Adrien Burlacot

Anne Sawyer

Stéphan Cuiné

Pascaline Auroy-Tarrago

Stéphanie Blangy

Thomas Happe

Gilles Peltier
email: gilles.peltier@cea.fr

Flavodiiron-mediated O2 photoreduction links H2 production with CO2 fixation during the anaerobic induction of photosynthesis

INTRODUCTION

Chlamydomonas reinhardtii is a unicellular green alga capable of an extraordinary metabolic versatility depending on the environmental conditions it encounters. As a photosynthetic organism, it can grow photoautotrophically, using sunlight as an energy source, by fixing atmospheric carbon dioxide (CO 2 ) and producing oxygen (O 2 ) from the photolysis of water. In the presence of organic substrates such as acetate, C. reinhardtii may grow either heterotrophically or mixotrophically. Like many soil microorganisms subjected to frequent shifts in O 2 availability, C. reinhardtii is a facultative anaerobe, able to rely either on aerobic respiration or anaerobic fermentation to oxidize reduced equivalents produced by glycolysis [START_REF] Catalanotti | Fermentation metabolism and its evolution in algae[END_REF]. During anaerobic fermentation, C. reinhardtii cells express a set of enzymes involved in a complex network of fermentative pathways, resulting in the production of acetate, formate, ethanol and hydrogen (H 2 ) as major end products [START_REF] Gfeller | Fermentative metabolism of Chlamydomonas reinhardtii. 1. Analysis of fermentative products from starch in dark and light[END_REF][START_REF] Milrad | Anaerobic acclimation in Chlamydomonas reinhardtii -Anoxic gene expression, hydrogenase induction, and metabolic pathways[END_REF]. Hydrogen can be produced in the dark by fermentation via the pyruvate:formate lyase pathway [START_REF] Noth | Pyruvate:Ferredoxin oxidoreductase is coupled to light-independent hydrogen production in Chlamydomonas reinhardtii[END_REF], but also in the light due to the coupling of photosynthesis and two plastidial [FeFe]-hydrogenases, HYDA1 and HYDA2, which use reduced ferredoxin as an electron donor for the reduction of protons [START_REF] Florin | A novel type of iron hydrogenase in the green alga Scenedesmus obliquus is linked to the photosynthetic electron transport chain[END_REF]. During H 2 photoproduction, photolysis of water at photosystem II (PSII) supplies electrons to the photosynthetic electron transport chain, resulting in the reduction of ferredoxin and production of H 2 [START_REF] Hemschemeier | Hydrogen production by Chlamydomonas reinhardtii: an elaborate interplay of electron sources and sinks[END_REF]. This process, often referred to as the direct biophotolysis pathway, is usually transient and limited by the ability of O 2 -consuming processes, such as mitochondrial respiration, to maintain an anaerobic environment in the vicinity of the O 2 -sensitive [FeFe]-hydrogenases [START_REF] Melis | Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii[END_REF][START_REF] Hemschemeier | Hydrogen production by Chlamydomonas reinhardtii: an elaborate interplay of electron sources and sinks[END_REF][START_REF] Burlacot | Photosynthetic electron transfer pathways during hydrogen photoproduction in green algae: mechanisms and limitations[END_REF].

When dark-adapted anaerobic cultures of C. reinhardtii are exposed to sunlight, their metabolism changes dramatically. During the first minutes of illumination, a transient burst of H 2 occurs (Cournac et al., 2002), then the O 2 produced by photosynthesis initiates a switch from fermentative metabolism to oxidative decarboxylation and oxidative phosphorylation by respiration [START_REF] Happe | Hydrogenases in green algae: do they save the algae's life and solve our energy problems?[END_REF], and triggers an irreversible inhibition of the [FeFe]-hydrogenases [START_REF] Erbes | Inactivation of hydrogenase in cell-free extracts and whole cells of Chlamydomonas reinhardi by oxygen[END_REF][START_REF] Stripp | How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms[END_REF]. Kessler observed that algal species devoid of hydrogenases experience a delay in the induction of photosynthesis and proposed that H 2 photoproduction would enable algae to oxidize photosynthetic electron acceptors and thereby activate the photosynthetic chain after anaerobic incubation [START_REF] Kessler | Effect of anaerobiosis on photosynthetic reactions and nitrogen-metabolism of algae with and without hydrogenase[END_REF]. However, the selective advantage conferred by the presence of O 2sensitive [FeFe]-hydrogenases in a photosynthetic organism producing O 2 remains to be demonstrated [START_REF] Ghysels | Function of the chloroplast hydrogenase in the microalga Chlamydomonas: The role of hydrogenase and state transitions during photosynthetic activation in anaerobiosis[END_REF].

During photosynthesis, O 2 can act as an electron acceptor, either through a non-enzymatic process (called the Mehler reaction) or through an enzymatic process involving flavodiiron proteins (Flvs) [START_REF] Peltier | Auxiliary electron transport pathways in chloroplasts of microalgae[END_REF]. In cyanobacteria, Flvs have been shown to catalyze the reduction of O 2 into water using NADPH as an electron donor [START_REF] Helman | Genes encoding a-type flavoproteins are essential for photoreduction of O2 in cyanobacteria[END_REF] and to play a critical role during growth under fluctuating light regimes [START_REF] Allahverdiyeva | Flavodiiron proteins Flv1 and Flv3 enable cyanobacterial growth and photosynthesis under fluctuating light[END_REF]. In C. reinhardtii, Flvs have been demonstrated to catalyze a massive and transient O 2 reduction during the induction of photosynthesis (Chaux et al., 2017). Despite their efficiency in reducing O 2 during a light transient (Chaux et al., 2017), Flvs do not compete with CO 2 fixation (Chaux et al., 2017;[START_REF] Wada | Flavodiiron protein substitutes for cyclic electron flow without competing CO2 assimilation[END_REF]. It was proposed that Flvs are mainly functioning where the chloroplast stroma reaches a high reducing state (Chaux et al., 2017), conditions which are obtained when illuminating dark anaerobic-adapted cells [START_REF] Alric | Cyclic electron flow around photosystem I in unicellular green algae[END_REF].

