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 .

Vitamin B6 is a collective term for a group of six interconvertible compounds: pyridoxine, pyridoxal, pyridoxamine and their phosphorylated derivatives. Vitamin B6 plays essential roles as a cofactor in a range of biochemical reactions. In addition, vitamin B6 is able to quench reactive oxygen species in vitro, and exogenously applied vitamin B6 protects plant cells against cell death induced by singlet oxygen (1O2). These results raise the important question as to whether plants employ vitamin B6 as an antioxidant to protect themselves against reactive oxygen species.

Photosynthetic activities were analyzed in Chlamydomonas reinhardtii mitochondrial mutants affected in different complexes (I, III, IV, I + III, and I + IV) of the respiratory chain. Oxygen evolution curves showed a positive relationship between the apparent yield of photosynthetic linear electron transport and the number of active proton-pumping sites in mitochondria. Although no significant alterations of the quantitative relationships between major photosynthetic complexes were found in the mutants, 77 K fluorescence spectra showed a preferential excitation of photosystem I (PSI) compared with wild type, which was indicative of a shift toward state 2. This effect was correlated with high levels of phosphorylation of light-harvesting complex II polypeptides, indicating the preferential association of light-harvesting complex II with PSI. The transition to state 1 occurred in untreated wild-type cells exposed to PSI light or in 3-(3,4-dichlorophenyl)-1,1-dimethylureatreated cells exposed to white light. In mutants of the cytochrome pathway and in double mutants, this transition was only observed in white light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. This suggests higher rates of nonphotochemical plastoquinone reduction through the chlororespiratory pathway, which was confirmed by measurements of the complementary area above the fluorescence induction curve in dark-adapted cells. Photo-acoustic measurements of energy storage by PSI showed a stimulation of PSI-driven cyclic electron flow in the most affected mutants. The present results demonstrate that in C. reinhardtii mutants, permanent defects in the mitochondrial electron transport chain stabilize state 2, which favors cyclic over linear electron transport in the chloroplast.

Protochlorophyllide (Pchlide) reductases are key enzymes in the process of chlorophyll biosynthesis. In this review, current knowledge on the molecular organization, substrate specificity and assembly of the light-dependent reduced nicotinamide adenine dinucleotide phosphate:Pchlide oxidoreductases are discussed. Characteristics of light-independent enzymes are also described briefly, and the possible reasons for the selection of light-dependent enzymes during the course of evolution are discussed.