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. 2018 Jul 5;177(4):1639–1649. doi: 10.1104/pp.18.00721

Flavodiiron-Mediated O2 Photoreduction Links H2 Production with CO2 Fixation during the Anaerobic Induction of Photosynthesis1,[OPEN]

Adrien Burlacot a, Anne Sawyer b,2, Stéphan Cuiné a, Pascaline Auroy-Tarrago a, Stéphanie Blangy a, Thomas Happe b, Gilles Peltier a,3
PMCID: PMC6084654  PMID: 29976836

Flavodiiron proteins recycle large amounts of O2 during the anaerobic induction of photosynthesis and act as a relay of hydrogenases in priming CO2 fixation in Chlamydomonas reinhardtii.

Abstract

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.

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 (CO2) and producing oxygen (O2) 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 O2 availability, C. reinhardtii is a facultative anaerobe, able to rely either on aerobic respiration or anaerobic fermentation to oxidize reduced equivalents produced by glycolysis (Catalanotti et al., 2013). 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 (H2) as major end products (Gfeller and Gibbs, 1984; Mus et al., 2007). H2 can be produced in the dark by fermentation via the pyruvate:formate lyase pathway (Noth et al., 2013) 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 (Florin et al., 2001). During H2 photoproduction, the photolysis of water at PSII supplies electrons to the photosynthetic electron transport chain, resulting in the reduction of ferredoxin and the production of H2 (Hemschemeier et al., 2008). This process, often referred to as the direct biophotolysis pathway, is usually transient and limited by the ability of O2-consuming processes, such as mitochondrial respiration, to maintain an anaerobic environment in the vicinity of the O2-sensitive [FeFe]-hydrogenases (Melis et al., 2000; Hemschemeier et al., 2008; Burlacot and Peltier, 2018).

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 H2 occurs (Cournac et al., 2002), then the O2 produced by photosynthesis initiates a switch from fermentative metabolism to oxidative decarboxylation and oxidative phosphorylation by respiration (Happe et al., 2002), which triggers an irreversible inhibition of the [FeFe]-hydrogenases (Erbes et al., 1979; Stripp et al., 2009). Kessler (1973) observed that algal species devoid of hydrogenases experience a delay in the induction of photosynthesis and proposed that H2 photoproduction would enable algae to oxidize photosynthetic electron acceptors and, thereby, activate the photosynthetic chain after anaerobic incubation. However, the selective advantage conferred by the presence of O2-sensitive [FeFe]-hydrogenases in a photosynthetic organism producing O2 remains to be demonstrated (Ghysels et al., 2013).

During photosynthesis, O2 can act as an electron acceptor, either through a nonenzymatic process (called the Mehler reaction) or through an enzymatic process involving flavodiiron proteins (Flvs; Peltier et al., 2010). In cyanobacteria, Flvs have been shown to catalyze the reduction of O2 into water using NADPH as an electron donor (Helman et al., 2003) and to play a critical role during growth under fluctuating light regimes (Allahverdiyeva et al., 2013). In C. reinhardtii, Flvs have been demonstrated to catalyze a massive and transient O2 reduction during the induction of photosynthesis (Chaux et al., 2017a). Despite their efficiency in reducing O2 during a light transient (Chaux et al., 2017a), Flvs do not compete with CO2 fixation (Chaux et al., 2017a; Wada et al., 2018). It was proposed that Flvs function mainly where the chloroplast stroma reaches a high reducing state (Chaux et al., 2017a), conditions that are obtained when illuminating dark anaerobiosis-adapted cells (Alric, 2010).

In the nitrogen-fixing cyanobacterium Anabaena sp. PCC7120, Flv3B was shown to protect the O2-sensitive nitrogenase (Gallon, 1992) from O2 attack (Ermakova et al., 2014). Recently, based on the continuous production of H2 in air-grown C. reinhardtii (Liran et al., 2016), it was concluded that micro-oxic niches allow the [FeFe]-hydrogenases to function in the presence of O2, and it was proposed that Flvs may be involved in this process. However, O2-consuming processes have been considered as minor electron sinks under H2 production conditions (Godaux et al., 2015; Milrad et al., 2018), and the existence of a possible O2-reducing process remains to be demonstrated.

