During oxygenic photosynthesis, the reducing power generated by light energy conversion is mainly used to reduce carbon dioxide. In bacteria and archae, flavodiiron (Flv) proteins catalyze O 2 or NO reduction, thus protecting cells against oxidative or nitrosative stress. These proteins are found in cyanobacteria, mosses, and microalgae, but have been lost in angiosperms. Here, we used chlorophyll fluorescence and oxygen exchange measurement using [18 O]-labeled O 2 and a membrane inlet mass spectrometer to characterize Chlamydomonas reinhardtii flvB insertion mutants devoid of both FlvB and FlvA proteins. We show that Flv proteins are involved in a photo-dependent electron flow to oxygen, which drives most of the photosynthetic electron flow during the induction of photosynthesis. As a consequence, the chlorophyll fluorescence patterns are strongly affected in flvB mutants during a light transient, showing a lower PSII operating yield and a slower nonphotochemical quenching induction. Photoautotrophic growth of flvB mutants was indistinguishable from the wild type under constant light, but severely impaired under fluctuating light due to PSI photo damage. Remarkably, net photosynthesis of flv mutants was higher than in the wild type during the initial hour of a fluctuating light regime, but this advantage vanished under longterm exposure, and turned into PSI photo damage, thus explaining the marked growth retardation observed in these conditions. We conclude that the C. reinhardtii Flv participates in a Mehler-like reduction of O 2 , which drives a large part of the photosynthetic electron flow during a light transient and is thus critical for growth under fluctuating light regimes.

Author contributions: A.B. and G.P. designed the research; A.B., O.D., P.A., S.C., and G.P. performed research; A.B. and G.P. contributed new reagents/analytic tools; A.B. and G.P. analyzed data; A.B. and G.P. wrote the paper with inputs from Y.L.-B.

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 () of results in dramatic alterations not only in peroxisomal fatty acid breakdown but also in chloroplast starch metabolism and photosynthesis. mutants accumulated 50% more storage lipid and 2-fold more starch than the wild type during nitrogen deprivation. In parallel, showed increased photosystem II yield and photosynthetic CO fixation. Metabolite analyses revealed a >60% reduction in malate, together with increased levels of NADPH and HO in Similar phenotypes were found upon high light exposure. Furthermore, based on the lack of starch accumulation in a knockout mutant of the HO-producing peroxisomal and on the effects of HO supplementation, we propose that peroxisome-derived HO 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 HO-based redox signaling.

Nitrous oxide (N2O), a potent greenhouse gas in the atmosphere, is produced mostly from aquatic ecosystems, to which algae substantially contribute. However, mechanisms of N2O production by photosynthetic organisms are poorly described. Here we show that the green microalga Chlamydomonas reinhardtii reduces NO into N2O 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 N2O-producing hot spots. Our results provide a mechanistic understanding of N2O production in eukaryotic phototrophs and represent an important step toward a comprehensive assessment of greenhouse gas emission by aquatic ecosystems.

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

Use of microbes to produce liquid transportation fuels is not yet economically viable. A key point to reduce production costs is the design a cell factory that combines the continuous production of drop-in fuel molecules with the ability to recover products from the cell culture at low cost. Medium-chain hydrocarbons seem ideal targets because they can be produced from abundant fatty acids and, due to their volatility, can be easily collected in gas phase. However, pathways used to produce hydrocarbons from fatty acids require two steps, low efficient enzymes and/or complex electron donors. Recently, a new hydrocarbon-forming route involving a single enzyme called fatty acid photodecarboxylase (FAP) was discovered in microalgae. Here, we show that in illuminated E. coli cultures coexpression of FAP and a medium-chain fatty acid thioesterase results in continuous release of volatile hydrocarbons. Maximum hydrocarbon productivity was reached under low/medium light while higher irradiance resulted in decreased amounts of FAP. It was also found that the production rate of hydrocarbons was constant for at least 5 days and that 30% of total hydrocarbons could be collected in the gas phase of the culture. This work thus demonstrates that the photochemistry of the FAP can be harnessed to design a simple cell factory that continuously produces hydrocarbons easy to recover and in pure form.

Since the first great oxygenation event, photosynthetic microorganisms have continuously shaped the Earth's atmosphere. Studying biological mechanisms involved in the interaction between microalgae and cyanobacteria with the Earth's atmosphere requires the monitoring of gas exchange. Membrane inlet mass spectrometry (MIMS) has been developed in the early 1960s to study gas exchange mechanisms of photosynthetic cells. It has since played an important role in investigating various cellular processes that involve gaseous compounds (O 2 , CO 2 , NO, or H 2) and in characterizing enzymatic activities in vitro or in vivo. With the development of affordable mass spectrometers, MIMS is gaining wide popularity and is now used by an increasing number of laboratories. However, it still requires an important theory and practical considerations to be used. Here, we provide a practical guide describing the current technical basis of a MIMS setup and the general principles of data processing. We further review how MIMS can be used to study various aspects of algal research and discuss how MIMS will be useful in addressing future scientific challenges.

Microalgae have emerged as a promising platform for production of carbon- and energy- rich molecules, notably starch and oil. Establishing an economically viable algal biotechnology sector requires a holistic understanding of algal photosynthesis, physiology, cell cycle and metabolism. Starch/oil productivity is a combined effect of their cellular content and cell division activities. Cell growth, starch and fatty acid synthesis all require carbon building blocks and a source of energy in the form of ATP and NADPH, but with a different requirement in ATP/NADPH ratio. Thus, several cellular mechanisms have been developed by microalgae to balance ATP and NADPH supply which are essentially produced by photosynthesis. Major energy management mechanisms include ATP production by the chloroplast-based cyclic electron flow and NADPH removal by water-water cycles. Furthermore, energetic coupling between chloroplast and other cellular compartments, mitochondria and peroxisome, is increasingly recognized as an important process involved in the chloroplast redox poise. Emerging literature suggests that alterations of energy management pathways affect not only cell fitness and survival, but also influence biomass content and composition. These emerging discoveries are important steps towards diverting algal photosynthetic energy to useful products for biotechnological applications.