In the nitrogen-fixing cyanobacterium Anabaena sp. PCC7120, Flv3B was shown to protect the O 2 -sensitive nitrogenase [START_REF] Gallon | Reconciling the incompatible: N2 fixation and O2[END_REF] from O 2 attack [START_REF] Ermakova | Heterocyst-specific flavodiiron protein Flv3B enables oxic diazotrophic growth of the filamentous cyanobacterium Anabaena sp. PCC 7120[END_REF]. Recently, based on the continuous production of H 2 in air-grown C. reinhardtii, [START_REF] Liran | Microoxic niches within the thylakoid stroma of air-grown Chlamydomonas reinhardtii protect [FeFe]hydrogenase and support hydrogen production under fully aerobic environment[END_REF] it was concluded that micro-oxic niches allow the [FeFe]-hydrogenases to function in the presence of O 2 and it was proposed that Flvs may be involved in this process. However, O 2 -consuming processes have been considered as minor electron sinks under H 2 production conditions [START_REF] Godaux | Induction of photosynthetic carbon fixation in anoxia relies on hydrogenase activity and Proton-Gradient Regulation-Like1-mediated cyclic electron flow in Chlamydomonas reinhardtii[END_REF][START_REF] Milrad | Anaerobic acclimation in Chlamydomonas reinhardtii -Anoxic gene expression, hydrogenase induction, and metabolic pathways[END_REF] and, the existence of a possible O 2 reducing process remains to be demonstrated.

In this context, we questioned the role of Flv-mediated O 2 photoreduction during the anaerobic induction of photosynthesis and its possible involvement in the formation of micro-oxic niches. By simultaneously measuring chlorophyll fluorescence and oxygen exchange, we identified an O 2 reduction mechanism during a light transient in anaerobically adapted C. reinhardtii cells and further demonstrate the involvement of Flvs in this process. We show that Flv-mediated O 2 reduction is critical for a fast induction of CO 2 fixation, but does not protect the [FeFe]-hydrogenase from O 2 inhibition by creating micro-oxic niches. We propose that Flvs act as a relay of hydrogenases to promote the rapid induction of photosynthesis when O 2 starts to become available as an electron acceptor and the [FeFe]-hydrogenases are inhibited by O 2 .

RESULTS

Combined gas exchanges and chlorophyll fluorescence measurements as a tool to study the photosynthetic activity of anaerobically-adapted C. reinhardtii cells. During the induction of photosynthesis in anaerobically-acclimated cells, O 2 is produced by PSII while H 2 is produced by photosystem I (PSI). The presence of O 2 inside the chloroplast may lead to O 2 photoreduction at the PSI acceptor side, thus resulting in O 2 recycling. In order to explore the interplay between H 2 and O 2 exchange during the anaerobic induction of photosynthesis, we designed a method to detect the existence of such O 2 recycling in the light. Oxygen uptake in the light has been measured in the past using [ 18 O]-labeled O 2 and a membrane inlet mass spectrometer (MIMS) [START_REF] Hoch | A mass spectrometer inlet system for sampling gases dissolved in liquid phases[END_REF], but this technique cannot be used under anaerobic conditions. However, O 2 uptake rates can be inferred by subtracting the net O 2 evolution from the gross O 2 produced by PSII, the latter determined from chlorophyll fluorescence measurements [START_REF] Kitajima | Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone[END_REF]. By performing simultaneous measurements of chlorophyll fluorescence and gas exchange, using a pulse amplitude modulated (PAM) fluorimeter and a MIMS with [ 18 O]-labeled O 2 , respectively (Cournac et al., 2002) (Supplemental Fig. S1A,B), we first established the relationship under aerobic conditions between the chlorophyll fluorescence parameter ΔF/F M ' (Supplemental Fig. S2) and the quantum yield of O 2 evolution by PSII. Since our experimental set-up was not designed to measure light absorption in a cell suspension, experiments were performed at similar chlorophyll concentrations, assuming a constant light absorption during the time-course of the experiment. As reported previously in leaves of land plants [START_REF] Genty | The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence[END_REF], a linear relationship was observed under a wide range of light intensities (Fig. 1A). For the highest quantum yields (measured at low light intensities), a non-linearity was observed which was unaffected by the chlorophyll concentration of the sample (Supplemental Fig. S3), indicating that it likely did not result from a light heterogeneity in the cuvette. Further experiments aiming at determining O 2 production by PSII from chlorophyll fluorescence measurements were performed at light intensities corresponding to the linear range of the relationship (Supplemental Fig. S4).