In this context, we questioned the role of Flv-mediated O2 photoreduction during the anaerobic induction of photosynthesis and its possible involvement in the formation of micro-oxic niches. By simultaneously measuring chlorophyll fluorescence and O2 exchange, we identified an O2 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 O2 reduction is critical for a fast induction of CO2 fixation but does not protect the [FeFe]-hydrogenase from O2 inhibition by creating micro-oxic niches. We propose that Flvs act as a relay of hydrogenases to promote the rapid induction of photosynthesis when O2 starts to become available as an electron acceptor and the [FeFe]-hydrogenases are inhibited by O2.

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, O2 is produced by PSII while H2 is produced by PSI. The presence of O2 inside the chloroplast may lead to O2 photoreduction at the PSI acceptor side, thus resulting in O2 recycling. In order to explore the interplay between H2 and O2 exchange during the anaerobic induction of photosynthesis, we designed a method to detect the existence of such O2 recycling in the light. O2 uptake in the light has been measured in the past using 18O-labeled O2 and a membrane inlet mass spectrometer (MIMS; Hoch and Kok, 1963), but this technique cannot be used under anaerobic conditions. However, O2 uptake rates can be inferred by subtracting the net O2 evolution from the gross O2 produced by PSII, the latter determined from chlorophyll fluorescence measurements (Kitajima and Butler, 1975). By performing simultaneous measurements of chlorophyll fluorescence and gas exchange, using a pulse amplitude-modulated (PAM) fluorimeter and a MIMS with 18O-labeled O2, respectively (Cournac et al., 2002; Supplemental Fig. S1, A and B), we first established the relationship under aerobic conditions between the chlorophyll fluorescence parameter ΔF/FM′ (Supplemental Fig. S2) and the quantum yield of O2 evolution by PSII. Since our experimental setup 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 (Genty et al., 1989), 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 nonlinearity was observed that 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 O2 production by PSII from chlorophyll fluorescence measurements were performed at light intensities corresponding to the linear range of the relationship (Supplemental Fig. S4).

Figure 1.

Figure 1.

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Combined measurements of gas exchange and chlorophyll fluorescence show the existence of O2 recycling during the first minutes of illumination of anaerobically acclimated C. reinhardtii. A, Relationship between the chlorophyll fluorescence parameter ΔF/FM′ and the apparent quantum yield of gross O2 evolution established from simultaneous measurements of chlorophyll fluorescence by a PAM fluorimeter and O2 exchange by MIMS in the presence of 18O-enriched O2. The red line shows the linear regression for the ΔF/FM′ values lower than 0.58. The chlorophyll concentration was 20 µg mL−1. Data were recorded after 10 min of dark adaptation. B, Combined measurements of H2 and O2 exchange during a dark-to-light (660 µmol photon m−2 s−1 green light) transient in 1.5-h anaerobically acclimated wild-type cells. Measured net O2 and H2 evolution rates are shown as solid circles (blue and green, respectively). Gross O2 evolution rates (black circles) were determined from ΔF/FM′ measurements based on the relationship established in A, and O2 uptake rates (red circles) were determined from the difference between gross and net O2 evolution rates. The box plots at right show gross O2 evolution and O2 uptake rates determined after 3 min of illumination (means ± sd; n = 5 biological replicates).

The PAM-MIMS setup was then used to determine H2 and O2 exchange rates during a dark-to-light transient in anaerobically adapted C. reinhardtii cells (Fig. 1B). As reported previously (Cournac et al., 2002), net H2 and O2 production rates increased rapidly upon illumination, with maximal values being observed after about 1 min of illumination (Fig. 1B). Gross O2 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 between PSI and PSII, a phenomenon associated with changes in the maximal fluorescence level (FM′; Wollman and Delepelaire, 1984). Therefore, we were careful to restrict the use of the linear relationship established in Figure 1A to the first 3 min of illumination, conditions in which no major change in FM′ was observed (Supplemental Fig. S5). From the difference between the gross O2 evolution rate (determined from chlorophyll fluorescence measurements) and net O2 evolution (measured by MIMS), we inferred the existence of an O2 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 O2 production rate (Fig. 1, B and C). It is noteworthy that O2 uptake started roughly when the maximum H2 and net O2 production rates were reached (Fig. 1B). We conclude from this experiment that O2 recycling occurs in C. reinhardtii cells upon the 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 O2 Recycling

