Functional redundancy between flavodiiron proteins and NDH-1 in Synechocystis sp. PCC 6803.

In oxygenic photosynthetic organisms excluding angiosperms, flavodiiron proteins (FDPs) catalyze light-dependent reduction of O2 to H2 O. This alleviates electron pressure on the photosynthetic apparatus and protects it from photodamage. In Synechocystis sp. PCC 6803, four FDP isoforms function as hetero-oligomers of Flv1 and Flv3 and/or Flv2 and Flv4. An alternative electron transport pathway mediated by the NAD(P)H dehydrogenase-like complex (NDH-1) also contributes to redox hemostasis and the photoprotection of photosynthesis. Four NDH-1 types have been characterized in cyanobacteria: NDH-11 and NDH-12 , which function in respiration; and NDH-13 and NDH-14 , which function in CO2 uptake. All four types are involved in cyclic electron transport. Along with single FDP mutants (∆flv1 and Δflv3) and the double NDH-1 mutants (∆d1d2, which is deficient in NDH-11,2 and ∆d3d4, which is deficient in NDH-13,4 ), we studied triple mutants lacking either one of Flv1 or Flv3, and NDH-11,2 or NDH-13,4 . We show that the presence of either Flv1/3 or NDH-11,2 , but not NDH-13,4 , is indispensable for survival during changes in growth conditions from high CO2 /moderate light to low CO2 / high light. Our results demonstrate functional redundancy between FDPs and NDH-11,2 under the studied conditions. We suggest that ferredoxin likely functions as a primary electron donor to both FDPs and NDH-11,2 , allowing their functions to be dynamically coordinated for efficient oxidation of PSI and for photoprotection under variable CO2 and light availability.


INTRODUCTION
Photosynthetic organisms have evolved a variety of different regulatory mechanisms, which are important for the protection of the photosynthetic machinery during rapid changes in environmental conditions. Fluctuating light intensities present particular risk to the photosystems due to over-reduction of the photosynthetic electron transport chain (PETC), particularly photosystem (PS) I (Allahverdiyeva et al., 2015;Tiwari et al., 2016;Shimakawa et al., 2016). To counter this, cyanobacteria, algae and plants (excluding angiosperms) employ C-type flavodiiron proteins (FDPs) as a strong photoprotective electron sink, directing excess photosynthetic electrons from downstream of PSI to O 2 (Helman et al., 2003;Allahverdiyeva et al., 2013;Gerotto et al., 2016;Ilik et al., 2017;Chaux et al., 2017;Jokel et al., 2018;Alboresi et al., 2019). This process is referred to as the Mehler-like reaction and, in contrast to the Mehler reaction (Mehler, 1957), does not produce harmful reactive oxygen species (Vicente et al., 2002;Brown et al., 2019).
The genome of the b-cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) encodes four isoforms of FDPs, namely Flv1-4, which function in O 2 photoreduction mainly as hetero-oligomers consisting of either Flv1 and Flv3 or Flv2 and Flv4 (Zhang et al., 2004;Mustila et al., 2016;Santana-Sanchez et al., 2019). Recently, we showed that under air level [CO 2 ], Flv1/3 and Flv2/4 hetero-oligomers function in a coordinated and interdependent manner: Flv1/ 3 stimulates strong but transient O 2 photoreduction upon the onset of illumination or during increases in light intensity, while Flv2/4 hetero-oligomers catalyze a slower and limited steady-state reduction of O 2 , also using electrons downstream of PSI (Santana-Sanchez et al., 2019). In elevated [CO 2 ] and in air [CO 2 ] at alkaline pH 9, the flv4-flv2 operon encoding Flv2, Flv4 and the Sll0218 protein is downregulated (Zhang et al., 2009;Santana-Sanchez et al., 2019). Nevertheless, a low level of Flv1/3 in elevated [CO 2 ] stimulates strong steady-state O 2 photoreduction, whereas in air [CO 2 ] at pH 9, Flv1/3 is solely responsible for strong but transient O 2 photoreduction (Santana-Sanchez et al., 2019). Whilst in vitro assays showed that homo-oligomers of recombinant Flv1, Flv3 and Flv4 can reduce O 2 with NADH and/or NADPH (Vicente et al., 2002;Shimakawa et al., 2015;Brown et al., 2019), ferredoxin-NADP + -reductase (FNR) and reduced ferredoxin (Fd) are yet to be considered as possible donors to FDPs. Moreover, the use of Synechocystis mutants solely overexpressing Flv1 or Flv3 clearly demonstrated that, in contrast to in vitro experiments, homo-oligomers of Flv3 or Flv1 are not involved in O 2 photoreduction in vivo (Mustila et al., 2016). Thus, the electron donor to FDPs remains to be elucidated in vivo.
