[en] In photosynthetic eukaryotes, nonphotochemical plastoquinone (PQ) reduction is important for the regulation of photosynthetic electron flow. In green microalgae where this process has been demonstrated, the chloroplastic enzyme that catalyses nonphotochemical PQ reduction has not been identified yet. Here, we show by an RNA interference (RNAi) approach that the NDA2 gene, belonging to a type II NAD(P)H dehydrogenases family in the green microalga Chlamydomonas reinhardtii, encodes a chloroplastic dehydrogenase that functions to reduce PQ nonphotochemically in this alga. Using a specific antibody, we show that the Nda2 protein is localized in chloroplasts of wild-type cells and is absent in two Nda2-RNAi cell lines. In both mutant cell lines, nonphotochemical PQ reduction is severely affected, as indicated by altered chlorophyll fluorescence transients after saturating illumination. Compared with wild type, change in light excitation distribution between photosystems ('state transition') upon inhibition of mitochondrial electron transport is strongly impaired in transformed cells because of inefficient PQ reduction. Furthermore, the amount of hydrogen produced by Nda2-RNAi cells under sulfur deprivation is substantially decreased compared with wild type, which supports previous assumptions that endogenous substrates serve as source of electrons for hydrogen formation. These results demonstrate the importance of Nda2 for nonphotochemical PQ reduction and associated processes in C. reinhardtii.
Bennoun P (1982) Evidence for a respiratory chain in the chloroplast. Proc Natl Acad Sci USA 79:4352-4356.
Godde D, Trebst A (1980) NADH as electron donor for the photosynthetic membrane of Chlamydomonas reinhardtii. Arch Microbiol 127:245-252.
Peltier G, Cournac L (2002) Chlororespiration Annu Rev Plant Biol 53:523-550.
Shimada H, Ugiura M (1991) Fine structural features of the chloroplast genome: Comparison of the sequenced chloroplast genomes. Nucleic Acids Res 19:983-995.
Shinozaki K, et al. (1986) The complete nucleotide sequence of the tobacco chloroplast genome: Its gene organization and expression. EMBO J 5:2043-2049.
Munekage Y, et al. (2004) Cyclic electron flow around photosystem I is essential for photosynthesis. Nature 429:579-582.
Sazanov LA, Burrows PA, Nixon PJ (1998) The chloroplast Ndh complex mediates the dark reduction of the plastoquinone pool in response to heat stress in tobacco leaves. FEBS Lett 429:115-118.
Rumeau D, Peltier G, Cournac L (2007) Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response. Plant Cell Environ 30:1041-1051.
Carol P, et al. (1999) Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation. Plant Cell 11:57-68.
Cournac L, et al. (2000) Electron flow between photosystem II and oxygen in chloroplasts of photosystem I-deficient algae is mediated by a quinol oxidase involved in chlororespiration. J Biol Chem 275:17256-17262.
Robbens S, Derelle E, Ferraz C, Wuyts J, Moreau H, Van de Peer Y (2007) The complete chloroplast and mitochondrial DNA sequence of Ostreococcus tauri: Organelle genomes of the smallest eukaryote are examples of compaction. Mol Biol Evol 24:956-968.
Mus F, Cournac L, Cardettini V, Caruana A, Peltier G (2005) Inhibitor studies on non-photochemical plastoquinone reduction and H(2) photoproduction in Chlamydomonas reinhardtii. Biochim Biophys Acta 1708:322-332.
Rasmusson AG, Geisler DA, Moller IM (2008) The multiplicity of dehydrogenases in the electron transport chain of plant mitochondria. Mitochondrion 8:47-60.
Michalecka AM, et al. (2003) Arabidopsis genes encoding mitochondrial type II NAD(P)H dehydrogenases have different evolutionary origin and show distinct responses to light. Plant Physiol 133:642-652.
Elhafez D, Murcha MW, Clifton R, Soole KL, Day DA, Whelan J (2006) Characterization of mitochondrial alternative NAD(P)H dehydrogenases in Arabidopsis: Intraorganelle location and expression. Plant Cell Physiol 47:43-54.
Wollman FA (2001) State transitions reveal the dynamics and flexibility of the photosynthetic apparatus. EMBO J 20:3623-3630.
Rochaix JD (2007) Role of thylakoid protein kinases in photosynthetic acclimation. FEBS Lett 581:2768-2775.
