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See detailGrowth of Chlorella in the presence of organic carbon: A photobioreactor study
Miazek, Krystian ULg; Goffin, Dorothée ULg; Richel, Aurore ULg et al

Scientific conference (2014, June 19)

In this study, the effect of organic carbon supplementation at low light intensity on Chlorella sorokiniana growth was evaluated. Addition of 1 g/L of acetate to media gave the highest growth rate and ... [more ▼]

In this study, the effect of organic carbon supplementation at low light intensity on Chlorella sorokiniana growth was evaluated. Addition of 1 g/L of acetate to media gave the highest growth rate and provided stable high biomass culture during prolonged cultivation time. Glucose at 1 – 5 g/L also improved biomass growth rate, although stability of high biomass culture could not be achieved. Overall, the presence of organic carbon can considerably enhance Chlorella growth when low light intensity is applied. [less ▲]

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See detailApplications des microalgues
Franck, Fabrice ULg; Remacle, Claire ULg

Conference (2014, May 22)

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See detailLack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth.
Plancke, Charlotte; Vigeolas, Hélène ULg; Hohner, Ricarda et al

in The Plant journal : for cell and molecular biology (2014), 77(3), 404-417

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the ... [more ▼]

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO2 is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data show that isocitrate lyase is required for growth in darkness on acetate (heterotrophic conditions), as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). Under these latter conditions, reduced acetate assimilation and concomitant reduced respiration occur, and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by 14 N/15 N labelling was performed, and more than 1600 proteins were identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis in parallel with a shift of the TCA cycle towards amino acid synthesis, accompanied by an increase in free amino acids. The decrease of the glyoxylate cycle and gluconeogenesis, as well as the decrease in enzymes involved in beta-oxidation of fatty acids in the icl mutant are probably major factors that contribute to remodelling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase, and increased resistance to paraquat. [less ▲]

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See detailThe mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae.
Perez, Emilie ULg; Lapaille, Marie; Degand, Herve et al

in Mitochondrion (2014)

The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential ... [more ▼]

The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential therapeutic target. Recently, numerous additional subunits of the respiratory-chain complexes have been described in Trypanosoma brucei and Trypanosoma cruzi. Since these subunits had apparently no counterparts in other organisms, they were interpreted as potentially associated with the parasitic trypanosome lifestyle. Here we used two complementary approaches to characterise the subunit composition of respiratory complexes in Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes. First, we developed a phylogenetic pipeline aimed at mining sequence databases for identifying homologs to known respiratory-complex subunits with high confidence. Second, we used MS/MS proteomics after two-dimensional separation of the respiratory complexes by Blue Native- and SDS-PAGE to both confirm in silico predictions and to identify further additional subunits. Altogether, we identified 41 subunits that are restricted to E. gracilis, T. brucei and T. cruzi, along with 48 classical subunits described in other eukaryotes (i.e. plants, mammals and fungi). This moreover demonstrates that at least half of the subunits recently reported in T. brucei and T. cruzi are actually not specific to Trypanosomatidae, but extend at least to other Euglenozoa, and that their origin and function are thus not specifically associated with the parasitic lifestyle. Furthermore, preliminary biochemical analyses suggest that some of these additional subunits underlie the peculiarities of the respiratory chain observed in Euglenozoa. [less ▲]

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See detailInteractions of subunits Asa2, Asa4 and Asa7 in the peripheral stalk of the mitochondrial ATP synthase of the chlorophycean alga Polytomella sp.
Miranda-Astudillo, Hector; Cano-Estrada, Araceli; Vazquez-Acevedo, Miriam et al

in Biochimica et Biophysica Acta-Bioenergetics (2014), 1837

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See detailInactivation of genes coding for mitochondrial Nd7 and Nd9 complex I subunits in Chlamydomonas reinhardtii. Impact of complex I loss on respiration and energetic metabolism.
Massoz, Simon; Larosa, Véronique ULg; Plancke, Charlotte et al

in Mitochondrion (2013)

In Chlamydomonas, unlike in flowering plants, genes coding for Nd7 (NAD7/49kDa) and Nd9 (NAD9/30kDa) core subunits of mitochondrial respiratory-chain complex I are nucleus-encoded. Both genes possess all ... [more ▼]

In Chlamydomonas, unlike in flowering plants, genes coding for Nd7 (NAD7/49kDa) and Nd9 (NAD9/30kDa) core subunits of mitochondrial respiratory-chain complex I are nucleus-encoded. Both genes possess all the features that facilitate their expression and proper import of the polypeptides in mitochondria. By inactivating their expression by RNA interference or insertional mutagenesis, we show that both subunits are required for complex I assembly and activity. Inactivation of complex I impairs the cell growth rate, reduces the respiratory rate, leads to lower intracellular ROS production and lower expression of ROS scavenging enzymes, and is associated to a diminished capacity to concentrate CO2 without compromising photosynthetic capacity. [less ▲]

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See detailTransformation of the mitochondrial genome.
Larosa, Véronique ULg; Remacle, Claire ULg

in International Journal of Developmental Biology (2013), 57

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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 detailRespiratory-deficient mutants of the unicellular green alga Chlamydomonas: A review.
Salinas, Thalia; Larosa, Véronique ULg; Cardol, Pierre ULg et al

in Biochimie (2013)

Genetic manipulation of the unicellular green alga Chlamydomonas reinhardtii is straightforward. Nuclear genes can be interrupted by insertional mutagenesis or targeted by RNA interference whereas random ... [more ▼]

Genetic manipulation of the unicellular green alga Chlamydomonas reinhardtii is straightforward. Nuclear genes can be interrupted by insertional mutagenesis or targeted by RNA interference whereas random or site-directed mutagenesis allows the introduction of mutations in the mitochondrial genome. This, combined with a screen that easily allows discriminating respiratory-deficient mutants, makes Chlamydomonas a model system of choice to study mitochondria biology in photosynthetic organisms. Since the first description of Chlamydomonas respiratory-deficient mutants in 1977 by random mutagenesis, many other mutants affected in mitochondrial components have been characterized. These respiratory-deficient mutants increased our knowledge on function and assembly of the respiratory enzyme complexes. More recently some of these mutants allowed the study of mitochondrial gene expression processes poorly understood in Chlamydomonas. In this review, we update the data concerning the respiratory components with a special focus on the assembly factors identified on other organisms. In addition, we make an inventory of different mitochondrial respiratory mutants that are inactivated either on mitochondrial or nuclear genes. [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 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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