Flvs were identified recently in C. reinhardtii as the main drivers of O2 photoreduction during the induction of photosynthesis under aerobic conditions (Chaux et al., 2017a). To test the possible involvement of Flvs during a light induction in anaerobically adapted cells, we performed experiments similar to those in Figure 1B in two independent C. reinhardtii flvB mutants lacking both FlvB and FlvA under aerobic and anaerobic conditions (Chaux et al., 2017a; Supplemental Fig. S6A). While O2 uptake started in the wild type 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 O2 uptake during the first 2 min of illumination (Fig. 2). After 3 min of illumination, the O2 uptake rate in the flvB mutants was much lower than in the wild type (Fig. 2B), such a late increase likely reflecting a growing contribution of other O2 uptake processes such as mitochondrial respiration. We conclude from this experiment that Flvs are the major actors involved in the O2 recycling occurring upon the illumination of anaerobically acclimated cells.

Figure 2.

Figure 2.

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Determination of O2 uptake rates during the first minutes of illumination of anaerobically adapted cells. After 1.5 h of dark anaerobic acclimation, algal suspensions of the wild type (WT; red) and two flvB mutants (flvB-208 in black and flvB-308 in gray) were illuminated (660 µmol photons m−2 s−1 green light). Gas exchange was measured using a MIMS, and PSII yields were determined using a PAM fluorimeter. Gross O2 evolution was determined from PSII yields. The minimal gross O2 uptake was calculated by subtracting the net O2 evolution from the calculated gross O2 evolution. A, Representative traces of minimal O2 uptake in the wild type and mutant strains. B, Mean values of minimal O2 uptake after 1, 2, and 2.5 min of illumination. Data shown are mean values ± sd (n = 3 for the mutants, n = 6 for the wild type). Asterisks mark significant differences (P < 0.05) based on ANOVA (Tukey’s adjusted P value).

H2 Photoproduction Is Enhanced in flvB Mutants

H2 photoproduction can be limited in vivo by different factors, including (1) the O2 sensitivity of the [FeFe]-hydrogenases (Erbes et al., 1979), O2 being produced in the light by PSII, and/or (2) competition for electrons at the acceptor side of PSI (Godaux et al., 2015). Flvs could be involved in both aspects, either by competing for electrons on the PSI acceptor side or by protecting the [FeFe]-hydrogenases from O2 attack. Indeed, the existence of intracellular micro-oxic niches protecting the [FeFe]-hydrogenases from O2 has been proposed (Liran et al., 2016), and Flvs have been suggested to be involved (Liran et al., 2016; Burlacot and Peltier, 2018). H2 production rates of the wild type and flvB mutant strains were thus compared during a dark-to-light transient. Enhanced H2 photoproduction was observed in flvB mutants as compared with the wild type between 1 and 3 min of illumination (Fig. 3), and this effect was not due to a difference in initial hydrogenase activities (Supplemental Fig. S7A). The O2 concentration in the cell suspension was slightly higher in the flvB mutants compared with the wild type 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 with the wild type (Supplemental Fig. S7B). Therefore, we conclude from this experiment that Flv-mediated O2 uptake competes for electrons with the [FeFe]-hydrogenases. A possible role of Flvs in the protection of the [FeFe]-hydrogenases from O2 attack could not be evidenced.

Figure 3.

Figure 3.

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H2 photoproduction in C. reinhardtii wild type and two flvB mutants. After 1.5 h of dark anaerobic acclimation, wild-type (WT; red) and two flvB mutant (flvB-208 in black and flvB-308 in gray) cell suspensions were illuminated (660 µmol photons m−2 s−1 green light), and H2 production was recorded with a MIMS. A, Representative traces of H2 production. B, H2 production rates during the first 3 min of illumination. Due to some variability in absolute gas exchange values between biological replicates, the data shown are expressed relative to the wild-type mean level ± sd (n = 3 for the mutants, n = 6 for the wild type). Asterisks indicate significant difference between the wild type and the mutant strains (P < 0.05) based on ANOVA (Tukey’s adjusted P value).

