SUMMARYIsocitrate 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 CO 2 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 b-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.

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of enzymes belonging to various fermentative pathways. Among them, oxygen-sensitive hydrogenases (HydA1/2) catalyze the synthesis of molecular hydrogen from protons and reduced ferredoxin in the stroma. In this work, by analyzing wild type and mutants affected in H2 production, we show that maximal PSII photosynthetic electron transfer during the first seconds of illumination after a prolonged darkanaerobiosis period is linearly related to hydrogenase capacity. Based on the specific chlorophyll fluorescence induction kinetics typical of hydrogenase-deficient mutants, we set up an in vivo fluorescence imaging screening protocol allowing to isolate mutants impaired in hydrogenase expression or activity, as well as mutants altered in related metabolic pathways required for energy production in anaerobiosis. Compared to previously described screens for mutants impaired in H2 production, our screening method is remarkably fast, sensitive and non invasive. Out of 3000 clones from a small-sized insertional mutant library, five mutants were isolated and the most affected one was analyzed and shown to be defective for the hydrogenase HydG assembly factor.Pierre Cardol, PhD, FNRS research associate Génétique des microorganismes -Institut de Botanique B22 Boulevard du Rectorat, 27 -Université de Liège-B-4000 Liège, Belgique Tel/Fax +32 4 3663840 e-mail: Pierre.cardol@ulg.ac.be Liège, 07 november 2012 Dear Editor, Please find here enclosed a revised version of our manuscript entitled 'A novel screening method for hydrogenase-deficient mutants in Chlamydomonas reinhardtii based on in vivo chlorophyll fluorescence and photosystem II quantum yield.' that had received a positive feedback from the first round of reviewing.We are grateful to you for having encouraged us to resubmit quickly a modified manuscript. We have addressed all the comments raised by the reviewers on our first submission. A point to point answer has been submitted along with the MS. Main changes are : (i) the addition of a new experimental set of data (hydrogen evolution measurements in all mutant strain and some control experiments) and (ii) all data that were previously not shown are now included in the manuscript in main tables or as supplemental figures.We hope that we have now improved our manuscript so you'll find it suitable for publication in International Journal of Hydrogen Energy. Sincerely yours,On behalf all coauthors, Pierre Cardol *Cover LetterReviewer #1:Major points:I. Please measure and show, respectively, the hydrogenase activity of the wild types or control strains (like respiratory mutants, pfl1-mutant) in your setup. Available literature shows that absolute values can differ significantly depending on exact experimental setup and strain. As the correlation between PSII quantum yield and hydrogenase activity is the major point of your study, this data is essential, and citing hydrogenase activities reported in literature is not sufficient.Answer : the...

Type-II NAD(P)H dehydrogenases form a multigene family that comprise six members in the green microalga Chlamydomonas. To date, only one enzyme (Nda2) located in the chloroplast has been characterized in this alga and demonstrated to participate in the reduction of the plastoquinone pool. We present here the functional characterization of Nda1. The enzyme is located on the inner face of the inner mitochondrial membrane. Its downregulation leads to a slight decrease of NADH:ferricyanide activity and of dark whole cell respiration. To determine whether the reduction of Nda1 combined with the lack of complex I would affect mitochondrial processes, double mutants affected in both Nda1 and complex I were isolated. Respiration and growth rates in heterotrophic conditions were significantly altered in the double mutants investigated, suggesting that Nda1 plays a role in the oxidation of matrix NADH in the absence of complex I.

SummaryPhylloquinone (PhQ), or vitamin K1, is an essential electron carrier (A1) in photosystem I (PSI). In the green alga Chlamydomonas reinhardtii, which is a model organism for the study of photosynthesis, a detailed characterization of the pathway is missing with only one mutant deficient for MEND having been analyzed. We took advantage of the fact that a double reduction of plastoquinone occurs in anoxia in the A1 site in the mend mutant, interrupting photosynthetic electron transfer, to isolate four new phylloquinone‐deficient mutants impaired in MENA,MENB,MENC (PHYLLO) and MENE. Compared with the wild type and complemented strains for MENB and MENE, the four men mutants grow slowly in low light and are sensitive to high light. When grown in low light they show a reduced photosynthetic electron transfer due to a specific decrease of PSI. Upon exposure to high light for a few hours, PSI becomes almost completely inactive, which leads in turn to lack of phototrophic growth. Loss of PhQ also fully prevents reactivation of photosynthesis after dark anoxia acclimation. In silico analyses allowed us to propose a PhQ biosynthesis pathway in Chlamydomonas that involves 11 enzymatic steps from chorismate located in the chloroplast and in the peroxisome.