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See detailFunction of the chloroplastic hydrogenase in the microalga Chlamydomonas reinhardtii: A trvel from dark to light
Godaux, Damien ULg

Doctoral thesis (2014)

The decreasing availability of fossil energy stocks and the eventuality of tragic climate changes caused by greenhouse gases lead to search for alternative renewable energy sources. Biological hydrogen ... [more ▼]

The decreasing availability of fossil energy stocks and the eventuality of tragic climate changes caused by greenhouse gases lead to search for alternative renewable energy sources. Biological hydrogen might be one promising renewable energy carrier. A specific and restricted group of microalgae developed the ability to produce hydrogen based on an oxygen-sensitive hydrogenase enzyme coupled to the photosynthetic pathway, acting as a putative valve for excess electrons in conditions where other electron acceptors are scarce. The unicellular green alga Chlamydomonas reinhardtii is widely regarded as a model organism for various biological processes, especially for photosynthesis. Moreover, the capacity of Chlamydomonas hydrogenase is claimed as the highest recorded in literature. Less than twenty years ago, a group of American scientists designed a new approach for sustained photobiological production of hydrogen, based on a two-stage protocol that temporally separates photosynthetic O2 evolution from the H2 production phase (Melis et al., 2000). The transition occurs upon sulfur deprivation of the culture and leads to an operating continuous production for several days, opening new possibilities in the aim of an economically rentable bioproduction. For these reasons, hydrogen photoproduction in Chlamydomonas reinhardtii has been extensively examined in the last decade as extension of photosynthesis research entailing the understanding of hydrogen metabolism in microalgae (for reviews, see Hankamer et al., 2007; Ghirardi et al., 2009; Ghysels and Franck, 2010). Despite the attractive trait of generating a renewable fuel from nature’s most plentiful resources, i.e. light and water, the physiological significance of such oxygen-sensitive enzyme coupled to oxygenic photosynthesis has been poorly investigated with the exception of some old studies (Kessler, 1973; Schreiber and Vidaver, 1974). In this work, hydrogenase implication in photosynthetic reactivation from dark and anoxic environment is investigated. In the first part of the work, by analyzing several strains affected in hydrogen metabolism (e.g. nda2-RNAi (Jans et al., 2008), pfl1 (Philipps et al., 2011), dum11 (Dorthu et al., 1992)), we show that the PSII–dependent photosynthetic electron flow upon dark to light shift is linearly related to the activity of hydrogenase, both for short and long-terms adaptation (Publication I). In agreement with this conclusion, a hydrogenase-deficient strain for the HydEF maturation factor (hydef, Posewitz et al., 2004) shows peculiar chlorophyll fluorescence induction kinetics after adaptation to dark and anoxia. Based on these findings, a novel imaging screening method is developed, allowing rapid identification of strains impaired in hydrogen metabolism. Compared to existing screens (for review, see Hemschemeier et al., 2009), our protocol is remarkably fast, sensitive and non-invasive. At this stage, application of this new screening method allowed us to isolate several hydrogenase-deficient strains, among which one was impaired for the hydrogenase maturation protein HydG (hydg-2 mutant). Chlamydomonas reinhardtii might frequently encounter period of dark and anoxia in its natural habitat, especially during the night when the microbial community respires the available oxygen. In the second part of my work, the physiological importance of hydrogenase is investigated in the context of photosynthesis induction at the onset of light upon anoxia. In such conditions, the plastoquinone pool is known as being overreduced. This triggers the process of state transitions which is described as allowing the redistribution of light capture between both photosystems to manage the redox poise of the photosynthetic pathway (for review, see Lemeille and Rochaix, 2010). We therefore revisit the impact of both state transitions and hydrogenase activity on the reactivation of photosynthetic electron flow (Publication II). Here we show that, in presence of hydrogenase, photosynthesis reactivation is slightly faster in stt7 mutant locked in state 1 (Depege et al., 2003) compared to wild type which is in state 2. However, photosynthesis reactivation is delayed in hydef stt7-9 double mutant compared to hydef mutant. This indicates that, in a hydrogenase-deficient context, state 2 promotes photosynthesis reactivation. Considered for a long time as being tightly interconnected (Finazzi et al., 1999; Finazzi et al., 2002; Finazzi and Forti, 2004), state transitions and PSI-CEF have recently been revealed as unrelated to each other (Takahashi et al., 2013). Nonetheless, the increasing of PSI antenna size in state 2 could even though enhance the PSI-CEF rate, in an indirect way, by enhancing PSI energy capture (Cardol et al., 2009; Alric, 2014). This reasonably raises the question of a possible involvement of PSI-CEF in photosynthesis induction. This possibility is further studied in the third and last part of the work. Thanks to mutants devoid of PSI-CEF (i.e. pgrl1 mutant (Tolleter et al., 2011)) and hydrogenase activity (i.e. hydg-2 mutant (Publication I)), we investigate the role played by PSI-CEF along with hydrogenase during photosynthesis reactivation during a shift from dark anoxia to light (Publication III). Herein, we demonstrate that Calvin cycle reactivation is proton gradient-dependent, most likely due to ATP requirement for carbon dioxide fixation. By measuring the PSI/PSII efficiency ratio during the re- illumination period, we point out the physiological occurrence of PSI-CEF within the first minutes of ilumination. We therefore propose a schematic model that assesses the electron flow through hydrogenase, PSI-CEF and Calvin cycle in function of the illumination period in all studied strains. Although lack of PSI-CEF does not appear to be essential for cell survival, photosynthesis reactivation is delayed in pgrl1 mutants. We also isolate a pgrl1 hydg-2 double mutant and demonstrate that the combination of both defects prevents any photosynthetic activity and strongly impairs growth. This highlights the importance for algae to keep both pathways in the course of evolution, being critical for the survival of Chlamydomonas reinhardtii in its natural environment. [less ▲]

