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See detailInduction of photosynthetic electron transfer upon anoxia in Chlamydomonas: role of hydrogenase activity and PSI-cyclic electron flow
Godaux, Damien ULg; Berne, Nicolas ULg; Remacle, Claire ULg et al

Poster (2013)

In Chlamydomonas reinhardtii, anoxic environment leads to the expression of various fermentative/anaerobic pathways. Among them, oxygen-sensitive hydrogenases catalyze the reduction of protons from ... [more ▼]

In Chlamydomonas reinhardtii, anoxic environment leads to the expression of various fermentative/anaerobic pathways. Among them, oxygen-sensitive hydrogenases catalyze the reduction of protons from reduced ferredoxin resulting in the production of molecular hydrogen. A possible role of chloroplast hydrogenase in the anaerobic induction of photosynthesis has been suggested forty years ago (Kessler, 1973) but never further explored. H2 evolution is a minor and transient phenomenon which is often considered as a safety mechanism to protect photosynthetic chain from overreduction (Melis and Happe, 2001; Hemschemeier et al., 2009). Recent data about hydrogen production in a pgrl1 (Proton Gradient Regulation like1) mutant with limited capacity for PSI-cyclic electron flow (CEF) also suggested a participation of CEF in photosynthesis reactivation after short dark-anoxic periods (Tolleter et al., 2011). Because H2 evolution is improved in pgrl1 mutant, authors came to the conclusion that H+ gradient generated by CEF strongly prevents electron supply to the hydrogenase and is thus a limitating factor for hydrogen production. The aim of our work is to further study the role of hydrogenase and CEF in the photosynthesis reactivation process after short (~1h) or long (>18h) dark-anoxic periods. We take advantage of the availability of hydrogenase-deficient mutants (hydEF, hydG) (Posewitz et al., 2005; Godaux et al., 2013) and above-mentioned CEF-deficient pgrl1 mutant. Light-induced photosynthetic electron transfer is studied by measuring hydrogen and oxygen evolution, as well as by following kinetics of chlorophyll fluorescence emission and P700 oxidoreduction. Firstly, we show that during the induction of photosynthesis after long dark-anoxic periods, there is a linear relationship between hydrogen evolution, PSI and PSII activities, meaning that an hydrogenase- dependent photosynthetic linear electron flow (LEF) mainly operates. Moreover, PSI and PSII photochemical yield are almost null in hydrogenase-deficient mutants. We conclude that hydrogenase is the main sink for photosynthetic electrons upon illumination after prolonged anoxia. Similarly, a linear correlation can be established between hydrogen evolution, hydrogenase expression/activity, and PSI or PSII photochemical yields upon adaptation to anoxia. In the next part of our work, we focus our attention on the role of PSI-CEF in the induction of photosynthesis upon anoxia. Combined measurements of PSI/PSII activities and O2/H2 evolution show that induction of photosynthesis is delayed in a Pgrl1-deficient strain. In absence of Pgrl1 protein, the H+ gradient is also lower and we thus propose that a lack of ATP is responsible for the delayed Calvin cycle reactivation, so that hydrogen production can be achieved for a longer time without inactivation of hydrogenase activity by evolved O2. These results are in good agreement with other results obtained by our group, demonstrating that state transition is a critical process for induction of photosynthesis in anoxia (Ghysels et al., accepted). In conclusion, a Pgrl1-dependent CEF seems to be in first importance to photosynthesis induction after one hour of dark-anaerobiosis adaptation, acting together with an hydrogenase dependant LEF to set favourable conditions for Calvin cycle activation. [less ▲]

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See detailMitochondrial Translation in Green Algae and Higher Plants
Salinas, Thalia; Remacle, Claire ULg; Maréchal-Drouard, Laurence

in Duchêne, A-M (Ed.) Translation in mitochondria and other organelles (2013)

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See detailLight induced photosynthetic electron transfer upon anaerobiosis in Chlamydomonas: Kinetics, electron sinks and setup of a fluorescence screen to identify new players
Godaux, Damien ULg; Emonds-Alt, Barbara ULg; Alric, Jean et al

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 ▲]

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See detailThe MicroH2 project:an association of four laboratories to improve theknowledge on biohydrogen production precesses
Beckers, Laurent ULg; Calusinska, Magdalena ULg; Hamilton, Christopher ULg et al

Poster (2012, June 04)

This poster presents a collaborative research project (MicroH2) held at the University of Liège (Belgium) since 2007 (www.microh2.ulg.ac.be) and involving four different research groups. The project aims ... [more ▼]

