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ChlamyStation : Chlamydomonas Photosynthetic Mutant Collection

PredAlgo : multi-subcellular localization prediction tool dedicated to Algae

IBPC : Institut de Biologie Physico-Chimique

LabEx DYNAMO

Research Domains

french version

   We study the chloroplast in vivo. That is, in a context in which it develops the full complexity of its intracellular interactions. The photosynthetic apparatus is both the object of our studies and a probe for chloroplast physiology. Its’ constitutive elements allow us to observe the energetic or redox state of the organelle as well as nuclear-chloroplast coordination in the expression of photosynthetic proteins. Our laboratory is profoundly multidisciplinary for two reasons :

  • The photosynthetic function (the collection of redox reactions governed by light capture within certain living organisms) finds itself at the crossroads of physics, chemistry and biology.

  • Our laboratory must deal with the development of all methodologies related to the study of this integrated macromolecular system; from classical genetics to biophysics, including genomics, molecular genetics, biochemistry and cellular biology of proteins.

   We are addressing several issues which are briefly outlined below and generally illustrated by two publications, one highlighting work from 2000-2010 and the other from 2010-today to be found under the following headings :

 1. Functional and Genetic Diversity of Photosynthesis

 2. Biogenesis of the Photosynthetic Apparatus in Chlamydomonas

 

1. Functional and Genetic Diversity of Photosynthesis

   Photosynthesis converts light energy into a difference in transmembrane electrochemical potential which serves as the motive force for ATP synthesis. This conversion requires, in series, a succession of electron transfers between the different redox cofactors making up the chain. These electron transfers are generally coupled to the translocation of protons from one side of the membrane to the other. Although these basic principles suffer no exception, a glance at the biodiversity of photosynthetic organisms allows one to realize that there are many variations on these common themes. Understanding this genetic and functional diversity provides means to address the integration of the photosynthetic function to the physiology of the cell as well as the various regulatory processes that tunes the photosynthetic function to match the metabolic demands of the cells. We address these issues following two main streams: the Physiological Regulations of Photosynthesis and the Genomics and Bioinformatics of Photosynthetic Metabolism.

 1.1. Physiological Regulations of Photosynthesis

   This issue is dealt with through the lens of the photosynthetic function and its modulation/regulation in response to the fluctuations it undergoes within or amongst its natural environment(s). Our strategy is, on the one hand, to deepen our understanding of the catalysis sustained by all the individual complexes that make the bioenergetic chain and, on the other hand, to rely on this knowledge to design original approaches or observables allowing the study of the integration of photosynthetic function to the cell metabolism and its environmental modulations. The main topics that we address are:

What are the thermodynamic parameters and kinetics of the electron transfer reaction? What redox co-factors are involved?

  • Li, Y., A. van der Est, M. G. Lucas, V. M. Ramesh, F. Gu, A. Petrenko, S. Lin, A. N. Webber, F. Rappaport, and K. Redding. 2006. Directing electron transfer within Photosystem I by breaking H-bonds in the cofactor branches. Proc Natl Acad Sci U S A, 103:2144-2149.

  • Rappaport, F., N. Ishida, M. Sugiura, and A. Boussac (2011) Ca(2+) determines the entropy changes associated with the formation of transition states during water oxidation by Photosystem II. Energy & Environmental Sciences 4:2520-2524.

By which mechanism is proton transfer coupled to electron transfer ?

What are the functional consequences of the adaptations or acclimations of the photosynthetic function to various environments?

   But the photosynthetic function is not the mere addition of the catalysis performed by its individual components. As an example, in most photosynthetic eukaryotes, the lateral distribution of these components in the thylakoid membrane is heterogeneous and the ultrastructural organization of the membrane determines well defined domains with specific proteins compositions. In addition, this ultrastructural heterogeneity may undergo massive reorganization in response to changing environmental conditions and these reorganization impacts the function of the chain, which prompted us to address the following issues:

What are the conditions and molecular mechanisms promoting cyclic electron flow?

What are the functional consequences of the peculiar ultrastructural organization of the bioenergetic electron transfer chain?

How do the respiratory, chlororespiratory and photosynthetic electron transfer chains interplay to meet the cellular metabolic demand in reducing power and ATP?

 1.2. Genomic and Bioinformatics approaches to Photosynthetic Metabolism

   Photosynthesis is a fully integrated biological function anchored in a complex genetic program. Thanks to the extraordinary development of genomic tools in land plants as well as in algae, it has become possible to identify genes involved in photosynthesis and in the biogenesis of the photosynthetic apparatus. Both forward and reverse genetic approaches are used in our lab to create mutants in the genes of interest.

What is the gene repertoire of Chlamydomonas ?

What can we learn by studying the genome of other algae?

  • Tourasse, N. J., T. Barbi, J. C. Waterhouse, N. Shtaida, S. Leu, S. Boussiba, S. Purton and O. Vallon (2014). The complete sequence of the chloroplast genome of the green microalga Lobosphaera (Parietochloris) incisa. Mitochondrial DNA: 1-3.

  • Tourasse, N. J., Y. Choquet and O. Vallon (2013). PPR proteins of green algae. RNA Biology 10(9): 1526-1542.

What proteins are addressed to the chloroplast?