The PAM-MIMS set-up was then used to determine H 2 and O 2 exchange rates during a dark to light transient in anaerobically-adapted C. reinhardtii cells (Fig. 1B). As previously reported (Cournac et al., 2002), net H 2 and O 2 production rates rapidly increased upon illumination, with maximal values being observed after about 1 min of illumination (Fig. 1B). Gross O 2 evolution rates were determined from chlorophyll fluorescence measurements with the assumption that linearity in the relationship established under aerobic conditions (Fig. 1A) was valid under anaerobic conditions. However, PSII absorbance may vary when switching from anaerobic to aerobic conditions due to the movement of light harvesting complex proteins (LHCs) between PSI and PSII, a phenomenon associated with changes in the maximal fluorescence level F M ' [START_REF] Wollman | Correlation between changes in light energy distribution and changes in thylakoid membrane polypeptide phosphorylation in Chlamydomonas reinhardtii[END_REF]. We were therefore careful to restrict the use of the linear relationship established in Fig. 1A to the first 3 min of illumination, conditions in which no major change in F M ' was observed (Supplemental Fig. S5). From the difference between the gross O 2 evolution rate (determined from chlorophyll fluorescence measurements) and net O 2 evolution (measured by MIMS), we inferred the existence of an O 2 uptake process, starting after about 50 s of illumination and reaching a maximal value after 2 min of illumination, where it was estimated to be about 40% of the gross O 2 production rate (Fig. 1B andC).

It is noteworthy that O 2 uptake roughly started when the maximum H 2 and net O 2 production rates were reached (Fig. 1B). We conclude from this experiment that O 2 recycling occurs in C. reinhardtii cells upon illumination of anaerobically adapted cells and is concomitant with dramatic changes in the use of electrons at the PSI acceptor side.

Role of Flvs in O 2 recycling. Flvs have been recently identified in C. reinhardtii as the main drivers

of O 2 photoreduction during the induction of photosynthesis under aerobic conditions (Chaux et al., 2017). To test the possible involvement of Flvs during a light induction in anaerobically adapted cells, we performed similar experiments as in Fig. 1B in two independent C. reinhardtii flvB mutants lacking both FlvB and FlvA under aerobic and anaerobic conditions (Chaux et al., 2017) (Supplemental Fig. S6A). While O 2 uptake started in the wild type (WT) after about 50 s of illumination and reached a maximal value after 2 min, both flvB-208 and flvB-308 mutants showed a strongly reduced O 2 uptake during the first 2 min of illumination (Fig. 2A andB). After 3 min of illumination, the O 2 uptake rate in the flvB mutants was much lower than in the WT (Fig. 2 B), such a late increase likely reflecting a growing contribution of other O 2 uptake processes such as mitochondrial respiration. We conclude from this experiment that Flvs are the major actors involved in the O 2 recycling occurring upon illumination of anaerobically acclimated cells.

Hydrogen photoproduction is enhanced in flvB mutants. Hydrogen photoproduction can be limited in vivo by different factors, including: i. the O 2 -sensitivity of the [FeFe]-hydrogenases [START_REF] Erbes | Inactivation of hydrogenase in cell-free extracts and whole cells of Chlamydomonas reinhardi by oxygen[END_REF], O 2 being produced in the light by PSII, and/or, ii. competition for electrons at the acceptor side of PSI [START_REF] Godaux | Induction of photosynthetic carbon fixation in anoxia relies on hydrogenase activity and Proton-Gradient Regulation-Like1-mediated cyclic electron flow in Chlamydomonas reinhardtii[END_REF]. Flvs could be involved in both aspects, either by competing for electrons on the PSI acceptor side, or by protecting the [FeFe]-hydrogenases from O 2 attack. Indeed, the existence of intracellular micro-oxic niches protecting the [FeFe]-hydrogenases from O 2 has been proposed [START_REF] Liran | Microoxic niches within the thylakoid stroma of air-grown Chlamydomonas reinhardtii protect [FeFe]hydrogenase and support hydrogen production under fully aerobic environment[END_REF], and Flvs have been suggested to be involved [START_REF] Liran | Microoxic niches within the thylakoid stroma of air-grown Chlamydomonas reinhardtii protect [FeFe]hydrogenase and support hydrogen production under fully aerobic environment[END_REF][START_REF] Burlacot | Photosynthetic electron transfer pathways during hydrogen photoproduction in green algae: mechanisms and limitations[END_REF]. Hydrogen production rates of WT and flvB mutant strains were thus compared during a dark to light transient. Enhanced H 2 photoproduction was observed in flvB mutants as compared to the WT between 1 and 3 min of illumination (Fig. 3A andB), and this effect was not due to a difference in initial hydrogenase activities (Supplemental Fig. S7A). The O 2 concentration in the cell suspension was slightly higher in the flvB mutants compared to the WT during the first minutes of the light transient (Supplemental Fig. S8). However, no difference in hydrogenase inhibition was observed after 2 min of illumination in a flvB mutant as compared to the WT (Supplemental Fig. S7B). We therefore conclude from this experiment that Flv-mediated O 2 uptake competes for electrons with the [FeFe]-hydrogenases. A possible role of Flvs in the protection of the [FeFe]-hydrogenases from O 2 attack could not be evidenced.

Flv-mediated O 2 uptake primes CO 2 fixation during the transition from anaerobiosis to aerobiosis. Hydrogen photoproduction has been reported to prime net photosynthesis and to allow the fast induction of photosynthesis under anaerobic conditions [START_REF] Ghysels | Function of the chloroplast hydrogenase in the microalga Chlamydomonas: The role of hydrogenase and state transitions during photosynthetic activation in anaerobiosis[END_REF]. On the other hand, Flv-mediated O 2 uptake precedes but does not influence CO 2 fixation during the induction of aerobic photosynthesis (Chaux et al., 2017). To determine the extent to which Flv-mediated O 2 reduction participates in the anaerobic induction of photosynthesis, we measured O 2 and CO 2 exchange in flvB mutants. While no significant difference in net O 2 evolution and net CO 2 fixation were measured after the first 3 min of illumination, a 30% decrease in both parameters was observed in both flvB mutants compared to the WT (Fig. 4A andB; Supplemental Fig. S9). Remarkably, this decrease in net photosynthesis was observed after H 2 production stopped, which occurred after 3-4 min of illumination (Fig. 3A). We conclude from this experiment that O 2 uptake by Flvs allows a fast induction of photosynthesis in anaerobically acclimated cells and primes CO 2 fixation when H 2 production has ceased.