This poster presents a collaborative research project (MicroH2) held at the University of Liège (Belgium) since 2007 (www.microh2.ulg.ac.be) and involving four different research groups. The project aims to develop a center of excellence in the fields of photo- and dark- biohydrogen production. Our studies contribute to improve the knowledge of the processes involved in the microbiological production of hydrogen, from a fundamental and practical point of view. Some results are highlighted here. The research concerning photofermentation focuses on the interactions between respiration, photosynthesis and H2-producing pathways in algal microorganisms, by using mitochondrial mutants and genetically modified strains with modified ability for hydrogen production [1-2]. To study the metabolism of the hydrogen production by anaerobic bacteria, pure cultures and defined consortia are used and their production of biogas and soluble metabolites is measured. Moreover, we have developed and optimized molecular tools, like quantitative RT-PCR and FISH, to monitor the variations of bacterial populations in novel bioreactors for hydrogen production [3-4]. We have also mined the complete genomes of Clostridium spp. for putative hydrogenase genes and found a large diversity of them [5]. [less ▲]

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See detailMitochondrial Proteomics of a Secondary Green Alga
Perez, Emilie ULg; Degand, Hervé; Morsomme, Pierre et al

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 ▲]

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See detailCharacterization of an internal type-II NADH dehydrogenase from Chlamydomonas reinhardtii mitochondria
Remacle, Claire ULg

in 15th International Conference on the Cell & Molecular Biology of Chlamydomonas (2012, June)

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See detailReconstruction of a Human Mitochondrial Complex I Mutation in the Green Microalga Chlamydomonas
Remacle, Claire ULg

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

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See detailReconstruction of a human mitochondrial complex I mutation in the unicellular green alga Chlamydomonas.
Larosa, Véronique ULg; Coosemans, Nadine ULg; Motte, Patrick ULg et al

in Plant Journal (The) (2012), 70

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 ▲]

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See detailFunction of the chloroplastic NAD(P)H dehydrogenase Nda2 for H(2) photoproduction in sulphur-deprived Chlamydomonas reinhardtii.
Mignolet, Emmanuel; Lecler, Renaud; Ghysels, Bart ULg et al

in Journal of biotechnology (2012), 162(1), 81-8

The relative contributions of the PSII-dependent and Nda2-dependent pathways for H(2) 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 H(2) photoproduction were investigated in the green microalga Chlamydomonas reinhardtii after suphur-deprivation. For this purpose, H(2) 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 H(2) photoevolution rate observed shortly after the establishment of anoxia, and an acceleration of the decline of H(2) photoevolution rate with time. DCMU addition to Nda2-deficient cells completely inhibited H(2) photoproduction, showing that the PSII-independent H(2) 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 H(2) 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 H(2) photoevolution rate with time in Nda2-deficient cells revealed a more pronounced inhibition of H(2) photoproduction by accumulated H(2) in the absence of non-photochemical plastoquinone reduction. Nda2 is therefore important for linking H(2) photoproduction with catabolism of storage carbon compounds, and seems also involved in regulating the redox poise of the photosynthetic electron transport chain during H(2) photoproduction. [less ▲]

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See detailMitochondrial transformation and in vitro DNA delivery
Remacle, Claire ULg; Hamel, Patrice; Larosa, Véronique ULg et al

in Bock, R; Knoop, V (Eds.) Genomics of Chloroplasts and Mitochondria (2012)

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See detailComplexes I in the green lineage.
Remacle, Claire ULg; Hamel, Patrice; Larosa, Véronique ULg et al

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

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See detailGreen Algae Genomics: A Mitochondrial Perspective
Rodriguez-Salinas, E; Remacle, Claire ULg; Gonzalez-Halphen, Diego

in Maréchal-Drouard, Laurence (Ed.) Mitochondrial Genome Evolution (2012)

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See detailFinding the bottleneck: a research strategy for improved biomass production
Bassi, Roberto; Cardol, Pierre ULg; Choquet, Yves et al

in Posten, Clemens; Walter, Christian (Eds.) Microalgal Biotechnology: integration and economy (2012)

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See detailFunction of the chloroplastic NADP(H) dehydrogenase NDA2 for the H2 photoproduction in sulphur-deprived Chlamydomonas reinhardtii
Mignolet, Emmanuel ULg; Lecler, Renaud ULg; Ghysels, Bart ULg et al

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 ▲]

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See detailInsertional mutagenesis to select mutants for modified hydrogen photoproduction in Chlamydomonas reinhardtii
Godaux, Damien ULg; Emonds-alt, Barbara; Cardol, Pierre ULg et al

Poster (2011, September 18)

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. [less ▲]

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See detailInsertional mutagenesis to select mutants for modified hydrogen photoproduction in Chlamydomonas reinhardtii
Godaux, Damien ULg; Emonds-Alt, Barbara ULg; Cardol, Pierre ULg et al

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 ▲]

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