Flv-Mediated O2 Uptake Primes CO2 Fixation during the Transition from Anaerobiosis to Aerobiosis

H2 photoproduction has been reported to prime net photosynthesis and to allow the fast induction of photosynthesis under anaerobic conditions (Ghysels et al., 2013). On the other hand, Flv-mediated O2 uptake precedes but does not influence CO2 fixation during the induction of aerobic photosynthesis (Chaux et al., 2017a). To determine the extent to which Flv-mediated O2 reduction participates in the anaerobic induction of photosynthesis, we measured O2 and CO2 exchange in flvB mutants. While no significant difference in net O2 evolution and net CO2 fixation were measured after the first 3 min of illumination, a 30% decrease in both parameters was observed in both flvB mutants compared with the wild type (Fig. 4, A and B; Supplemental Fig. S9). Remarkably, this decrease in net photosynthesis was observed after H2 production stopped, which occurred after 3 to 4 min of illumination (Fig. 3A). We conclude from this experiment that O2 uptake by Flvs allows a fast induction of photosynthesis in anaerobically acclimated cells and primes CO2 fixation when H2 production has ceased.

Figure 4.

Figure 4.

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Net O2 and CO2 exchange rates during a light transient in C. reinhardtii wild type and two flvB mutants. After 1.5 h of dark anaerobic acclimation, wild-type (WT; in red) and two independent flvB mutant (flvB-208 in black and flvB-308 in gray) cell suspensions were illuminated (660 µmol photons m−2 s−1 green light). O2 and CO2 gas exchange were measured using a MIMS. A and B, Representative traces of net O2 evolution (A) and net CO2 evolution (B). C and D, Mean values of net O2 evolution (C) and net CO2 uptake (D) rates measured at 3 and 9 min of illumination. Due to some variability in absolute gas exchange values between biological replicates, the data shown are expressed in relative units (r.u.) to the wild-type mean level ± sd (n = 3 for the mutants, n = 6 for the wild type). Asterisks indicate significant differences between the wild type and the mutant strains (P < 0.05) based on ANOVA (Tukey’s adjusted P value).

Flv-Mediated O2 Uptake Primes CO2 Fixation Even in the Absence of the Hydrogenase

H2 photoproduction has been considered as the main process enabling algae to start photosynthesis following anaerobic incubation (Kessler, 1973). However, hydrogenases need at least 10 min of anaerobiosis to produce hydrogen in vivo (Cournac et al., 2002), and only a restricted group of microalgae possess a functional hydrogenase (Burlacot and Peltier, 2018). 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 (Meuser et al., 2012) and flvb (Chaux et al., 2017a); three independent triple mutant progeny (hydA1/A2 flvB-1, -2, and -3) were isolated (Supplemental Fig. S6) and analyzed further. As shown previously (Ghysels et al., 2013), the lack of both hydrogenases had a dramatic effect on the induction of photosynthesis upon the 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). CO2 fixation was more severely affected in the triple mutants as compared with the hydA1/A2 mutant (Fig. 5B). We conclude from this experiment that O2 uptake by Flvs allows a faster induction of photosynthesis after anaerobic incubation even in the absence of H2 production.

Figure 5.

Figure 5.

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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, CO2 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. Data shown are means ± sd (n = 3). Asterisks indicate significant difference between the wild type and flvB (on the left) and between triple mutants and the hydA1/A2 mutant (on the right) based on ANOVA (Tukey’s adjusted P value).

