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ChlamyStation : Chlamydomonas Photosynthetic Mutant Collection

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   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 1990-2000 and the other from 2001-2005 to be found under the following three headings :

 Transfer of electrons and protons associated with photosynthetic chain function

 Dynamics of the photosynthetic membrane supramolecular organisation

 Biogenesis and degradation of photosynthetic apparatus proteins

Transfer of electrons and protons associated with photosynthetic chain function

   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. Our investigations deal with all the protein complexes involved in photosynthetic function and have permitted us to provide some insights into the following questions:

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

• Guergova-Kuras, M., Boudreaux, B., Joliot, A., Joliot, P. and Redding, K. “Evidence for two active branches for electron transfer in photosystem I”Proc. Natl. Acad. Sci. U S A, 2001, 98, 4437-42.

• Baymann, F., Rappaport, F., Joliot, P. and Kallas, T. “Rapid electron transfer to photosystem I and unusual spectral features of cytochrome c₆ in Synechococcus sp. PCC 7002 in vivo” Biochemistry, 2001, 40, 10570-10577.

By which mechanism is proton transfer coupled to electron transfer ?

• F. Rappaport and J. Lavergne. « Charge recombination and proton transfer in manganese-depleted Photosystem II » Biochemistry , 1997, 36, 15294-15302.

• Rappaport, F. and Lavergne, J. "Coupling of proton and electron transfer in photosynthetic water oxidase" Biochim. Biophys. Acta, 2001, 1503, 246-259.

What are the thermodynamic and kinetic consequences of a difference in electrochemical potential on the electron and/or proton transfer reactions catalysed by the different protein complexes of the photosynthetic chain ?

• G. Finazzi and F. Rappaport. « In vivo characterization of the electrochemical proton gradient generated in darkness in green algae and its kinetic effects on cytochrome b₆f turnover » Biochemistry , 1998, 37, 9999-100005

• Rappaport, F., Cuni, A., Xiong, L., Sayre, R. and Lavergne, J. «Charge recombination and thermoluminescence in photosystem II» Biophys J, 2005, 88, 1948-1958.

Dynamics of the photosynthetic membrane supramolecular organisation

   The major photosynthetic machinery proteins can be observed as groups of individual macromolecules by electron microscopy or by biochemical methods. In most photosynthetic eukaryotes these different protein complexes have different lateral distributions in the grana (stacked thylakoids) and the stroma (lamellae regions where the thylakoids are in isolation). Their lateral distribution is therefore susceptible to modifications which contribute to the regulation of the photosynthetic function. This regulation is particularly spectacular when the light environment changes i.e. by modifying the intensity or spectral quality of the light; or in response to changes in chloroplast metabolism. For example, the transition between aerobic and anaerobic environments or when the intracellular ATP charge varies. We are working on the following questions:

What is the molecular mechanism controlling “State Transitions”, the process by which the peripheral antennas and the cytochrome b6f complex move between the two photosystems situated in different membrane domains?

• F. Zito, G. Finazzi, R. Delosme, W. Nitschke, D. Picot and F-A. Wollman. "The Qo site of cytochrome b₆f complexes controls the activation of the LHCII kinase". EMBO J., 1999, 18, 2961-2969.

• C. de Vitry, Y. Ouyang, G. Finazzi, F.-A. Wollman and T.Kallas.  «The chloroplast Rieske iron-sulfur protein. At the crossroad of electron transport and signal transduction » J. Biol. Chem., 2004, 279 , 44621-44627

When light absorption goes beyond the electron transfer capacity, what are the different photo-protection strategies ?

• M. Schroda, O. Vallon, F.A. Wollman and C. Beck. "A chloroplast-targeted HSP70 contributes to the photoprotection and repair of Photosystem II during and after photoinhibition". Plant Cell, 1999, 11, 1165-1178

• Finazzi G, Johnson GN, Dall'Osto L, Joliot P, Wollman FA, Bassi R. « A zeaxanthin-independent nonphotochemical quenching mechanism localized in the photosystem II core complex. » Proc Natl Acad Sci USA. , 2004, 101, 12375-12380

What are the conditions and molecular mechanisms adopted by the photosynthetic machinery for the linear or cyclic flow of electrons ?

• P. Joliot and A. Joliot. "Cyclic electron transfer in plant leaf" Proc.Natl..Acad..Sci.USA., 2002, 99, 10209-10214

• G. Finazzi, F. Rappaport, A. Furia, M. Fleischmann, J-D. Rochaix, F. Zito and G. Forti. "Involvement of state transitions in the switch between linear and cyclic electron flow in Chlamydomonas reinhardtii". EMBO Report , 2002, 3, 280-285

Biogenesis and degradation of photosynthetic apparatus proteins

   The photosynthetic membrane protein complexes are made up of a large number of subunits ranging from 10 for the b6f cytochrome complex to more than 20 for photosystem II. Certain are coded for by the chloroplast genome and others by the nuclear genome. These polypeptides, the products of different genetic origins, are associated with numerous cofactors making up multi-molecular complexes constructed in precise stochiometric ratios. We want to understand the biogenesis of these complex systems and to answer the following questions:

What are the mechanisms of chloroplast gene expression ?

• D. Drapier, H. Suzuki, H. Levy, B. Rimbault, K.L. Kindle, D.B. Stern and F-A. Wollman. « The chloroplast atpA gene cluster in Chlamydomonas reinhardtii: Functional analysis of polycistronic transcription unit » Plant Physiol., 1998, 117, 629-641.

• 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

In which ways can the nuclear genome control the expression of chloroplast genes ?

• Drapier D., Girard-Bascou J. and Wollman F.-A. "Evidence for nuclear control of the expression of the atpA and atpB chloroplast genes in Chlamydomonas". Plant Cell, 1992, 4, 283-295.

• Wostrikoff, K, Choquet, Y., Wollman, F.A. and Girard-Bascou, J. "TCA1, a single nuclear encoded translational activator specific for petA mRNA in Chlamydomonas reinhardtii chloroplast." Genetics, 2001, 159, 119-132.

How are the protein complex’ cofactors and polypeptide chains dispatched and assembled in the photosynthetic membrane?

• R. Kuras, C. de Vitry, Y. Choquet, J. Girard-Bascou, D. Culler, S. Büschlen, S. Merchant and F-A. Wollman. «Molecular genetic identification of a pathway for heme binding to cytochrome b6».J. Biol. Chem., 1997, 272, 32427-32435.

• G. Finazzi, C. Chasen, F-A. Wollman and C.de Vitry. « Thylakoid  targeting of Tat passenger proteins shows no ΔpH dependence in vivo ».EMBO J., 2003, 22, 807-815

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

• Kuras R. and Wollman F.-A. "The assembly of cytochrome b6f complexes: an approach using genetic transformation of the green alga Chlamydomonas reinhardtii". EMBO J., 1994, 13, 1019-1027.

• K. Wostrikoff, J.Girard-Bascou, F.-A. Wollman and Y. Choquet. “Biogenesis of PSI involves a cascade of autoregulation of translation in Chlamydomonas chloroplasts”. EMBO J., 2004, 23, 2696-2705

How are photosynthesis proteins degraded?

• Majeran W., Wollman F.A. and Vallon O. "Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b6f complex". Plant Cell, 2000, 12, 137-149

• Majeran, M., Olive, J., Drapier, D., Vallon, O. and Wollman, F.A. "The light sensitivity of ATP synthase mutants of Chlamydomonas reinhardtii". Plant Physiol., 2001, 126, 421-433.