In the current study, we aim to elucidate how the electron transport pathways mediated by FDPs and NDH-1 cooperate to allow the maintenance of redox poise between the PETC, respiration, CCM and CO 2 fixation in the Calvin-Benson-Bassham cycle (CBB) under variable light and C i availability. To this end, we employed both biophysical and biochemical methods to characterize various Synechocystis mutant strains with combined deficiencies of both FDP and NDH-1 pathways. Our results provide convincing evidence for the presence of either Flv1/3 or NDH-1 1,2 , but not NDH-1 3,4 , being indispensable for the survival of Synechocystis cells under transitions from high [CO 2 ] conditions to the combined stress conditions of air [CO 2 ] and high light. We show that the dynamically coordinated and cooperative function of Flv1/3 and NDH-1 1,2 is required for the photoprotection of the photosynthetic apparatus of Synechocystis cells and discuss the molecular mechanisms involved. We suggest that this coordination is enabled by a shared electron donor, as Fd probably functions as the main reductant of both Flv1/3 and NDH-1 1,2 , although the possible contribution of NADPH as a donor cannot be fully excluded.

RESULTS
Simultaneous inactivation of Flv1/3 and NDH-1 1,2 is lethal upon shift from high to air [CO 2

] and high light
To examine whether the simultaneous inactivation of NDH-1 and FDPs has an adverse effect on cell survival, we monitored the growth of Synechocystis wild-type (WT) and various mutant strains lacking (i) either Flv1 or Flv3 (Δflv1 and Δflv3, respectively; Helman et al., 2003), (ii) both NdhD1 and NdhD2 (Δd1d2, deficient in NDH-1 1 and NDH-1 2 ; Ohkawa et al., 2000b), (iii) both NdhD3 and NdhD4 (Δd3d4, deficient in NDH-1 3 and NDH-1 4 ; Ohkawa et al., 2000b), as well as triple mutants with combined deficiencies of both pathways (Δflv1 d1d2, Δflv3 d1d2, Δflv1 d3d4, Δflv3 d3d4) in conditions of differing CO 2 availability and light intensity. The triple mutants exhibited similar or slightly slower growth compared with the WT and other mutant strains under high [CO 2 ] (3% CO 2 ) and moderate light (ML, intensity of 50 lmol photons m À2 sec À1 of photosynthetically active radiance) (Figure 1a). We also detected no substantial difference in growth when the 3% CO 2 /ML pre-grown cells were diluted [optical density (OD) 750 = 0.1] and shifted to air [CO 2 ] conditions under constant light intensity (Figure 1b; Figure S1a,c). In contrast, when cultures pre-grown in 3% [CO 2 ]/ML were diluted (OD 750 = 0.1) and subjected simultaneously to air [CO 2 ] and high light stress conditions (HL, intensity of 220 lmol photons m À2 sec À1 ), the growth of both Δflv3 d1d2 and Δflv1 d1d2 strains was completely inhibited (Figure 1d). Importantly, Δflv1, Δflv3 and Δd1d2 strains demonstrated growth similar to WT cells under air [CO 2 ]/HL conditions. We next examined whether pre-adaptation to air [CO 2 ] would rescue the growth arrest. When cells previously adapted to air [CO 2 ]/ML were diluted (OD 750 = 0.1) and shifted to air [CO 2 ]/HL, no differences in growth patterns between the strains were detected ( Figure 1c). Importantly, the triple mutants lacking either Flv1 or Flv3 and NDH-1 3,4 (Δflv1 d3d4 and Δflv3 d3d4 strains) did not show a lethal phenotype after a shift from 3% [CO 2 ]/ML to air [CO 2 ]/HL, although somewhat reduced growth was observed compared with the control strains ( Figure S1b,d). This suggests that, under the studied conditions, cooperation of Flv1/3 with NDH-1 3,4 complexes is not as crucial as with NDH-1 1,2 . Our attempts to create M55/Δflv1 and M55/Δflv3 double mutants lacking either Flv1 or Flv3 and the central membrane component of the NDH-1 complex, NdhB, were both unsuccessful. We were unable to obtain any M55/Δflv1 colonies and the M55/Δflv3 mutant strain could not be segregated (Figure S1e), suggesting that the absence of both Flv1/3 and NDH-1 disrupts essential cell metabolism.