DISCUSSION

By performing simultaneous gas exchange and chlorophyll fluorescence measurements, we show here that O2 uptake occurs at the same time that H2 is produced upon the illumination of anaerobic C. reinhardtii cells. The study of flvB mutants allowed us to show the involvement of Flvs in this O2 uptake process. While different processes, including H2 production and oxidation (Gaffron and Rubin, 1942), CO2 fixation, and regulatory mechanisms such as state transitions (Ghysels et al., 2013) and cyclic electron flow (CEF; Godaux et al., 2015), have been shown to impact H2 photoproduction, little attention has been paid to the possible occurrence of O2 photoreduction, this mechanism being considered as a minor pathway for electrons in these conditions (Finazzi et al., 1999; Godaux et al., 2015). 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 intracellular O2 recycling mechanism during H2 production.

Flv-Mediated O2 Photoreduction Primes CO2 Fixation during the Transition from Anaerobiosis to Aerobiosis

Optimal photosynthetic CO2 fixation requires both ATP and NADPH and a fine-tuning of the NADPH/ATP ratio (Kramer and Evans, 2011). By reducing O2 at the acceptor side of PSI, Flvs divert electrons toward O2, thus limiting the NADPH supply; therefore, they participate in the establishment of a proton motive force that is used to produce ATP. Until now, negative effects observed under fluctuating light regimes in flv mutants were considered to result from photoinhibition (Gerotto et al., 2016; Chaux et al., 2017a; Shimakawa et al., 2017), suggesting that the Flvs play a role in protecting photosystems from long-term overreduction 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 (Yamamoto et al., 2016; Chaux et al., 2017a; Wada et al., 2018). 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 NADPH/ATP ratio for CO2 fixation, but repeated light transients induce a progressive imbalance in this ratio, thus resulting in PSI photoinhibition (Chaux et al., 2017a). Similar conditions of NADPH/ATP imbalance occur under anaerobic conditions (Clowez et al., 2015), 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 overreduction of PSI acceptors could have resulted in the production of reactive oxygen species, leading to PSI and/or PSII photoinhibition. However, no difference in PSI and PSII activities was observed between the wild type and flv mutants (Supplemental Fig. S10), showing that, if occurring, the production of reactive oxygen species 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., 2017a) and enable a proper induction of CO2 fixation in anaerobiosis. This function is particularly important when the buffering capacity of cells is exceeded, conditions that occur after several light transients under aerobiosis or after a single transient under anaerobiosis.

Flv-Mediated O2 Reduction Is a Relay between H2 Production and CO2 Fixation

During the switch from anaerobiosis to aerobiosis, H2 photoproduction is a transient process allowing the reoxidation of PSI acceptors and a fast induction of photosynthesis (Ghysels et al., 2013). Differences in the CO2 fixation rate between a flvB mutant and the wild type appeared after H2 production ceased (Supplemental Fig. S9B). Therefore, H2 photoproduction compensates for the loss of Flvs until inhibition of the [FeFe]-hydrogenase by O2. Flv-mediated O2 reduction, therefore, appears to act as a relay system, supporting the reoxidation of PSI acceptors when the [FeFe]-hydrogenase is no longer functional. We also have 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 (Meuser et al., 2011; Burlacot and Peltier, 2018). 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 O2 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 the illumination of anaerobically adapted cells (Finazzi et al., 1999) and is of great importance for the reactivation of photosynthesis under these conditions (Ghysels et al., 2013). Forti and Caldiroli (2005) showed that light-driven O2-dependent processes are involved in this process but could not dissect the molecular contributors. In our study, the increase in FM′, likely due to the transition from state II to state I (Supplemental Fig. S5), occurred after H2 production (Fig. 2A) and when Flv-dependent O2 uptake is maximal, highlighting the role of Flv-mediated O2 reduction in the transition to state I.

Flv-Mediated O2 Reduction versus CEF

Since both Flv-mediated O2 reduction and CEF produce ATP without generating NADPH, they are expected to have similar effects on photosynthesis (Shikanai and Yamamoto, 2017). Recent experiments have shown that these two processes can complement each other in various organisms (Dang et al., 2014; Gerotto et al., 2016; Yamamoto et al., 2016; Wada et al., 2018). However, this question has not been addressed in an H2-producing context, as O2 reduction processes have been neglected. Recently, Godaux et al. (2015) observed that the induction of photosynthesis in anaerobically acclimated cells without Glc oxidase was not affected by a defect in Proton Gradient Regulation Like1-dependent CEF, which indicates compensation by other mechanisms such as H2 production or Flv-dependent O2 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 (Alric, 2014; Fig. 1B). Thus, our data suggest that Flv-dependent O2 reduction is the main alternative electron route after H2 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 O2 Recycling