Flv-mediated O 2 uptake primes CO 2 fixation even in the absence of the hydrogenase. Hydrogen photoproduction has been considered as the main process enabling algae to start photosynthesis following anaerobic incubation [START_REF] Kessler | Effect of anaerobiosis on photosynthetic reactions and nitrogen-metabolism of algae with and without hydrogenase[END_REF]. However, hydrogenases need at least 10 min of anaerobiosis to become activated (Cournac et al., 2002) and only a restricted group of microalgae possess a functional hydrogenase [START_REF] Burlacot | Photosynthetic electron transfer pathways during hydrogen photoproduction in green algae: mechanisms and limitations[END_REF]. Given that most unicellular green algae harbor Flvs (Supplemental Fig. S10), the involvement of Flvs in the start of photosynthesis after anaerobic incubation in the absence of an active hydrogenase was questioned. A triple mutant (hydA1/A2 flvB) lacking both hydrogenases and both Flvs was obtained by crossing two existing mutants: hydA1/A2 [START_REF] Meuser | Genetic disruption of both Chlamydomonas reinhardtii [FeFe]hydrogenases: Insight into the role of HYDA2 in H2 production[END_REF] and flvb (Chaux et al., 2017); three independent triple mutant progenies (hydA1/A2 flvB 1, 2 and 3) were isolated (Supplemental Fig. S6) and further analyzed. As previously shown [START_REF] Ghysels | Function of the chloroplast hydrogenase in the microalga Chlamydomonas: The role of hydrogenase and state transitions during photosynthetic activation in anaerobiosis[END_REF], the lack of both hydrogenases had a dramatic effect on the induction of photosynthesis upon illumination of anaerobically-acclimated cells (Fig. 5A). While photosynthesis started after a 3 min illumination in the hydA1/A2 mutant, no change in the PSII yield was detected in triple hydA1/A2 flvB mutants during the first 10 min of illumination (Fig. 5A). CO 2 fixation was more severely affected in the triple mutants as compared to the hydA1/A2 mutant (Fig. 5B). We conclude from this experiment that O 2 uptake by Flvs allows a faster induction of photosynthesis after anaerobic incubation even in the absence of H 2 production.

DISCUSSION

By performing simultaneous gas exchange and chlorophyll fluorescence measurements, we show here that O 2 uptake occurs at the same time as H 2 is produced upon illumination of anaerobic C. reinhardtii cells. The study of flvB mutants allowed us to show the involvement of Flvs in this O 2 uptake process.

While different processes including H 2 production and oxidation [START_REF] Gaffron | Fermentative and photochemical production of hydrogen in algae[END_REF], CO 2 fixation, and regulatory mechanisms such as state transitions [START_REF] Ghysels | Function of the chloroplast hydrogenase in the microalga Chlamydomonas: The role of hydrogenase and state transitions during photosynthetic activation in anaerobiosis[END_REF] or cyclic electron flow [START_REF] Godaux | Induction of photosynthetic carbon fixation in anoxia relies on hydrogenase activity and Proton-Gradient Regulation-Like1-mediated cyclic electron flow in Chlamydomonas reinhardtii[END_REF] have been shown to impact H 2 photoproduction, little attention has been paid to the possible occurrence of O 2 photoreduction, this mechanism being considered as a minor pathway for electrons in these conditions [START_REF] Finazzi | State transitions, cyclic and linear electron transport and photophosphorylation in Chlamydomonas reinhardtii[END_REF][START_REF] Godaux | Induction of photosynthetic carbon fixation in anoxia relies on hydrogenase activity and Proton-Gradient Regulation-Like1-mediated cyclic electron flow in Chlamydomonas reinhardtii[END_REF]. Here, we have shown that Flv-mediated photoreduction can drive up to 40% of the total linear electron flow during the induction of photosynthesis in anaerobic cells (Fig. 1B), supplying experimental evidence for the existence of an important intra-cellular O 2 recycling mechanism during H 2 production.

Flv-mediated O 2 photoreduction primes CO 2 fixation during the transition from anaerobiosis to

aerobiosis. Optimal photosynthetic CO 2 fixation requires both ATP and NADPH and a fine tuning of the NADPH/ATP ratio [START_REF] Kramer | The importance of energy balance in improving photosynthetic productivity[END_REF]. By reducing O 2 at the acceptor side of PSI, Flvs divert electrons towards O 2 , thus limiting the NADPH supply, and therefore participate in the establishment of a proton motive force which is used to produce ATP. Until now, negative effects observed under fluctuating light regimes in flv mutants were considered to result from photoinhibition [START_REF] Gerotto | Flavodiiron proteins act as safety valve for electrons in Physcomitrella patens[END_REF]Chaux et al., 2017;[START_REF] Shimakawa | The Liverwort, Marchantia, drives alternative electron flow using a flavodiiron protein to protect PSI[END_REF], suggesting that the Flvs play a role in protecting photosystems from long-term over-reduction that could lead to photoinhibition.

However, the loss of Flvs in algae or the expression in angiosperms of Flvs (which do not naturally contain Flvs) had no effect on net photosynthesis during a dark to light transient (Chaux et al., 2017;[START_REF] Wada | Flavodiiron protein substitutes for cyclic electron flow without competing CO2 assimilation[END_REF][START_REF] Yamamoto | Artificial remodelling of alternative electron flow by flavodiiron proteins in Arabidopsis[END_REF]. This apparent discrepancy may be due to the fact that the metabolic buffering capacity of cells can compensate for the loss of Flvs during steady state conditions by maintaining a favorable NADHP/ATP ratio for CO 2 fixation, but that repeated light transients induce a progressive imbalance in this ratio, thus resulting in PSI photoinhibition (Chaux et al., 2017).

