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 ▲]

Mitochondrial Proteomics of a Secondary Green Alga

Poster (2012, June)

Euglena gracilis is an alga that derives from a secondary endosymbiosis with a green alga. Our general objective is to study the interactions established between the chloroplast and the mitochondrion ... [more ▼]

Euglena gracilis is an alga that derives from a secondary endosymbiosis with a green alga. Our general objective is to study the interactions established between the chloroplast and the mitochondrion during the endosymbiosic event and to determine the phylogenetic origin of the genes encoding the proteins involved in these interactions. As a first step, we performed a high-throughput analysis of the mitochondrial proteome of Euglena gracilis. Our MS/MS experiments mostly recover mitochondrial proteins representing 15 mitochondrial pathways, which indicates that our mitochondrial extracts are relatively pure, but the phylogenetic origins of the corresponding genes are surprisingly diverse. [less ▲]

Reconstruction of a Human Mitochondrial Complex I Mutation in the Green Microalga Chlamydomonas

in Mitochondria in life, death and disease (2012, May)

Reconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas.

in Plant Journal : for Cell & Molecular Biology (2012)

Defects in complex I (NADH:ubiquinone oxidoreductase) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate ... [more ▼]

Defects in complex I (NADH:ubiquinone oxidoreductase) are the most frequent cause of human respiratory disorders. The pathogenicity of a given human mitochondrial mutation can be difficult to demonstrate because the mitochondrial genome harbors large numbers of polymorphic base changes that have no pathogenic significance. In addition, mitochondrial mutations are usually found in the heteroplasmic state, which could hide the biochemical effect of the mutation. We propose that the unicellular green alga Chlamydomonas could be used to study such mutations because (1) respiratory-deficient mutants are viable and mitochondrial mutations are found in the homoplasmic state, (2) transformation of the mitochondrial genome is feasible, (3) Chlamydomonas complex I is close to that of humans. To illustrate that, we have introduced a Leu157Pro substitution in the Chlamydomonas ND4 subunit of complex I of two different recipient strains by biolistic transformation, demonstrating that site-directed mutagenesis of the Chlamydomonas mitochondrial genome is possible. This substitution did not lead to any respiratory enzyme defect when it is present in the heteroplasmic state in a patient presenting chronic progressive external ophthalmoplegia. When present in the homoplasmic state in the alga, the mutation does not prevent the assembly of the 950 kDa whole complex I which conserves nearly all the NADH dehydrogenase activity of the peripheral arm. However, the NADH:duroquinone oxidoreductase activity is strongly reduced, suggesting that the substitution could affect ubiquinone fixation to the membrane domain. The in vitro defects are correlated in vivo with a decrease in dark respiration and growth rate. [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 ▲]

Characterization of an internal type-II NADH dehydrogenase from Chlamydomonas reinhardtii mitochondria

in Current Genetics (2012), 58

Complexes I in the green lineage.

in Sazanov, Leonid (Ed.) A structural perspective on complex I. (2012)

Insertional mutagenesis to select mutants for modified hydrogen photoproduction in Chlamydomonas reinhardtii

Poster (2011, May 17)

The unicellular green alga Chlamydomonas reinhardtii has evolved the ability to redirect electrons from the photosynthetic chain to drive hydrogen production via chloroplast oxygen-sensitive hydrogenases ... [more ▼]

The unicellular green alga Chlamydomonas reinhardtii has evolved the ability to redirect electrons from the photosynthetic chain to drive hydrogen production via chloroplast oxygen-sensitive hydrogenases. This process occurs under anaerobic conditions and provides a biological basis for solar-driven hydrogen production. Nevertheless, the yield is a major limitation for an economic viability and fundamental knowledge is still needed in order to have a better understanding of the process. In 2000, Melis and co-worker defined a protocol allowing a sustainable hydrogen production in sulfur deprivation condition. By adjustment of an existent protocol called the Winkler test, we are trying to isolate mutants with an attenuated photosynthesis to respiration capacity ratio (P/R ratio). This kind of mutants could be able to reach anoxia needed for hydrogenases activity without the stressful impact of sulfur deprivation. An insertional mutagenesis of Chlamydomonas has been carried out with an hygromycin resistance cassette and about 2500 transformants have generated and screened by the adapted Winkler test. We have isolated several oxygen-consuming mutants and the most promising one is subject to functional, molecular and genetic characterization. To discover new genes involved in hydrogenases activity, we are also planning to screen the same insertional library for mutants with attenuated levels of hydrogen photoproduction, using sensitive chemochromic sensor films which turn in blue in presence of hydrogen. We are currently making the chemochromic sensor WO3 films by dip-coating which is on the brink of being useable. [less ▲]

Characterization of a knock-down mutant deficient for isocitrate lyase in Chlamydomonas reinhardtii

in Microorganisms for bio-fuel production from sunlight, ESF conference (2011)

Functional analysis of hydrogen photoproduction in respiratory-deficient mutants of Chlamydomonas reinhardtii

in International Journal of Hydrogen Energy (2011)

In this paper, mitochondrial mutants of Chlamydomonas reinhardtii defective for respiratory complex I (NADH:ubiquinone oxidoreductase), complex III (ubiquinol cytochrome c oxidoreductase) and both ... [more ▼]

In this paper, mitochondrial mutants of Chlamydomonas reinhardtii defective for respiratory complex I (NADH:ubiquinone oxidoreductase), complex III (ubiquinol cytochrome c oxidoreductase) and both complexes I and III were analyzed for H2 photoproduction. Several parameters were followed during the S-deficiency stage and the anaerobic stage leading to H2 photoproduction. At the early aerobic S-deficiency stage, starch and neutral lipids accumulated in all strains but their amount was significantly decreased in mutants compared to wild type. During the H2 photoproduction process, whereas starch content strongly decreased in all strains, neutral lipid amount remained nearly unchanged, suggesting that starch degraded by glycolysis is the preferential substrate for energy production during anaerobiosis. The mutants displayed a decrease in H2 photoproduction correlating to the number of active mitochondrial proton-pumping sites lost in the strains. Our results thus highlight the critical role of oxidative phosphorylation during the first (aerobic) stage of S-starvation when carbon resources are accumulated. [less ▲]