Finazzi G, Furia A, Barbagallo RP, Forti G (1999) State transitions, cyclic and linear electron transport and photophosphorylation in Chlamydomonas reinhardtii. Biochim Biophys Acta 1413:117-129.
Cardol P, Gloire G, Havaux M, Remacle C, Matagne R, Franck F (2003) Photosynthesis and state transitions in mitochondrial mutants of Chlamydomonas reinhardtii affected in respiration. Plant Physiol 133:2010-2020.
Gaffron H, Rubin J (1942) Fermentative and photochemical production of hydrogen in algae. J Gen Physiol 26:219-240.
Happe T, Naber JD (1993) Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii. Eur J Biochem 214:475-481.
Melis A, Zhang L, Forestier M, Ghirardi ML, Seibert M (2000) Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol 122:127-136.
Ghirardi ML, Togasaki RK, Seibert M (1997) Oxygen sensitivity of algal H2-production. Appl Biochem Biotech 63:141-151.
Melis A (2007) Photosynthetic H2 metabolism in Chlamydomonas reinhardtii (unicellular green algae). Planta 226:1075-1086.
Zhang L, Happe T, Melis A (2002) Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green alga). Planta 214:552-561.
Merchant SS, et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245-250.
Bennoun P (2001) Chlororespiration and the process of carotenoid biosynthesis. Biochim Biophys Acta 1506:133-142.
Joet T, Genty B, Josse EM, Kuntz M, Cournac L, Peltier G (2002) Involvement of a plastid terminal oxidase in plastoquinone oxidation as evidenced by expression of the Arabidopsis thaliana enzyme in tobacco. J Biol Chem 277:31623-31630.
Bulté L, Gans P, Rebeillé F, Wollman FA (1990) ATP control on state transitions in vivo in Chlamydomonas reinhardtii. Biochim Biophys Acta 1020:72-80.
Rebeille F, Gans P (1988) Interaction between Chloroplasts and Mitochondria in Microalgae: Role of Glycolysis. Plant Physiol 88:973-975.
Wollman FA, Delepelaire P (1984) Correlation between changes in light energy distribution and changes in thylakoid membrane polypeptide phosphorylation in Chlamydomonas reinhardtii. J Cell Biol 98:1-7.
Rasmusson AG, Svensson AS, Knoop V, Grohmann L, Brennicke A (1999) Homologues of yeast and bacterial rotenone-insensitive NADH dehydrogenases in higher eukaryotes: Two enzymes are present in potato mitochondria. Plant J 20:79-87.
Kerscher SJ (2000) Diversity and origin of alternative NADH:ubiquinone oxidoreductases. Biochim Biophys Acta 1459:274-283.
Martin W, Herrmann RG (1998) Gene transfer from organelles to the nucleus: How much, what happens, and Why? Plant Physiol 118:9-17.
Posewitz MC, Smolinski SL, Kanakagiri S, Melis A, Seibert M, Ghirardi ML (2004) Hydrogen photoproduction is attenuated by disruption of an isoamylase gene in Chlamydomonas reinhardtii. Plant Cell 16:2151-2163.
Melis A, Happe T (2001) Hydrogen production. Green algae as a source of energy. Plant Physiol 127:740-748.
Harris E (1989) The Chlamydomonas sourcebook (Academic, San Diego, California).
Loppes R, Radoux M (2001) Identification of short promoter regions involved in the transcriptional expression of the nitrate reductase gene in Chlamydomonas reinhardtii. Plant Mol Biol 45:215-227.
Shimogawara K, Fujiwara S, Grossman A, Usuda H (1998) High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 148:1821-1828.
Berthold P, Schmitt R, Mages W (2002) An engineered Streptomyces hygroscopicus aph 7″ gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist 153:401-412.
Remacle C, Cardol P, Coosemans N, Gaisne M, Bonnefoy N (2006) High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes. Proc Natl Acad Sci USA 103:4771-4776.
Cardol P, Matagne RF, Remacle C (2002) Impact of mutations affecting ND mitochondria-encoded subunits on the activity and assembly of complex I in Chlamydomonas. Implication for the structural organization of the enzyme. J Mol Biol 319:1211-1221.
Goldschmidt-Clermont M, Malnoë P, Rochaix JD (1989) Preparation of Chlamydomonas chloroplasts for the in vitro import of polypeptide precursors. Plant Physiol 89:15-18.
Patel AM, Dunn SD (1995) Degradation of Escherichia coli uncB mRNA by multiple endonucleolytic cleavages. J Bacteriol 177:3917-3922.