Similar conditions of NADPH/ATP imbalance occur under anaerobic conditions [START_REF] Clowez | The involvement of hydrogen-producing and ATP-dependent NADPH-consuming pathways in setting the redox poise in the chloroplast of Chlamydomonas reinhardtii in anoxia[END_REF], resulting in a delay in the induction of photosynthesis when Flvs are absent (Fig. 4). An alternative explanation of the delayed induction in the flv mutants is that over-reduction of PSI acceptors could have resulted in the production of reactive oxygen species (ROS), leading to PSI and/or PSII photoinhibition. However, no difference in PSI and PSII activities was observed between WT and flv mutants (Supplemental Fig. S10), showing that if occurring, the production of ROS had no impact on photosystem activity at this time scale. We conclude that rather than protecting photosystems from photoinhibition, Flvs are involved in the priming of photosynthesis likely by rebalancing the NADPH/ATP ratio. By finely tuning the NADPH/ATP ratio, Flvs protect photosystems from photoinhibition in oxic conditions (Chaux et al., 2017), and enable a proper induction of CO 2 fixation in anaerobiosis. This function is particularly important when the buffering capacity of cells is exceeded, conditions which occur after several light transients under aerobiosis, or after a single transient under anaerobiosis.

Flv-mediated O 2 reduction is a relay between H 2 production and CO 2 fixation.

During the switch from anaerobiosis to aerobiosis, H 2 photoproduction is a transient process allowing the reoxidation of PSI acceptors and a fast induction of photosynthesis [START_REF] Ghysels | Function of the chloroplast hydrogenase in the microalga Chlamydomonas: The role of hydrogenase and state transitions during photosynthetic activation in anaerobiosis[END_REF]. Differences in the CO 2 fixation rate between a flvB mutant and the WT appeared after H 2 production ceased (Supplemental Fig. S9B). H 2 photoproduction therefore compensates the loss of Flvs until inhibition of the [FeFe]hydrogenase by O 2 . Flv-mediated O 2 reduction therefore appears to act as a relay system, supporting the re-oxidation of PSI acceptors when the [FeFe]-hydrogenase is no longer functional. We have also shown that Flvs act as the main drivers for the induction of photosynthesis in mutants devoid of hydrogenase (Fig. 5). Interestingly, although Flvs are present in most green algae (Supplemental Fig. S11), hydrogenases are restricted to the core Chlorophytes [START_REF] Meuser | Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A[END_REF][START_REF] Burlacot | Photosynthetic electron transfer pathways during hydrogen photoproduction in green algae: mechanisms and limitations[END_REF]. If Flvs alone may allow a transition from anaerobiosis to aerobiosis in the light in most green algae, the presence of hydrogenases may have conferred a selective advantage in specific biotopes from the core chlorophytes by allowing a faster photosynthetic induction when O 2 is not yet available.

Interplay with state transitions. During anaerobiosis, mobile light harvesting antennae are phosphorylated, detach from PSII and become associated with PSI. This process, called state transitions, is reversible and cells in state II (antennae associated with PSI) can return to state I (antennae associated with PSII) in more oxidizing conditions. A transition to state I occurs upon illumination of anaerobically adapted cells [START_REF] Finazzi | State transitions, cyclic and linear electron transport and photophosphorylation in Chlamydomonas reinhardtii[END_REF] and is of great importance for the reactivation of photosynthesis under these conditions [START_REF] Ghysels | Function of the chloroplast hydrogenase in the microalga Chlamydomonas: The role of hydrogenase and state transitions during photosynthetic activation in anaerobiosis[END_REF]. Forti and Caldiroli [START_REF] Forti | State transitions in Chlamydomonas reinhardti. The role of the mehler reaction in state 2-to-state 1 transition[END_REF] showed that light-driven O 2 -dependent processes are involved in this process but could not dissect the molecular contributors. In our study, the increase in F M ' likely due to the transition from state II to state I (Supplemental Fig. S5) occurred after H 2 production (Fig. 2A), and when Flv-dependent O 2 uptake is maximal, highlighting the role of Flv-mediated O 2 reduction in the transition to state I.

Flv-mediated O 2 reduction versus Cyclic Electron Flow (CEF).

Since both Flv-mediated O 2 reduction and CEF produce ATP without generating NADPH, they are expected to have similar effects on photosynthesis [START_REF] Shikanai | Contribution of cyclic and pseudo-cyclic electron transport to the formation of proton motive force in chloroplasts[END_REF]. Recent experiments have shown that these two processes can complement each other in various organisms [START_REF] Dang | Combined increases in mitochondrial cooperation and oxygen photoreduction compensate for deficiency in cyclic electron flow in Chlamydomonas reinhardtii[END_REF][START_REF] Gerotto | Flavodiiron proteins act as safety valve for electrons in Physcomitrella patens[END_REF][START_REF] Yamamoto | Artificial remodelling of alternative electron flow by flavodiiron proteins in Arabidopsis[END_REF][START_REF] Wada | Flavodiiron protein substitutes for cyclic electron flow without competing CO2 assimilation[END_REF]. However, this question has not been addressed in a H 2 -producing context, as O 2 reduction processes have been neglected. Recently, [START_REF] Godaux | Induction of photosynthetic carbon fixation in anoxia relies on hydrogenase activity and Proton-Gradient Regulation-Like1-mediated cyclic electron flow in Chlamydomonas reinhardtii[END_REF] observed that induction of photosynthesis in anaerobically-acclimated cells without glucose oxidase was not affected by a defect in Proton Gradient Regulation Like 1 (PGRL1)-dependent CEF, which indicates compensation by other mechanisms such as H 2 production or Flv-dependent O 2 reduction.

