A novel screening method for hydrogenase-deficient mutants in Chlamydomonas reinhardtii based on in vivo chlorophyll fluorescence and photosystem II quantum yield

Abstract

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...

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii

“…In the hydg-2 strain, J H2 and ETR PSII were below detection during the first 2 min of illumination (Fig. 1C), as previously reported Godaux et al, 2013), but ETR PSII increased later. In the pgrl1 mutant, J H2 decreased very slowly, in agreement with previous observations (Tolleter et al, 2011), together with a slow increase of ETR PSII (Fig.…”

“…In this range of light intensities, growth and photosynthesis of aerated cultures were similar for all the strains (Table I). After 1 h of acclimation to dark and anoxia, a time required for proper expression and activity of hydrogenases (Forestier et al, 2003;Godaux et al, 2013;Supplemental Fig. S2), we measured the hydrogen evolution rate (J H2 ) and the quantum yield of PSII (f PSII ) and PSI (f PSI ), as well as the mean photochemical rate (R ph ) based on electrochromic shift (ECS) measurements at the onset of light (10 s; Table II).…”

“…In anoxia, both PQ and PSI acceptor pools are almost fully reduced (Bennoun, 1982;Ghysels et al, 2013;Godaux et al, 2013;Takahashi et al, 2013), which might hamper the putative limiting step of PSI-CEF electron transfer (i.e. NADPH to PQ; Alric, 2014), as well as the PSI electron transfer due to acceptor side limitation (Takahashi et al, 2013).…”

“…Aliquots of the liquid culture were then collected every 24 h, and about 300 cells were platted on solid TAP media in the light to allow the growth of single-cell colonies. The proportions of each phenotype were analyzed on the basis of peculiar hydrogenasedeficient-related chlorophyll fluorescence kinetics by video imaging according to Godaux et al (2013). The ratio of pgrl1 hydg-2 was plotted against time.…”

The Involvement of Hydrogen-producing and ATP-dependent NADPH-consuming Pathways in Setting the Redox Poise in the Chloroplast of Chlamydomonas reinhardtii in Anoxia

“…Strains and Growth Conditions-We used the following C. reinhardtii wild-type strain T222ϩ, the double mutant pfl1-1 adh1 (33), which lacks the pyruvate formate-lyase and the alcohol dehydrogenase involved in fermentation pathways; the ATP synthase mutant Fud50.ORϩ (34), the ⌬rbcL 1-7.5 mutant, which lacks the large subunit of Rubisco (35); the plastocyanin mutant ac208 (36), the so-called pewy mutant, which accumulates the b 6 f complex but is impaired in its quinol oxidation site (37); the ⌬petA mutant, which lacks b 6 f complex (38); the dum22 mutant lacks both the mitochondrial complex I and bc 1 complexes (39), and the HydEF (40) and HydG (41) mutants that lack assembly factors required for hydrogenase maturation. Cells were grown to mid-log phase (3-5 10 6 cells per ml) in Tris/acetate/phosphate medium (42) at 25°C at 50 mol photons m Ϫ2 s Ϫ1 .…”