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In photosynthetic eukaryotes, the redox state of the plastoquinone (PQ) pool is an important sensor for mechanisms that regulate the photosynthetic electron transport. In higher plants, a multimeric nicotinamide adenine dinucleotide (phosphate) (NAD(P))H dehydrogenase (NDH) complex and a plastid terminal oxidase (PTOX) are involved in PQ redox homeostasis in the dark. We recently demonstrated that in the microalgae Chlamydomonas reinhardtii, which lacks the multimeric NDH complex of higher plants, non-photochemical PQ reduction is mediated by a monomeric type-II NDH (Nda2). In this study, we further explore the nature and the importance of non-photochemical PQ reduction and oxidation in relation to redox homeostasis in this alga by recording the 'dark' chlorophyll fluorescence transients of pre-illuminated algal samples. From the observation that this fluorescence transient is modified by addition of propyl gallate, a known inhibitor of PTOX, and in a Nda2-deficient strain we conclude that it reflects post-illumination changes in the redox state of PQ resulting from simultaneous PTOX and Nda2 activity. We show that the post-illumination fluorescence transient can be used to monitor changes in the relative rates of the non-photochemical PQ reduction and reoxidation in response to different physiological situations. We study this fluorescence transient in algae acclimated to high light and in a mutant deficient in mitochondrial respiration. Some of our observations indicate that the chlororespiratory pathway participates in redox homeostasis in C. reinhardtii.

The relative contributions of the PSII-dependent and Nda2-dependent pathways for H₂ photoproduction were investigated in the green microalga Chlamydomonas reinhardtii after suphur-deprivation. For this purpose, H₂ gas production was compared for wild-type and Nda2-deficient cells with or without DCMU (a PSII-inhibitor) in the same experimental conditions. Nda2-deficiency caused a 30% decrease of the maximal H₂ photoevolution rate observed shortly after the establishment of anoxia, and an acceleration of the decline of H₂ photoevolution rate with time. DCMU addition to Nda2-deficient cells completely inhibited H₂ photoproduction, showing that the PSII-independent H₂ photoproduction relies on the presence of Nda2, which feeds the photosynthetic electron transport chain with electrons derived from oxidative catabolism. Nda2-protein abundance increased as a result of sulphur deprivation and further during the H₂ photoproduction process, resulting in high rates of non-photochemical plastoquinone reduction in control cells. Nda2-deficiency had no significant effect on photosynthetic and respiratory capacities in sulphur-deprived cells, but caused changes in the cell energetic status (ATP and NADPH/NADP+ ratio). The rapid decline of H₂ photoevolution rate with time in Nda2-deficient cells revealed a more pronounced inhibition of H₂ photoproduction by accumulated H₂ in the absence of non-photochemical plastoquinone reduction. Nda2 is therefore important for linking H₂ photoproduction with catabolism of storage carbon compounds, and seems also involved in regulating the redox poise of the photosynthetic electron transport chain during H₂ photoproduction.

In photosynthetic eukaryotes, nonphotochemical plastoquinone (PQ) reduction is important for the regulation of photosynthetic electron flow. In green microalgae where this process has been demonstrated, the chloroplastic enzyme that catalyses nonphotochemical PQ reduction has not been identified yet. Here, we show by an RNA interference (RNAi) approach that the NDA2 gene, belonging to a type II NAD(P)H dehydrogenases family in the green microalga Chlamydomonas reinhardtii , encodes a chloroplastic dehydrogenase that functions to reduce PQ nonphotochemically in this alga. Using a specific antibody, we show that the Nda2 protein is localized in chloroplasts of wild-type cells and is absent in two Nda2-RNAi cell lines. In both mutant cell lines, nonphotochemical PQ reduction is severely affected, as indicated by altered chlorophyll fluorescence transients after saturating illumination. Compared with wild type, change in light excitation distribution between photosystems (‘state transition’) upon inhibition of mitochondrial electron transport is strongly impaired in transformed cells because of inefficient PQ reduction. Furthermore, the amount of hydrogen produced by Nda2-RNAi cells under sulfur deprivation is substantially decreased compared with wild type, which supports previous assumptions that endogenous substrates serve as source of electrons for hydrogen formation. These results demonstrate the importance of Nda2 for nonphotochemical PQ reduction and associated processes in C. reinhardtii .

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.