On the other hand, we have shown in this work that the anaerobic induction of photosynthesis is impaired in flvB mutants. However, the maximum capacity of each alternative electron route can account for up to 40% of the linear electron flow [START_REF] Alric | Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii: (II) Involvement of the PGR5-PGRL1 pathway under anaerobic conditions[END_REF] (Fig. 1B). Our data thus suggest that Flv-dependent O 2 reduction is the main alternative electron route after H 2 photoproduction upon illumination in anaerobically adapted C. reinhardtii, and that in these conditions it exceeds the capacity of CEF in tuning the NADPH/ATP ratio.

Intracellular O 2 recycling. Upon its production by PSII, O 2 can diffuse from thylakoid membranes towards the extra-cellular medium. During the diffusion process, O 2 can be reduced by cellular metabolic pathways, thus resulting in intra-cellular O 2 recycling. This phenomenon has been reported to involve mitochondrial respiration [START_REF] Lavergne | Mitochondrial responses to intracellular pulses of photosynthetic oxygen[END_REF], and may potentially involve other O 2 photoreduction pathways such as those linked to the Plastid Terminal Oxidase (PTOX) or the enzymatic activities of Flvs. Because a hydrogenase deficient mutant shows PSII activity (Fig. 5A) while not producing O 2 in the medium (not shown), and because this phenomenon depends on the presence of Flvs (absent in a Flv and hydrogenase deficient mutant), we conclude here that Flvs take part in such an intracellular O 2 recycling.

Flvs are not involved in protecting the [FeFe]-hydrogenase in micro-oxic niches. Since the report that some algae can produce H 2 in the presence of atmospheric concentrations of O 2 [START_REF] Liran | Microoxic niches within the thylakoid stroma of air-grown Chlamydomonas reinhardtii protect [FeFe]hydrogenase and support hydrogen production under fully aerobic environment[END_REF], the possibility for intra-cellular micro-oxic niches allowing hydrogenases to be active at high O 2 concentrations has been considered. However, although the molecular players involved in such micro-oxic niches have not been identified so far, Flvs have been proposed as possible candidates [START_REF] Liran | Microoxic niches within the thylakoid stroma of air-grown Chlamydomonas reinhardtii protect [FeFe]hydrogenase and support hydrogen production under fully aerobic environment[END_REF]. We have shown here that H 2 production is not negatively impacted and is in fact higher in the absence of Flvs (Fig. 2) and that the activity of the [FeFe]-hydrogenase is not affected (Supplemental Fig. S5). Based on these data we conclude that at least in the experimental conditions that have been tested, Flvs are not involved in the establishment of intracellular micro-oxic niches that would protect the [FeFe]-hydrogenase. We instead showed that the enhancement of H 2 production in flvB mutants likely results from an increased electron flux towards [FeFe]-hydrogenases. This directly supports previous data showing that the competition for electrons at the acceptor side of PSI plays a key role in H 2 photoproduction during a light transient [START_REF] Godaux | Induction of photosynthetic carbon fixation in anoxia relies on hydrogenase activity and Proton-Gradient Regulation-Like1-mediated cyclic electron flow in Chlamydomonas reinhardtii[END_REF][START_REF] Milrad | Anaerobic acclimation in Chlamydomonas reinhardtii -Anoxic gene expression, hydrogenase induction, and metabolic pathways[END_REF]. In aerobic condition, Flvs have their highest activity during light transitions (Chaux et al., 2017), conditions that we have explored in anaerobiosis here. However, no protective effect of the [FeFe]hydrogenases could be observed under these conditions. The possibility that Flvs might protect the [FeFe]-hydrogenases from O 2 in steady state low light, as proposed by [START_REF] Liran | Microoxic niches within the thylakoid stroma of air-grown Chlamydomonas reinhardtii protect [FeFe]hydrogenase and support hydrogen production under fully aerobic environment[END_REF], seems rather unlikely given their reduced activity during steady state illumination (Chaux et al., 2017). Further investigations will be needed to determine the possible molecular mechanisms involved in the protection of [FeFe]-hydrogenases from O 2 in microalgae.

MATERIALS AND METHODS

Chlamydomonas strains and cultures. The C. reinhardtii WT strain CC-4533 and flvB mutants (fvlB-208 and flvB-308) were previously described (Chaux et al., 2017). Cells were grown photoautotrophically in flasks at 25°C in HSM medium (pH 7.2) under dim light (30-40 µmol photons m -2 s -1 ). For some experiments (Figs. 234), cells were grown in photobioreactors [START_REF] Dang | Combined increases in mitochondrial cooperation and oxygen photoreduction compensate for deficiency in cyclic electron flow in Chlamydomonas reinhardtii[END_REF]Chaux et al., 2017), under a light intensity of 66 µmol photons m -2 s -1 by bubbling air in HSM medium (pH 6.2). C. reinhardtii double mutants devoid of both hydrogenases (hydA1/A2) and triple mutants (hydA1/A2 flvB) were grown in TAP medium (Fig. 5.) Cells were cultivated under low light and harvested during the exponential phase. Experiments presented throughout this manuscript were performed on three independent single colony-derived lines for the WT, for each of the two strains carrying insertions in the FLVB gene and for each of the three hydA1/A2 flvB strains, thus SDs account for standard errors of biological triplicates calculated with Prism (GraphPad Softwares, USA).