We also studied the growth of the mutant strains on agar plates containing BG-11 ( Figure 1e). For these experiments, 3% CO 2 /ML-grown cells were diluted and grown on plates under either air [CO 2 ]/ML or air [CO 2 ]/HL conditions for 7 days. The Δflv1 and Δflv3 and Δd1d2 mutants did not exhibit any visible differences in growth capacity under these conditions in comparison with the WT. In contrast, the growth of the Δflv1 d1d2 and Δflv3 d1d2 triple mutants was strongly reduced under air [CO 2 ]/ ML, while no growth was detected under air [CO 2 ]/HL (Figure 1e). The Δflv1 d3d4 and Δflv3 d3d4 triple mutants demonstrated a slow growth phenotype, similarly to that previously reported for Δd3d4 on agar plates (Ohkawa et al., 2000b).
As the growth phenotypes of Δflv1 and Δflv3, Δflv1 d1d2 and Δflv3 d1d2 were similar under all conditions, we only included Δflv3 and Δflv3 d1d2 in the majority of subsequent experiments.
Either Flv1/3 or NDH-1 1,2 is required for the oxidation of PSI during sudden increases in light intensity  (Helman et al., 2003;Allahverdiyeva et al., 2011Allahverdiyeva et al., , 2013 and by NDH-1 1,2 complex-mediated respiration and CET (Ohkawa et al., 2000b;Bernat et al., 2011) suggests a functional redundancy between these electron transport pathways. To investigate this possibility further, we used membrane inlet mass spectrometry (MIMS) to examine the kinetics of O 2 and CO 2 exchange upon HL illumination of dark-adapted WT and mutant cells grown for 4 days in air [CO 2 ]/ML. To distinguish O 2 uptake from photosynthetic gross O 2 evolution under illumination, we enriched cell suspensions with 18 O 2 before the measurements. In dark-adapted WT cells, a transient peak in O 2 uptake occurs during the first minute of illumination ( Figure 2a). We showed recently that this transient peak is attributed to the Mehler-like reaction catalyzed predominantly by Flv1/3 hetero-oligomers, while Flv2/4 hetero-oligomers mainly contribute to steady-state light-induced O 2 reduction in an interdependent manner (Santana-Sanchez et al., 2019). In Δflv3 cells, light-induced O 2 reduction was almost abolished and only a slight impairment of the rate of photosynthetic gross O 2 evolution was observed in comparison with WT cells (Figure 2b). This result is in agreement with previous studies (Helman et al., 2003;Allahverdiyeva et al., 2013;Santana-Sanchez et al., 2019). In contrast to the WT, Δd1d2 cells lacked a fast decay phase of light-induced O 2 uptake during the first minute of illumination, demonstrating sustained O 2 photoreduction at high levels [approximately 75-100 µmol O 2 mg chlorophyll (Chl) À1 h À1 ] throughout the illumination period. Meanwhile, gross O 2 evolution was diminished nearly two-fold compared with the WT (Figure 2c). Thus, sustained O 2 photoreduction in Δd1d2 is not due to increased electron flow from PSII, but most probably due to increased activity of FDPs. Indeed, Δflv3 d1d2 mutant cells showed only minor light-induced O 2 uptake transiently during the first minute of illumination ( Figure 2d). The Δflv3 d1d2 cells demonstrated slower induction of photosynthetic O 2 evolution following two-component kinetics, and slightly impaired steady-state gross O 2 evolution compared with WT (Figure 2d).
