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

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.