No abstract

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.

Background: Shift to anoxia promotes the over-reduction of photosystem I electron acceptors. Results: This over-reduction can be relieved by the activation of several pathways. Conclusion: The two mains pathways are the ATP-dependent CO 2 fixation pathway and the ATP-independent hydrogenase. Significance: We disentangle the role of the various NADPH-consuming pathways in setting the redox poise in the chloroplast of unicellular photosynthetic algae.

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...

No abstract