The initial peak in CO 2 uptake rate at the onset of illumination probably reflects activation of the CCM (Liran et al., 2018). Accordingly, this initial peak is absent in the M55 mutant deficient in NDH-1 3,4 in addition to NDH-1 1,2 , as well as in WT cells grown at pH 6 ( Figure S2). No salient impairment of CCM was observed in any of the studied strains under air [CO 2 ]/ML, with CO 2 uptake rates peaking at approximately 1.5 mM CO 2 mg Chl À1 h À1 . In WT as well as in Δflv3, CO 2 fixation is then induced and the respiratory compensation point surpassed after approximately 1 min of illumination (Figure 2a,b). However, in Δd1d2, induction of CO 2 fixation is severely delayed and the cells only reached the compensation point at the end of the 5-min illumination period ( Figure 2c). Intriguingly, although a slight delay was observed also in Δflv3 d1d2, induction of CO 2 fixation was largely recovered in the triple mutant ( Figure 2d).
These MIMS results indicated that combined deficiency of Flv1/3 and NDH-1 1,2 under standard growth conditions, air [CO 2 ]/ML, slightly perturbs photosynthetic electron transfer and the redox poise between the PETC and cytosolic sink reactions. Next, we examined the ability of the mutant strains to adjust their photosynthetic activity to variable light conditions by measuring Chl a fluorescence simultaneously with P700 redox changes in conditions where light intensity periodically fluctuated between low (25 lmol photons m À2 sec À1 , LL) and high irradiance (530 lmol photons m À2 sec À1 , HL). Cells lacking Flv3 suffered from transient PSI acceptor side limitation Y(NA) upon sudden increases in light intensity, resulting in delayed oxidation of PSI (Figure 3a), which is in line with Figure 2. O 2 and CO 2 exchange rates in wild-type and mutant strains. Cells were grown in air [CO 2 ]/ moderate light for 4 days, after which the cells were harvested and chlorophyll (Chl) a concentration adjusted to 10 lg ml À1 with fresh BG-11. Cells were dark-adapted for 15 min, and gas exchange was monitored by membrane inlet mass spectrometry over a 5-min illumination period with 500 lmol photons m À2 sec À1 of white actinic light. Before the measurements, samples were supplemented with 18 O 2 at an equivalent concentration to 16 O 2 to distinguish O 2 uptake from O 2 evolution, and with 1.5 mM NaHCO 3 . Dashed line in each panel indicates the compensation point of CO 2 fixation (uptake rate equals respiratory rate). The experiment was repeated with three independent biological replicates, of which representative measurements are shown.
© 2020 The Authors. previous studies (Helman et al., 2003;Allahverdiyeva et al., 2013). However, during subsequent 1 min cycles of LL/HL, the ability of Dflv3 to oxidize PSI in HL improved, suggesting that cells were able to acclimate to the fluctuating light condition via a compensatory mechanism that is distinct from Flv1/3 hetero-oligomers, such as, for example, NDH-1 mediated electron transport.
We detected no acceptor-side limitation during the HL phases in Dd1d2 ( Figure 3a) as PSI was oxidized similarly to the WT ( Figure 3b). However, interestingly, Dd1d2 cells exhibited slightly elevated acceptor-side limitation during the LL phases of the experiment (Figure 3a), suggesting a role for NDH-1 1,2 in maintaining photosynthetic redox poise in light-limited conditions. In the triple mutant strain Dflv3 d1d2, transitions from LL to HL as well as from dark to light caused severe limitation on the acceptor side of PSI, resulting in an inability to oxidize PSI during periodic 1-min HL illumination. Unlike the Dflv3 mutant, Dflv3 d1d2 did not show improvement in the PSI acceptor side limitation during subsequent cycles. This implies a compensating activity of NDH-1 in Dflv3 mutant under the studied conditions. Interestingly, diminished donor side limitation in Dflv3 d1d2 resulted in slightly elevated effective yield of PSI during the HL phases ( Figure 3c). This could indicate the attenuation of pH-dependent limitation of electron transfer at Cyt b 6 f, due to impairment of proton motive force (pmf) generation in CET and the Mehler-like reaction. The effective yield of PSII was slightly decreased in HL in Dflv3 and Dd1d2, but not in Dflv3 d1d2 (Figure 3d).