Upon its production by PSII, O2 can diffuse from thylakoid membranes toward the extracellular medium. During the diffusion process, O2 can be reduced by cellular metabolic pathways, thus resulting in intracellular O2 recycling. This phenomenon has been reported to involve mitochondrial respiration (Lavergne, 1989) and may potentially involve other O2 photoreduction pathways, such as those linked to the plastid terminal oxidase or the enzymatic activities of Flvs. Because a hydrogenase-deficient mutant shows PSII activity (Fig. 5A) while not producing O2 in the medium (data 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 O2 recycling.

Flvs Are Not Involved in Protecting the [FeFe]-Hydrogenase in Micro-Oxic Niches

Since the report that some algae can produce H2 in the presence of atmospheric concentrations of O2 (Liran et al., 2016), the possibility for intracellular micro-oxic niches allowing hydrogenases to be active at high O2 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 (Liran et al., 2016). We have shown here that H2 production is not negatively impacted and, in fact, is 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. Instead, we showed that the enhancement of H2 production in flvB mutants likely results from an increased electron flux toward [FeFe]-hydrogenases. This directly supports previous data showing that the competition for electrons at the acceptor side of PSI plays a key role in H2 photoproduction during a light transient (Godaux et al., 2015; Milrad et al., 2018). In aerobic conditions, Flvs have their highest activity during light transitions (Chaux et al., 2017a), 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 O2 in steady-state low light, as proposed by Liran et al. (2016), 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 O2 in microalgae.

MATERIALS AND METHODS

Chlamydomonas reinhardtii Strains and Cultures

The C. reinhardtii wild-type strain CC-4533 and flvB mutants (fvlB-208 and flvB-308) were described previously (Chaux et al., 2017a). 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. 2–4), cells were grown in photobioreactors (Dang et al., 2014; Chaux et al., 2017b) 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 Tris-acetate phosphate medium (Fig. 5). Cells were cultivated under low light and harvested during the exponential phase. The experiments presented throughout this article were performed on three independent single colony-derived lines for the wild type, for each of the two strains carrying insertions in the FLVB gene, and for each of the three hydA1/A2 flvB strains; thus, sd values account for se values of biological triplicates calculated with Prism (GraphPad Softwares).

The mutant strain mt− flvB-308 was back-crossed twice with a 137c strain (Tolleter et al., 2011), which is the parental strain of the hydA1/A2 mutant reference strain (Meuser et al., 2012). The mt− flvB mutant obtained was then crossed with the mt+ hydA1/A2 mutant. The progeny of this crossing were selected based on chlorophyll fluorescence to screen for the insertion in the FLVB gene and H2 production to screen for the insertion in HYDA1 and HYDA2. We isolated three independent progeny exhibiting a flvB mutant-like chlorophyll fluorescence transient (Chaux et al., 2017a; Supplemental Fig. S6B) that did not produce H2 after 1.5 h of dark anaerobic induction. The absence of the FlvB and FlvA proteins was then checked via immunodetection (Supplemental Fig. S6B). The three triple mutants obtained were named hydA1/A2 flvB-1, -2, and -3; CC-124 was used as a reference strain for these mutants.

Chlorophyll Fluorescence Measurements

Chlorophyll fluorescence measurements were performed using a PAM fluorimeter (Dual-PAM 100; Walz), the optic fiber of which was coupled to a Plexiglas optic guide used as a stopper for the measuring chamber (Supplemental Fig. S1). Detection pulses (10 µmol photons m−2 s−1 blue light) were supplied at a frequency of 100 Hz. Basal fluorescence was measured after a 1.5-h dark incubation under anaerobiosis and after 10 min of dark acclimation for aerobic samples. Red saturating flashes (8,000 µmol photons m−2 s−1, 600 ms) were delivered to measure FM (in dark-acclimated samples) and then every 15 s to measure FM′ (upon actinic light exposure). PSII quantum yields were calculated as (FM′ − Fs)/FM′ according to Genty et al. (1989). 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 O2 production was calculated assuming, as in aerobic conditions, that there is a linear relationship between the quantum yield of O2 evolution and the FV/FM ratio (Fig. 1A). Gross O2 evolution was then deduced linearly from the FV/FM ratio and was minimized by the MIMS-measured net O2 evolution to overcome any initial difference in PSII absorbance.