The mutant strain mt -flvB-308 was back-crossed twice with a 137c strain [START_REF] Tolleter | Control of hydrogen photoproduction by the proton gradient generated by cyclic electron flow in Chlamydomonas reinhardtii[END_REF] which is the parental strain of the hydA1/A2 mutant reference strain [START_REF] Meuser | Genetic disruption of both Chlamydomonas reinhardtii [FeFe]hydrogenases: Insight into the role of HYDA2 in H2 production[END_REF]. The mt -flvB mutant obtained was then crossed with the mt + hydA1/A2 mutant. The progeny of this crossing was selected based on chlorophyll fluorescence to screen for the insertion in the FLVB gene and H 2 production to screen for the insertion in HYDA1 and HYDA2. We isolated three independent progenies exhibiting a flvB-mutant-like chlorophyll fluorescence transient (Chaux et al., 2017) (Supplemental Fig. S6 B) that did not produce H 2 after 1.5 h of dark anaerobic induction. Absence of the FlvB and FlvA proteins was then checked via immunodetection (Supplemental Fig. S6 B). The three triple mutants obtained were named hydA1/A2 flvB (1, 2 and 3 respectively), CC-124 was used as a reference strain for these mutants.

Chlorophyll fluorescence measurements. Chlorophyll fluorescence measurements were performed using a pulse amplitude-modulated fluorimeter (Dual-PAM 100, Walz, Germany), the optic fiber of which was coupled to a Plexiglas optic guide used as a stopper for the measuring chamber (Supplemental Figure S1). Detection pulses (10 µmol photons m -2 s -1 blue light) were supplied at a 100 Hz frequency. Basal fluorescence (F 0 ) was measured after a 1.5h dark incubation under anaerobiosis and for aerobic samples after 10 min of dark acclimation. Red saturating flashes (8,000 µmol photons m -2 s -1 , 600 ms) were delivered to measure F M (in dark acclimated samples) and then every 15 s to measure F M ' (upon actinic light exposure). PSII quantum yields were calculated as (F M '-F s )/F M ' according to [START_REF] Genty | The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence[END_REF]). An actinic green light was chosen in order to limit light heterogeneity in the measuring chamber, delivered by three LEDs surrounding the measuring chamber and powered by a stabilized voltage generator. Anaerobic gross O 2 production was calculated assuming, like in aerobic conditions, that there is a linear relationship between the quantum yield of O 2 evolution and the F V /F M ratio (Fig. 1A). Gross O 2 evolution was then deduced linearly from the F V /F M ratio and was minimized by the MIMS-measured net O 2 evolution to overcome any initial difference in PSII absorbance.

Membrane Inlet Mass Spectrometry (MIMS) measurements. Gas exchanges were monitored inside a water-jacketed and thermoregulated (25°C) measuring chamber (modified Hansatech O 2 electrode chamber) containing 1.5 mL of cell suspension. The bottom of the chamber was sealed by a Teflon membrane (13 µm thickness) allowing dissolved gases to be introduced into the ion source of the mass spectrometer (model Prima δB, Thermo-Fischer) through a vacuum line containing a water trap cooled at -65°C. Cells were harvested from stabilized (at least 48 h) turbidostatic cultures in photobioreactors or exponential growth phase cultures in flasks, then centrifuged at 450 x g for 5 min and resuspended in fresh HSM medium (pH 6.2) or fresh TAP medium (pH 7.2) at a final concentration of 20 µg chl mL -1 . The cell suspension was introduced into the measuring chamber under constant stirring and the cuvette was closed with the optic guide stopper. Actinic green light was supplied by three green LEDs. For anaerobic measurements, self-anaerobiosis was reached inside the measuring chamber and cells were incubated for 1.5 h or 45 min in anaerobiosis. For aerobic measurements, [ 18 O]-enriched O 2 (99% 18 O 2 isotope content; Euriso-Top) was bubbled into the cell suspension until approximately equal concentrations of 16 O 2 and 18 O 2 were reached. The mass spectrometer sequentially scanned gas abundances (H 2 , N 2 , 16 O 2, 18 O 2 and CO 2 ) by automatically adjusting the magnet current to the corresponding mass peaks (m/z = 2; 28; 32; 36; 44, respectively).

The amperometric signal collected by the spectrometer was calibrated by bubbling pure gases in a cell-free solution. Gas exchange rates were determined after correction for the gas consumption by the mass spectrometer assuming a first order kinetic, and normalizing H 2 , O 2 and CO 2 concentrations to the N 2 concentration used as an internal reference. O 2 exchange rates were measured according to [START_REF] Peltier | Oxygen uptake in the light in Chlamydomonas. Evidence for persistent mitochondrial respiration[END_REF]. The apparent quantum yield of O 2 evolution was determined by dividing gross O 2 evolution rates by the incident light intensity integrated over the measuring chamber.

For H/D exchange rate measurements, D 2 was bubbled into the cell suspension until saturation. Then, the cuvette was closed and gas abundances (H 2 , HD, D 2 and N 2 ) were recorded (m/z=2; 3; 4; 28; respectively). The H/D exchange rate was determined according to [START_REF] Jouanneau | Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rhodopseudomonas capsulata[END_REF]. photons m -2 s -1 green light). Gas exchange was measured using a MIMS and PSII yields were determined using a PAM. Gross oxygen evolution was determined from PSII yields. The minimal gross oxygen uptake was calculated by subtracting the net oxygen evolution to the calculated gross oxygen evolution. (A) Representative traces of minimal O 2 uptake in the WT and mutant strains. (B) Mean values of minimal O 2 uptake after 1 min, 2 min and 2.5 min illumination. Shown are mean values (+/-SD, n=3 for the mutants, n=6 for the WT). A star marks significant differences (Pvalue <0.05) based on ANOVA analysis (Tukey adjusted P value).