The differences in photosynthetic electron transport reported above may also be contingent on altered redox states of the NADP+/NADPH pool. Therefore, to investigate the effect of Flv3 and/or NDH-1 1,2 deficiency on NADP+/ NADPH redox kinetics, we recorded NADPH fluorescence changes from dark-adapted cells simultaneously with Chl a fluorescence in conditions where actinic light intensity fluctuated between 25 (LL) and 530 lmol photons m À2 sec À1 (HL) similarly to Figure 3. Upon dark-to-LL transitions, NADPH rapidly accumulated close to a maximal amount in WT, Dflv3 and Dd1d2 cells, while in Dflv3d1d2 the NADP pool was highly reduced already in darkness. Reoxidation of NADPH then occurred in WT and Dflv3d1d2 cells, while in Dflv3 a slower oxidation phase preceded a transient rereduction phase during the first minute of illumination ( Figure 4a,b). Strong reduction of the NADP+ pool was also detected in Dflv3 during the second HL phase of the experiment. In Dd1d2 cells, very little oxidation of the NADPH pool occurred during illumination, even at HL-LL transitions (Figure 4c). Chl a fluorescence also remained at an elevated level (Figure 4c), suggesting a reduced PQ pool. This was possibly due to delayed activation of CO 2 fixation in the CBB cycle (Figure 2c), and impaired CET or dark respiration. In contrast, NADPH was strongly oxidized in Dflv3 d1d2 cells at HL-LL transitions (Figure 4d), but was predominantly reduced in dark-adapted cells (Figure 4d). Upon cessation of illumination, oxidation of the NADPH pool was followed by transient re-reduction after approximately 10 sec. As observed previously (Schreiber and Klughammer, 2009;Holland et al., 2015), the re-reduction peak coincided with a secondary post-illumination rise in Chl a fluorescence (PIFR), while oxidation of NADPH paralleled an initial PIFR. The post-illumination re-reduction of NADP+ was diminished and both Chl a PIFR peaks were missing in Dd1d2 ( Figure 4c). Therefore, the PIFR peaks  Klughammer and Schreiber (2008). Chlorophyll a fluorescence changes were measured simultaneously with PSI redox changes and used to calculate (d) effective yield of PSII. Cells were grown in air [CO 2 ]/moderate light for 4 days, after which the cells were harvested and chlorophyll a concentration adjusted to 10 lg ml À1 with fresh BG-11. Cells were dark-adapted for 10 min, and then illuminated with red actinic light intensity alternating between 25 and 530 lmol photons m À2 sec À1 in 1 min periods. Saturating pulses (500 msec of 5000 lmol photons m À2 sec À1 ) were provided every 15 sec. Values shown are averages from 3-4 independent biological replicates AE SE.
© 2020 The Authors.  ]/moderate light for 4 days, after which the cells were harvested and Chl a concentration adjusted to 5 lg ml À1 with fresh BG-11. Cells were dark-adapted for 20 min, and then subjected to illumination with red actinic light alternating between 25 and 530 lmol photons m À2 sec À1 in 1-min periods. NADPH redox changes were monitored with the Dual-PAM 100 spectrophotometer and its 9-AA/NADPH accessory module by measuring fluorescence changes between 420 and 580 nm induced by excitation at 365 nm, as described by (Schreiber and Klughammer, 2009) and (Kauny and Setif, 2014). Chl a fluorescence was recorded simultaneously.
(e) NADPH redox changes during illumination of dark-adapted cells with 530 lmol photons m À2 sec À1 . Other experimental details are as in a-d.
(f) Shows a magnification of the first 2 sec of illumination in (e). Values are normalized according to minimum and maximum fluorescence changes at the onset and cessation of illumination, respectively. Experiments were repeated with three independent biological replicates, of which representative measurements are shown. HL, high light/irradiance; LL, low light/irradiance; r.u., relative units.  Figure 2). No substantial differences were observed between the strains, as close to maximal reduction of NADP+ was obtained within approximately 0.25 sec ( Figure 4f) and maintained throughout a 40-sec illumination period (Figure 4e). This argues against NADPH being a primary electron donor to either Flv1/3 or NDH-1 1,2 , at least during fast time scales, although based on the current data, we cannot exclude the possibility of NADPH contribution during longer time scales or in steady-state conditions.
