Chlamydomonas flavodiiron proteins FlvA and FlvB massively drive the photosynthetic electron flow to O2 during the induction of photosynthesis and are critical

Nitrous oxide (N ₂ O), a potent greenhouse gas in the atmosphere, is produced mostly from aquatic ecosystems, to which algae substantially contribute. However, mechanisms of N ₂ O production by photosynthetic organisms are poorly described. Here we show that the green microalga Chlamydomonas reinhardtii reduces NO into N ₂ O using the photosynthetic electron transport. Through the study of C. reinhardtii mutants deficient in flavodiiron proteins (FLVs) or in a cytochrome p450 (CYP55), we show that FLVs contribute to NO reduction in the light, while CYP55 operates in the dark. Both pathways are active when NO is produced in vivo during the reduction of nitrites and participate in NO homeostasis. Furthermore, NO reduction by both pathways is restricted to chlorophytes, organisms particularly abundant in ocean N ₂ O-producing hot spots. Our results provide a mechanistic understanding of N ₂ O production in eukaryotic phototrophs and represent an important step toward a comprehensive assessment of greenhouse gas emission by aquatic ecosystems.

Plants and algae must tightly coordinate photosynthetic electron transport and metabolic activities given that they often face fluctuating light and nutrient conditions. The exchange of metabolites and signaling molecules between organelles is thought to be central to this regulation but evidence for this is still fragmentary. Here, we show that knocking out the peroxisome-located MALATE DEHYDROGENASE2 (MDH2) of Chlamydomonas reinhardtii results in dramatic alterations not only in peroxisomal fatty acid breakdown but also in chloroplast starch metabolism and photosynthesis. mdh2 mutants accumulated 50% more storage lipid and 2-fold more starch than the wild type during nitrogen deprivation. In parallel, mdh2 showed increased photosystem II yield and photosynthetic CO₂ fixation. Metabolite analyses revealed a >60% reduction in malate, together with increased levels of NADPH and H₂O₂ in mdh2. Similar phenotypes were found upon high light exposure. Furthermore, based on the lack of starch accumulation in a knockout mutant of the H₂O₂-producing peroxisomal ACYL-COA OXIDASE2 and on the effects of H₂O₂ supplementation, we propose that peroxisome-derived H₂O₂ acts as a regulator of chloroplast metabolism. We conclude that peroxisomal MDH2 helps photoautotrophs cope with nitrogen scarcity and high light by transmitting the redox state of the peroxisome to the chloroplast by means of malate shuttle- and H₂O₂-based redox signaling.

Some microalgae, such as Chlamydomonas reinhardtii, harbor a highly flexible photosynthetic apparatus capable of using different electron acceptors, including carbon dioxide (CO₂), protons, or oxygen (O₂), allowing survival in diverse habitats. During anaerobic induction of photosynthesis, molecular O₂ is produced at photosystem II, while at the photosystem I acceptor side, the reduction of protons into hydrogen (H₂) by the plastidial [FeFe]-hydrogenases primes CO₂ fixation. Although the interaction between H₂ production and CO₂ fixation has been studied extensively, their interplay with O₂ produced by photosynthesis has not been considered. By simultaneously measuring gas exchange and chlorophyll fluorescence, we identified an O₂ photoreduction mechanism that functions during anaerobic dark-to-light transitions and demonstrate that flavodiiron proteins (Flvs) are the major players involved in light-dependent O₂ uptake. We further show that Flv-mediated O₂ uptake is critical for the rapid induction of CO₂ fixation but is not involved in the creation of the micro-oxic niches proposed previously to protect the [FeFe]-hydrogenase from O₂. By studying a mutant lacking both hydrogenases (HYDA1 and HYDA2) and both Flvs (FLVA and FLVB), we show that the induction of photosynthesis is strongly delayed in the absence of both sets of proteins. Based on these data, we propose that Flvs are involved in an important intracellular O₂ recycling process, which acts as a relay between H₂ production and CO₂ fixation.