Figure legends Figure 1 .

 legends1 Figure legends Figure 1. Combined measurements of gas exchange and chlorophyll fluorescence show the existence of O 2 recycling during the first minutes of illumination of anaerobically acclimated C. reinhardtii. (A) Relationship between the chlorophyll fluorescence parameter ΔF/F M and the apparent quantum yield of gross O 2 evolution established from simultaneous measurements of chlorophyll fluorescence by a PAM fluorimeter, and O 2 exchange by MIMS in the presence of [ 18 O]-enriched O 2 . The red line shows the linear regression for the ΔF/F M values lower than 0.58. The chlorophyll concentration was 20 µg Chl mL -1 . Data were recorded after 10 minutes dark adaptation. (B) Combined measurements of H 2 and O 2 exchange during a dark to light (660 µmol photon m -2 s -1 green light) transient in 1.5h anaerobically acclimated WT cells. Measured net O 2 and H 2 evolution rates are shown as filled circles (blue and green, respectively). Gross O 2 evolution rates (black empty circles) were determined from ΔF/F M measurements based on the relationship established in (A), and O 2 uptake rates (red empty circles) from the difference between gross and net O 2 evolution rates. Right panel: box plots show gross O 2 evolution and O 2 uptake rates determined after 3 min of illumination (mean +/-SD, n=5 biological replicates).

Figure 2 .

 2 Figure 2. Determination of oxygen uptake rates during the first minutes of illumination of anaerobically-adapted cells. After 1.5h of dark anaerobic acclimation, algal suspensions of wild type

Figure 3 .

 3 Figure 3. Hydrogen photoproduction in C. reinhardtii wild-type and two flvB mutants. After 1.5h of dark anaerobic acclimation, wild-type (WT, red) and two flvB mutants (flvB-208 in black, and flvB-308 in grey) cell suspensions were illuminated (660 µmol photons m -2 s -1 green light) and H 2 production was recorded with a MIMS. (A) Representative traces of H 2 production. (B) H 2 production rates during the first three minutes of illumination. Due to some variability in absolute gas exchange values between biological replicates, shown data are expressed relative to the WT mean level (+/-SD, n=3 for the mutants, n=6 for the WT). Stars indicate significant difference between WT and mutant strains (P-value < 0.05) based on ANOVA analysis (Tukey adjusted P value).

Figure 4 .

 4 Figure 4. Net O 2 and CO 2 exchange rates during a light transient in C. reinhardtii wild-type and two flvB mutants. After 1.5h of dark anaerobic acclimation, wild-type (WT, in red) and two independent flvB mutants (flvB-208 in black, and flvB-308 in grey) cell suspensions were illuminated (660 µmol photons m -2 s -1 green light). O 2 and CO 2 gas exchange were measured using a MIMS. (A, B) Representative traces of net O 2 evolution and net CO 2 evolution. (C, D) Mean values of net O 2 evolution (C) and net CO 2 uptake (D) rates measured at 3 min and 9 min of illumination. Due to some variability in absolute gas exchange values between biological replicates, shown data are expressed relative to the WT mean level (+/-SD, n=3 for the mutants, n=6 for the WT). Stars indicate significant differences between the wild-type and the mutant strains (P-value < 0.05) based on ANOVA analysis (Tukey adjusted P value).

Figure 5 .

 5 Figure 5. Induction of photosynthesis in anaerobically adapted C. reinhardtii cells devoid of hydrogenases and Flvs. Chlorophyll fluorescence and gas exchange rates were recorded under illumination (660 µmol photons m -2 s -1 ). (A) Operating PSII quantum yield. (B) CO 2 fixation rates measured after 9 min of illumination. Anaerobic acclimation was performed during 90 min for hydrogenase containing strains and 45 min for hydrogenase deficient strains. Shown are mean (+/-SD, n=3), a star marks a significant difference between WT and flvB (on the left) and triple mutants and the hydA1/A2 mutant (on the right) based on ANOVA analysis (Tukey adjusted P value).

The authors thank Drs. Bernard Genty and Jean Alric (CEA Cadarache) for helpful discussions and Frédéric Espanet (CEA Cadarache) and Charlie Mathiot for technical support. The authors are grateful to Dr. Pierre Richaud for critical reading of the manuscript. Experimental support was provided by the HélioBiotec platform, funded by the European Union (European Regional Development Fund), the Région Provence Alpes Côte d'Azur, the French Ministry of Research and the CEA. T.H. appreciates funding by the Volkswagen Stiftung (Design of [FeS]-cluster containing Metallo-DNAzymes (Az 93412)) and the Deutsche Forschungsgemeinschaft (GRK 2341: Microbial Substrate Conversion).This work was supported by the ERA-SynBio Sun2Chem, and by the A*MIDEX (ANR-11-IDEX-0001-02) projects.

Funding information. This work was supported by the ERA-SynBio project Sun2Chem, and by the A*MIDEX (ANR-11-IDEX-0001-02) project.

Accession Numbers

Genes studied on this articles can be found on https://phytozome.jgi.doe.gov/ under the loci Cre03.g199800 (HYDA1), Cre09.g396600 (HYDA2), Cre12.g531900 (FLVA) and Cre16.g691800 (FLVB).

Sequence data from this article can be found in the GenBank data library under accession numbers XM_001693324.1 (HYDA1), XM_001694451.1 (HYDA2), XM_001699293.1 (FLVA) and XM_001692864.1 (FLVB). S1. List of algae exhibiting a Flv in their genome. Acknowledgements

Supplemental