Redox status of PSI donor and acceptor side electron carriers and build-up of pmf during dark-to-light transitions depend on both Flv1/3 and NDH-1 1,2 To elucidate the molecular mechanism behind the photosynthetic phenotypes of the studied mutant strains, we next utilized a DUAL-KLAS-NIR spectrophotometer to distinguish between redox changes of plastocyanin (PC), P700 and Fd (Klughammer and Schreiber, 2016;Schreiber, 2017;Setif et al., 2019) upon exposure of dark-adapted cells to HL (503 lmol photons m À2 sec À1 ). Upon illumination of WT cells, rapid oxidation of P700 and PC occurred, followed by transient reduction of PC, P700, as well as Fd after approximately 0.2 sec (Figure 5a). Reoxidation of all three electron carriers then ensued after approximately 0.5 sec. In Dflv3 cells, P700 and PC remained mostly reduced after the initial oxidation transient until approximately 3 sec, when another transient and partial reoxidation peak of P700 and PC occurred at approximately 8 sec (Figure 5b). This was followed by re-reduction of P700, similar to that observed recently by Bulychev et al. (2018). After 15 sec in light, P700 and PC became gradually oxidized. In contrast to WT, Fd remained reduced for several seconds in light, and was only slowly reoxidized over the 30 sec illumination period (Figure 5b). These observations indicate the importance of Flv1/3 as an electron sink to O 2 , accepting electrons presumably from reduced Fd (after approximately 0.5 sec in light). In Dd1d2, redox changes of PC, P700 and Fd were similar to WT, except that the reduction of P700+ following initial oxidation as well as reduction of Fd occurred already after approximately 50 msec (Figure 5c). In Dflv3 d1d2, re-reduction of P700+ and reduction of Fd also occurred already after approximately 50 msec (Figure 5d), suggesting involvement of NDH-1 1,2 as an acceptor of electrons from Fd at that stage. Fd was reduced more quickly in Dflv3 d1d2 than in any other strains and was slowly reoxidized over 30 sec (Figure 5d), probably due to the shortage of electron acceptors (Figure 3a). In summary, these NIR-spectroscopic measurements revealed that Flv1/3 and NDH-1 1,2 control the redox poise between PSI, PC and primary electron acceptor Fd during specific time frames at transitions to HL. The results are congruent with Fd functioning as the electron donor to both NDH-1 1,2 and Flv1/3. To inspect whether the phenotypes observed above depend on differential build-up or regulation of pmf, we measured in vivo changes in the pmf by monitoring the absorbance change difference between 500 and 480 nm, which constitutes the electrochromic shift (ECS) in Synechocystis (Viola et al., 2019). In vivo measurement of lightinduced ECS revealed that after 1 sec of illumination of dark-adapted WT cells with 500 lmol photons m À2 sec À1 , high pmf level was transiently generated, followed by decline during the subsequent seconds ( Figure 5e). After approximately 10 sec of illumination, pmf again increased towards a steadier value (Figure 5e). Congruently with the strong reduction of P700 and Fd (Figure 5b), the initial pmf peak after the first second of illumination was heavily dependent on the presence of Flv1/3. In both Dflv3 and Dflv3 d1d2, pmf and thylakoid proton flux (vH+) were lower than in WT after 1 sec (Figure 5g). In Dflv3, pmf remained drastically lower than in WT during the first seconds of illumination, but differed only slightly from WT thereafter due to diminished conductivity of the thylakoid membrane (gH+) (Figure 5f), which is mainly determined by the activity of the adenosine triphosphate (ATP) synthase (Cruz et al., 2005;Viola et al., 2019). In Δd1d2, the thylakoid proton flux was similar to WT (Figure 5g), probably due to enhanced FDP activity (Figure 2c) compensating for impaired CET and respiration. However, pmf remained lower than in WT throughout the 1-min experiment (Figure 5e) because of elevated conductivity (Figure 5f). In contrast, despite drastically lower proton flux (Figure 5g), Δflv3 d1d2 cells maintained pmf close to the WT levels after the first seconds in light (Figure 5e) due to diminished conductivity (Figure 5f). This indicates that the increased acceptor side limitation of PSI in Δflv3 d1d2 in comparison with Δflv3 (Figure 3a) is not caused by lack of photosynthetic control as a consequence of lowered pmf generation in CET. Based on the comparison of ECS kinetics in single, double and triple mutant cells studied here, we conclude that both Flv1/3 and NDH-1 1,2 contribute to proton flux during transitions from dark to HL. However, deficiency in NDH-1 1,2 in Δd1d2 is mostly compensated by elevated FDP activity in terms of proton flux, while ATP synthase activity is increased, possibly as a response to the delay in activation of carbon fixation (Figure 2c). In contrast, impaired thylakoid proton flux in Δflv3 during  dark-to-light transitions cannot be compensated by NDH-1. Instead, downregulation of ATP-synthase activity lowers thylakoid conductivity and allows maintenance of pmf (apart from the first seconds of illumination) in Δflv3 and, even more dramatically, in Δflv3 d1d2.

