Bart Ghysels

A type II NAD(P)H dehydrogenase mediates light-independent plastoquinone reduction in the chloroplast of Chlamydomonas

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.

Hydrogen photo-evolution upon S deprivation stepwise: an illustration of microalgal photosynthetic and metabolic flexibility and a step stone for future biotechnological methods of renewable H2 production

The metabolic flexibility of some photosynthetic microalgae enables them to survive periods of anaerobiosis in the light by developing a particular photofermentative metabolism. The latter entails compounds of the photosynthetic electron transfer chain and an oxygen-sensitive hydrogenase in order to reoxidize reducing equivalents and to generate ATP for maintaining basal metabolic function. This pathway results in the photo-evolution of hydrogen gas by the algae. A decade ago, Melis and coworkers managed to reproduce such a condition in a laboratory context by depletion of sulfur in the algal culture media, making the photo-evolution by the algae sustainable for several days (Melis et al. in Plant Physiol 122:127–136, 2000). This observation boosted research in algal H2 evolution. A feature, which due to its transient nature was long time considered as a curiosity of algal photosynthesis suddenly became a phenomenon with biotechnological potential. Although the Melis procedure has not been developed into a biotechnological process of renewable H2 generation so far, it has been a useful tool for studying microalgal metabolic and photosynthetic flexibility and a possible step stone for future H2 production procedures. Ten years later most of the critical steps and limitations of H2 production by this protocol have been studied from different angles particularly with the model organism Chlamydomonas reinhardtii, by introducing various changes in culture conditions and making use of mutants issued from different screens or by reverse genomic approaches. A synthesis of these observations with the most important conclusions driven from recent studies will be presented in this review.

Proteomic and functional characterization of a Chlamydomonas reinhardtii mutant lacking the mitochondrial alternative oxidase 1.

In the present work, we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase 1 (AOX1). The AOX1-deficient strain displays a remarkable doubling of the cell volume and biomass without alteration of the generation time or change in total respiratory rate, with a significantly higher ROS production. To identify the molecular adaptation underlying these observations, we have carried out a comparative study of both the mitochondrial and the cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important quantitative modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways.

Function of the chloroplast hydrogenase in the microalga Chlamydomonas: the role of hydrogenase and state transitions during photosynthetic activation in anaerobiosis.

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.

Function of the chloroplastic NAD(P)H dehydrogenase Nda2 for H2 photoproduction in sulphur-deprived Chlamydomonas 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.

Analysis of PSII antenna size heterogeneity of Chlamydomonas reinhardtii during state transitions

PSII antenna size heterogeneity has been intensively studied in the past. Based on DCMU fluorescence rise kinetics, multiple types of photosystems with different properties were described. However, due to the complexity of fluorescence signal analysis, multiple questions remain unanswered. The number of different types of PSII is still debated as well as their degree of connectivity. In Chlamydomonas reinhardtii we found that PSIIα possesses a high degree of connectivity and an antenna 2-3 times larger than PSIIβ, as described previously. We also found some connectivity for PSIIβ in contrast with the majority of previous studies. This is in agreement with biochemical studies which describe PSII mega-, super- and core-complexes in Chlamydomonas. In these studies, the smallest unit of PSII in vivo would be a dimer of two core complexes hence allowing connectivity. We discuss the possible relationships between PSIIα and PSIIβ and the PSII mega-, super- and core-complexes. We also showed that strain and medium dependent variations in the half-time of the fluorescence rise can be explained by variations in the proportions of PSIIα and PSIIβ. When analyzing the state transition process in vivo, we found that this process induces an inter-conversion of PSIIα and PSIIβ. During a transition from state 2 to state 1, DCMU fluorescence rise kinetics are satisfactorily fitted by considering two PSII populations with constant kinetic parameters. We discuss our findings about PSII heterogeneity during state transitions in relation with recent results on the remodeling of the pigment-protein PSII architecture during this process.

Interplay between non-photochemical plastoquinone reduction and re-oxidation in pre-illuminated Chlamydomonas reinhardtii: a chlorophyll fluorescence study

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.