimation, STN7 is also involved in the long-term response (LTR). Protein kinases are highlighted in yellow.

SOLAR-H2: European Solar-Fuel Initiative. Renewable Hydrogen from Sun and Water. Science Linking Molecular Biomimetics and Genetics

This project involves 12 European research teams and integrates two frontline research topics: artificial photosynthesis in man-made biomimetic systems, and photobiological H2 production in living organisms. C. reinhardtii is able to evolve hydrogen under anaerobic conditions in the light. However this occurs only transiently because the hydrogenase is quickly inactivated by the oxygen produced through photosynthesis. To circumvent this problem we have developed a repressible chloroplast gene expression system in C. reinhardtii which allows us to turn off PSII. Under these conditions oxygen is consumed by respiration and hydrogen production is induced until all the reduced carbon sources are exhausted. We have taken advantage of the properties of the copper-sensitive cytochrome c6 promoter and of the nucleus-encoded Nac2 chloroplast protein. This protein is specifically required for the stable accumulation of the chloroplast psbD RNA and acts on its 5'UTR. We have constructed a strain containing the Nac2 coding sequence driven by the cytochrome c6 promoter so that psbD is expressed in copper-depleted but not in copper replete medium. Because psbD encodes the D2 reaction center polypeptide of photosystem II (PSII), the repression of psbD leads to the loss of PSII. We have tested this system for hydrogen production. Upon addition of copper to cells pre-grown in copper deficient medium, PSII levels declined to a level at which oxygen consumption by respiration exceeded oxygen evolution by PSII. The resulting anaerobic conditions led to the induction of hydrogenase activity. Because the Cyc6 promoter is also induced under anaerobic conditions, this system opens possibilities for sustained cycling hydrogen production. Moreover, this inducible gene expression system is applicable to any chloroplast gene by replacing its 5'UTR with the psbD 5'UTR in the same genetic background.

Repressible chloroplast gene expression

In order to address the function of chloroplast essential genes, we have developed another repressible chloroplast gene expression system which is based on the properties of the vitamin-sensitive B12 promoter and TPP-riboswitch characterized by Allison Smith and her colleagues and of the nucleus-encoded Nac2 chloroplast protein. A construct containing the Nac2 coding sequence fused to the B12 responsive promoter and TPP (thiamine pyrophosphate)-riboswitch was introduced into the nac2-26 mutant strain deficient in Nac2. In this transformant psbD is expressed in vitamin-depleted but not in vitamin replete medium. It is possible to fuse the psbD 5’UTR to any chloroplast gene for repressing its expression with vitamins. This has been achieved with the ycf1, rps12, rpoA and clpP genes which are essential for cell growth and survival. The aim is to use this repressible system to examine how the cell responds to the repression of these genes. Experiments are in progress with the ClpP repressible system to examine the early events in this response using proteomics and RNA seq technology.