Light induced photosynthetic electron transfer upon anaerobiosis in Chlamydomonas: Kinetics, electron sinks and setup of a fluorescence screen to identify new players

Conference (2012, June 15)

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of various fermentative pathways. Among them, oxygen-sensitive hydrogenases (hyd) catalyze the reduction of protons from ... [more ▼]

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of various fermentative pathways. Among them, oxygen-sensitive hydrogenases (hyd) catalyze the reduction of protons from reduced ferredoxin resulting in the production of molecular hydrogen. In this work, light-induced photosynthetic electron transfer after a prolonged dark-anaerobiosis period was studied by following the kinetics of chlorophyll fluorescence emission, P700 oxidation and proton-motive force formation and consumption during the first 3 seconds of illumination. We show that during the induction of photosynthesis, an hyd-dependent photosynthetic electron transfer operates at a maximal rate of 110 electrons per photosystem per second, that is about half the one measured in aerobiosis. The implication in this process of components of the linear, cyclic and chlororespiratory electron transfer pathways, as well as various electron sinks, are investigated thanks to the availability of mutants. In a next step, we screen an insertional mutant library (~3000 clones) on the basis of the fluorescence induction kinetics upon a shift from dark-anaerobiosis to light. Five mutants display the signature of mutants deficient for NADPH:PQ oxidoreductase or hyd activities. In particular, one is defective for hydrogenase HydG assembly factor. This mutant behaves exactly has the hydEF mutant, thus confirming that in vivo both the assembly factors are required for an efficient hydrogenase activity. [less ▲]

Function of the chloroplastic NADP(H) dehydrogenase NDA2 for the H2 photoproduction in sulphur-deprived Chlamydomonas reinhardtii

in Journal of Biotechnology (2012), 162

The relative contributions of the PSII-dependent and Nda2-dependent pathways for H2 photoproduction were investigated in the green microalga Chlamydomonas reinhardtii after suphur-deprivation. For this ... [more ▼]

The relative contributions of the PSII-dependent and Nda2-dependent pathways for H2 photoproduction were investigated in the green microalga Chlamydomonas reinhardtii after suphur-deprivation. For this purpose, H2 gas production was compared for wild-type and Nda2-deficient cells with or without DCMU (a PSII-inhibitor) in the same experimental conditions. Nda2-deficiency caused a 30 % decrease of the maximal H2 photoevolution rate observed shortly after the establishment of anoxia, and an acceleration of the decline of H2 photoevolution rate with time. DCMU addition to Nda2-deficient cells completely inhibited H2 photoproduction, showing that the PSII-independent H2 photoproduction relies on the presence of Nda2, which feeds the photosynthetic electron transport chain with electrons derived from oxidative catabolism. Nda2-protein abundance increased as a result of sulphur deprivation and further during the H2 photoproduction process, resulting in high rates of non-photochemical plastoquinone reduction in control cells. Nda2-deficiency had no significant effect on photosynthetic and respiratory capacities in sulphur-deprived cells, but caused changes in the cell energetic status (ATP and NADPH/NADP+ ratio). The rapid decline of H2 photoevolution rate with time in Nda2-deficient cells revealed a more pronounced inhibition of H2 photoproduction by accumulated H2 in the absence of non-photochemical plastoquinone reduction. Nda2 is therefore important for linking H2 photoproduction with catabolism of storage carbon compounds, and seems also involved in regulating the redox poise of the photosynthetic electron transport chain during H2 photoproduction. [less ▲]

Influence of the nitrogen source and of the mitochondrial alternative oxidase (AOX) on the response to sulphur deficiency in Chlamydomonas reinhardtii

Poster (2011, September 18)

La réponse photosynthétique d'une algue verte à la carence en soufre

Book published by Editions universitaires europeennes (2010)

Chlamydomonas reinhardtii possède la capacité de produire de l'hydrogène à la lumière en absence d'oxygène. Cette condition peut être obtenue en cultivant les algues dans un milieu carencé en soufre. La ... [more ▼]

Chlamydomonas reinhardtii possède la capacité de produire de l'hydrogène à la lumière en absence d'oxygène. Cette condition peut être obtenue en cultivant les algues dans un milieu carencé en soufre. La carence en soufre entraîne une forte diminution de l'activité du photosystème II tout en maintenant une respiration élevée, ce qui provoque un passage de cultures fermées en anoxie et induit la production d'hydrogène. Dans cette étude, nous avons caractérisé la réponse photosynthétique à la carence en soufre chez la souche sauvage et la souche déficiente en oxydase alternative mictochondriale (AOX) dans des milieux contenant de l'ammonium ou du nitrate comme source d'azote. L'AOX, inductible par le nitrate, fait partie de la chaîne de transport d'électrons mitochondriale et catalyse l'oxydation de l'ubiquinol en transférant directement ses électrons à l'oxygène. Ainsi l'AOX entre en compétition avec le complexe III et est impliquée dans une voie de dissipation du pouvoir réducteur en excès. [less ▲]

Hydrogen photo-evolution upon S deprivation stepwise: An illustration of microalgal photosynthetic and metabolic flexibility and a step stone for future biotechnological methods of renewable H2 production

in Photosynthesis Research (2010), 106

The metabolic flexibility of some photosynthetic microalgae enables them to survive periods of anaerobiosis in the light by developing a particular photofermentative metabolism. The latter entails ... [more ▼]

The metabolic flexibility of some photosynthetic microalgae enables them to survive periods of anaerobiosis in the light by developing a particular photofermentative metabolism. The latter entails compounds of the photosynthetic electron transfer chain and an oxygen-sensitive hydrogenase in order to reoxidise reducing equivalents and to generate ATP for maintaining basal metabolic function. This pathway results in the photo-evolution of hydrogen gas by the algae. A decade ago Melis and coworkers managed to reproduce such a condition in a laboratory context by depletion of sulfur in the algal culture media, making the photo-evolution by the algae sustainable for several days (Melis et al. 2000). This observation boosted research in algal H2 evolution. A feature, which due to its transient nature was long time considered as a curiosity of algal photosynthesis suddenly became a phenomenon with biotechnological potential. Although the Melis procedure has not been developed into a biotechnological process of renewable H2 generation so far, it has been a useful tool for studying microalgal metabolic and photosynthetic flexibility and a possible step stone for future H2 production procedures. Ten years later most of the critical steps and limitations of H2 production by this protocol have been studied from different angles particularly with the model organism C. reinhardtii, by introducing various changes in culture conditions and making use of mutants issued from different screens or by reverse genomic approaches. A synthesis of these observations with the most important conclusions driven from recent studies will be presented in this review. [less ▲]

A type II NAD(P) H dehydrogenase mediates light-independent plastoquinone reduction in the chloroplast of Chlamydomonas

in Proceedings of the National Academy of Sciences of the United States of America (2008), 105(51), 20546-20551

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 ... [more ▼]

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. [less ▲]