What can we do to facilitate the obtention and analysis of mutants in microalgae?

2. Biogenesis of the Photosynthetic Apparatus in Chlamydomonas

 2.1. Gene Expression and Protein Assembly in the Chloroplast

   Chloroplasts have evolved from free-living cyanobacteria engulfed by a primitive eukaryotic cell. Compared to their ancestors, chloroplasts have undergone massive genetic losses and retain only tiny genomes (50-200 respectively) that only code for a fraction of the factors required for their own expression or of the polypeptides of the multi-subunits photosynthetic protein complexes. Still, the functional assembly of photosynthetic protein requires the stoichiometric production of its subunits, even if encoded in two distinct genetic compartments.

   The machinery for genetic expression is still prokaryotic in nature, as it mostly involved the products of ancestral cyanobacterial genes transferred to the nucleus. Yet, organelles are not just bacteria that have transferred part of their genome to another compartment. Indeed, the nucleus tightly controls chloroplast gene expression by encoding gene-specific factors that bind chloroplast mRNAs – usually in the 5'UTR region - and govern their stability or translatability.

   We are interested in understanding the biogenesis of these macromolecular multimeric objects, by answering the following questions:

What are the mechanisms of chloroplast gene expression?

  • S. Eberhard, D. Drapier and F-A. Wollman. "Searching limiting steps in the expression of chloroplast-encoded proteins : relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii". Plant J., 2002, 31, 149-160

  • Eberhard S, Loiselay C, Drapier D, Bujaldon S, Kuras R, ChoquetY, Wollman F-A (2011) Dual functions of the nucleus-encoded factor TDA1 in trapping and translation activation of atpA transcripts in Chlamydomonas chloroplast. Plant J. 67(6):1055-66

How does the nucleus control the expression of chloroplast genes?

  • Raynaud C, Loiselay C, Wostrikoff K, Kuras R, Girard-Bascou J, Wollman FA, Choquet Y. (2007) “Evidence for regulatory function of nucleus-encoded factors on mRNA stabilization and translation in the chloroplast” Proc. Natl. Acad. Sci. U.S.A., 104, 9093-8.

  • Boulouis A, Drapier D, Razafimanantsoa H, Wostrikoff K, Tourasse NJ, Pascal K, Girard-Bascou J, Vallon O, Wollman F-A and Choquet Y (2015) Spontaneous dominant mutations in Chlamydomonas highlight ongoing evolution of by gene diversification. Plant Cell, in press.

Which mechanisms ensure the stoichiometric accumulation of subunits for a same protein complex?

  • Drapier D., Rimbault B., Vallon O., Wollman F.-A. and Choquet Y. (2007) Intertwined translational regulations set uneven stoichiometry of chloroplast ATP synthase subunits. EMBO J., 26, 3581-3591.

  • Boulouis A, Raynaud C, Bujaldon S, Aznar A, Wollman F-A, and Choquet Y (2011) The Nucleus-Encoded trans-Acting Factor MCA1 Plays a Critical Role in the Regulation of Cytochrome f Synthesis in Chlamydomonas Chloroplasts. Plant Cell 23, 333-49.

 2.2. Chloroplast post-translational modifications

   Protein post-translation modifications contribute greatly to the expression of the photosynthetic apparatus by degrading damaged proteins or by acting on regulatory proteins. We focus on: 1) proteolysis, 2) biogenesis proteins that catalyze cofactor binding and 3) various covalent modifications (phosphorylation, nitrosylation, glutathionylation, disulfide bridge formations, etc) that regulate the biogenesis, stability and function of the photosynthesis apparatus.

How are photosynthesis proteins degraded?

  • Majeran W, Wollman FA, Vallon O (2000) Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b6f complex. Plant Cell 12(1), 137-149.

  • Malnoë A, Wang F, Girard-Bascou J, Wollman FA, de Vitry C (2014) Thylakoid FtsH Protease Contributes to Photosystem II and Cytochrome b6f Remodeling in Chlamydomonas reinhardtii under Stress Conditions. Plant Cell 26(1), 373-390.

How are polypeptide chains and their cofactors targeted and assembled in the photosynthetic membrane?

  • Kuras R, Saint-Marcoux D, Wollman FA, de Vitry C. (2007) A specific c-type cytochrome maturation system is required for oxygenic photosynthesis. Proc Natl Acad Sci USA 104(23), 9906-9910.

  • Saint-Marcoux D, Wollman FA, de Vitry C (2009) Biogenesis of cytochrome b6 in photosynthetic membranes. J Cell Biol 185(7), 1195-1207.

Which regulations occur by covalent modifications of chloroplast proteins?

  • Giglione C, Vallon O, Meinnel T (2003) Control of protein life-span by N-terminal methionine excision. EMBO J, 22, 13-23.

  • Allorent G, Tokutsu R, Roach T, Peers G, Cardol P, Girard-Bascou J, Seigneurin-Berny D, Petroutsos D, Kuntz M, Breyton C, Franck F, Wollman FA, Niyogi KK, Krieger-Liszkay A, Minagawa J, Finazzi G (2013) A Dual Strategy to Cope with High Light in Chlamydomonas reinhardtii. Plant Cell 25, 545-557.

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