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See detailA forward genetic screen to identify hydrogenase-deficient mutants in the unicellular green alga Chlamydomonas reinhardtii
Emonds-Alt, Barbara ULg; Godaux, Damien ULg; Cardol, Pierre ULg et al

Poster (2014, June 15)

The ability of the unicellular green alga Chlamydomonas reinhardtii to evolve molecular hydrogen (H2) is due to the presence of oxygen-sensitive Fe-hydrogenases (HydA1/2), expressed in anoxic conditions ... [more ▼]

The ability of the unicellular green alga Chlamydomonas reinhardtii to evolve molecular hydrogen (H2) is due to the presence of oxygen-sensitive Fe-hydrogenases (HydA1/2), expressed in anoxic conditions that drive the photosynthetic electron flow to reduce protons into H2. In order to identify new players involved in H2 photoproduction in Chlamydomonas, an insertion mutant library was generated using cassettes conferring resistance to hygromycin or paromomycin. Hydrogenase activity is physiologically relevant during a transition from dark anoxia to light. In dark anoxic conditions, the cellular redox poise is high and the photosynthetic electron transport chain is fully reduced. Upon illumination, hydrogenase activity allows the reoxidation of photosynthetic intersystem electron carriers until oxic conditions and carbon fixation ability are restored. We thus designed an in vivo fluorescence imaging screen based on the different kinetics of photosynthesis induction between wild type and hydrogenase-deficient mutants [1]. At this stage, three putative hydrogenase mutants have been identified on 10,000 transformants. Molecular characterization of the insertion site of the resistance cassette by TAIL-PCR and genetic analyses of the linkage between the antibiotic resistance and the fluorescence phenotype showed that one mutant was untagged with the resistance while two tagged mutants were deficient for the HydG assembly factor. [less ▲]

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See detailFunction of the Chloroplast Hydrogenase in the Microalga Chlamydomonas: The Role of Hydrogenase and State Transitions during Photosynthetic Activation in Anaerobiosis
Ghysels, Bart ULg; Godaux, Damien ULg; Matagne, René-Fernand ULg et al

in PLoS ONE (2013), 8(5), 64161

Like a majority of photosynthetic microorganisms, the green unicellular alga Chlamydomonas reinhardtii may encounter O2 deprived conditions on a regular basis. In response to anaerobiosis or in a ... [more ▼]