MIMS Measurements

Gas exchanges were monitored inside a water-jacketed and thermoregulated (25°C) measuring chamber (modified Hansatech O2 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-Fisher) 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 450g for 5 min and resuspended in fresh HSM medium (pH 6.2) or fresh Tris-acetate phosphate medium (pH 7.2) at a final concentration of 20 µg chlorophyll 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, 18O-enriched O2 (99% 18O2 isotope content; Euriso-Top) was bubbled into the cell suspension until approximately equal concentrations of 16O2 and 18O2 were reached. The mass spectrometer sequentially scanned gas abundances (H2, N2, 16O2, 18O2, and CO2) by automatically adjusting the magnet current to the corresponding mass peaks (m/z = 2, 28, 32, 36, and 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 H2, O2, and CO2 concentrations to the N2 concentration used as an internal reference. O2 exchange rates were measured according to Peltier and Thibault (1985). The apparent quantum yield of O2 evolution was determined by dividing gross O2 evolution rates by the incident light intensity integrated over the measuring chamber.

For H/D exchange rate measurements, D2 was bubbled into the cell suspension until saturation. Then, the cuvette was closed and gas abundances (H2, HD, D2, and N2) were recorded (m/z = 2, 3, 4, and 28, respectively). The H/D exchange rate was determined according to Jouanneau et al. (1980).

Accession Numbers

Genes studied in this article can be found at https://phytozome.jgi.doe.gov/ under 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).

Supplemental Data

The following supplemental materials are available.

  • Supplemental Figure S1. Experimental device used for combined measurements of gas exchange by using MIMS and chlorophyll fluorescence with a PAM fluorimeter.

  • Supplemental Figure S2. Representative chlorophyll fluorescence recordings obtained during a dark-to-light transient in anaerobically acclimated C. reinhardtii cells.

  • Supplemental Figure S3. Relationship between the quantum yield of O2 evolution and ΔF/FM′ in aerobiosis at a low chlorophyll concentration.

  • Supplemental Figure S4. Saturation curve of net photosynthesis with green light.

  • Supplemental Figure S5. FM′ upon a shift from dark anaerobiosis to light as a control for assessing PSII absorbance changes.

  • Supplemental Figure S6. Characterization of the mutant C. reinhardtii cells.

  • Supplemental Figure S7. Hydrogenase activity assayed in vivo by H/D exchange.

  • Supplemental Figure S8. Differences in O2 concentration present in the MIMS chamber containing flvB mutants compared with the wild type during a dark-to-light transient in anaerobically acclimated C. reinhardtii.

  • Supplemental Figure S9. Net O2 and CO2 exchange rates in the light in C. reinhardtii wild type and two flvB mutants.

  • Supplemental Figure S10. PSI and PSII activities as measured by electrochromic shift upon the illumination of anaerobically acclimated C. reinhardtii cells.

  • Supplemental Figure S11. Repartition of Flvs and [Fe-Fe]-hydrogenase on the evolutionary tree of eukaryotic microalgae.

  • Supplemental Table S1. List of algae exhibiting a Flv in their genomes.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

Acknowledgments

We thank Drs. Bernard Genty and Jean Alric (Commissariat à l’Energie Atomique [CEA] Cadarache) for helpful discussions and Frédéric Espanet (CEA Cadarache) and Charlie Mathiot for technical support. We are grateful to Dr. Pierre Richaud for critical reading of the article. 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.

Footnotes

1

This work was supported by the ERA-SynBio project Sun2Chem (ERASYNBIO1-062) and by the A*MIDEX project (ANR-11-IDEX-0001-02). 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).

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