Like a majority of photosynthetic microorganisms, the green unicellular alga Chlamydomonas reinhardtii may encounter O2 deprived conditions on a regular basis. In response to anaerobiosis or in a respiration defective context, the photosynthetic electron transport chain of Chlamydomonas is remodeled by a state transition process to a conformation that favours the photoproduction of ATP at the expense of reductant synthesis. In some unicellular green algae including Chlamydomonas, anoxia also triggers the induction of a chloroplast-located, oxygen sensitive hydrogenase, which accepts electrons from reduced ferredoxin to convert protons into molecular hydrogen. Although microalgal hydrogen evolution has received much interest for its biotechnological potential, its physiological role remains unclear. By using specific Chlamydomonas mutants, we demonstrate that the state transition ability and the hydrogenase function are both critical for induction of photosynthesis in anoxia. These two processes are thus important for survival of the cells when they are transiently placed in an anaerobic environment. [less ▲]

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See detailA novel screening method for hydrogenase-deficient mutants in Chlamydomonas reinhardtii based on in vivo chlorophyll fluorescence and photosystem II quantum yield
Godaux, Damien ULg; Emonds-Alt, Barbara ULg; Berne, Nicolas ULg et al

in International Journal of Hydrogen Energy (2013), 38

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of enzymes <br />Received 30 August 2012 belonging to various fermentative pathways. Among them, oxygen-sensitive hydrogenases ... [more ▼]

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of enzymes <br />Received 30 August 2012 belonging to various fermentative pathways. Among them, oxygen-sensitive hydrogenases <br />Received in revised form (HydA1/2) catalyze the synthesis of molecular hydrogen from protons and reduced ferre- <br />12 November 2012 doxin in the stroma. In this work, by analyzing wild type and mutants affected in H2 <br />Accepted 16 November 2012 production, we show that maximal PSII photosynthetic electron transfer during the first <br />Available online 21 December 2012 seconds of illumination after a prolonged dark-anaerobiosis period is linearly related to <br />hydrogenase capacity. Based on the specific chlorophyll fluorescence induction kinetics <br />Keywords: typical of hydrogenase-deficient mutants, we set up an in vivo fluorescence imaging <br />Chlamydomonas reinhardtii screening protocol allowing to isolate mutants impaired in hydrogenase expression or <br />Anaerobic photosynthesis activity, as well as mutants altered in related metabolic pathways required for energy <br />Hydrogenase production in anaerobiosis. Compared to previously described screens for mutants <br />Chlorophyll fluorescence impaired in H2 production, our screening method is remarkably fast, sensitive and non- <br />Microalgae invasive. Out of 3000 clones from a small-sized insertional mutant library, five mutants <br />Hydrogen photoproduction were isolated and the most affected one was analyzed and shown to be defective for the <br />hydrogenase HydG assembly factor. [less ▲]

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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 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 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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See detailFunctional analysis of hydrogen photoproduction in respiratory-deficient mutants of Chlamydomonas reinhardtii
Lecler, Renaud ULg; Godaux, Damien ULg; Vigeolas, Hélène ULg et al

in International Journal of Hydrogen Energy (2011), 36

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 ▲]

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See detail(Functionnal) analysis of hydrogen production in Chlamydomonas reinhardtii mitochondrial mutants
Lecler, Renaud ULg; Godaux, Damien ULg; Hamilton, Christopher ULg et al

Poster (2010, June 27)

Mitochondrial Chlamydomonas mutants for respiratory complexes present a decreased dark respiration and apparent yield of photosynthetic linear electron flow. They accumulate reducing power such as NAD(P)H ... [more ▼]

Mitochondrial Chlamydomonas mutants for respiratory complexes present a decreased dark respiration and apparent yield of photosynthetic linear electron flow. They accumulate reducing power such as NAD(P)H and show lower levels of ATP. Under restrictive conditions, like sulfur depletion and anoxia, Chlamydomonas is able to produce hydrogen towards the activation of a chloroplatic O2-sensitive Fe-hydrogenase which catalyses the reduction of electrons to H2. In this study we used an adapted Melis protocol to analyse hydrogen evolution of mitochondrial mutants. For this aim a simple-flask system was built with gaz collecting tubes. A parallel flask was used for GC analyses. [less ▲]

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