PSI content is diminished in triple mutants deficient in Flv1/3 and NDH-1 1,2 shifted to air [CO 2 ] and HL
The results from the growth assays, along with the realtime gas exchange, P700 redox change and ECS measurements suggested that in Dd1d2 cells, an increase in the activity of FDPs compensates for the absence of a functional NDH-1 1,2 complex, allowing efficient oxidation of PSI under HL. Conversely, in Dflv1 and Dflv3 mutants, overreduction of the electron transport chain occurs initially upon exposure to HL, but another mechanism(s) eventually allow acclimation and survival in high [CO 2 ] and high light (Helman et al., 2003). Under air [CO 2 ] Flv1/3, hetero-oligomers catalyze transient O 2 photoreduction, which is why no strong growth phenotype is observed in Dflv1 or Dflv3 under constant HL (Allahverdiyeva et al., 2013;Santana-Sanchez et al., 2019). However, when both Flv1/3 and NDH-1 1,2 are absent and the electron sink capacity of the cytosol is not elevated by high [CO 2 ], cells are unable to oxidize PSI in high light, possibly resulting in lethal photodamage to PSI. To assess this hypothesis, we employed immunoblotting to provide estimates of the protein content of PSII (D1) and PSI (PsaB) subunits, orange carotenoid protein (OCP), large isoform of the Fd-NADP+ oxidoreductase (FNR L ), FDPs, as well as NdhD3 and the bicarbonate transporter SbtA after 24 h exposure to different growth conditions. WT and Dflv1 or Dflv3 cells grown at 3% [CO 2 ]/ML and shifted to air [CO 2 ]/ML at OD = 0.1, accumulated similar levels of the PSII reaction center protein D1 and the PSI subunit PsaB. However, we detected a moderate decrease in the amount of both D1 and PsaB in the Dflv1 d1d2 and Dflv3 d1d2 triple mutants, while an increased amount of both proteins was detected in Dd1d2 (Figure 6a). After 24 h exposure to air [CO 2 ]/HL, PsaB amount had decreased even further in Dflv1 d1d2 and Dflv3 d1d2 (Figure 6c). Furthermore, 77K fluorescence spectra revealed that while Dflv3 d1d2 and WT cells grown under air [CO 2 ]/ML had similar PSI/PSII ratios (Figure 6d), the 24-h exposure to air [CO 2 ]/ HL caused a dramatic decrease in the relative PSI fluorescence cross-section ( Figure 6e). This strongly supports the hypothesis that loss of PSI contributes to the lethality of the shift to air [CO 2 ]/HL. Interestingly, a substantial decrease in D1 content in air [CO 2 ]/HL was also observed in the triple mutants (Figure 6b,c).
Importantly, the Dflv1 d1d2 and Dflv3 d1d2 strains were unable to induce substantial accumulation of the proteins encoded by the flv4-2 operon after a shift from 3% to air [CO 2 ] (Figure 6a,c). Only when pre-grown for 4 days in air [CO 2 ]/ML before being shifted to higher irradiance, the expression of the operon was induced (Figure 6b). Similar to flv4-2 operon proteins, both NdhD3 and SbtA failed to accumulate after shifts from 3% to air [CO 2 ]/HL in the triple mutants, which probably impairs CCM and contributes to the lethal phenotype of the triple mutants in those conditions. Only small amounts of NdhD3 and SbtA were detected also in triple mutant cells shifted from 3% [CO 2 ] to air [CO 2 ]/ML (Figure 6a). However, while closer to the WT amount of NdhD3, only a small amount of SbtA was detected after 24 h in air [CO 2 ]/HL (shifted from air [CO 2 ]/ ML) (Figure 6b). In the Dd1d2 mutant, the NdhD3 content was similar to WT, but Flv3 as well as the flv4-2 operon proteins (Flv2, Flv4 and Sll0218) were upregulated in all conditions tested (Figure 6), which probably contributes to the increased rate of O 2 photoreduction in that strain (Figure 2c). The amounts of OCP and of FNR L were unchanged in the mutant strains in all conditions ( Figure 6).

DISCUSSION
Flv1/3 hetero-oligomers have been shown to be essential for photoreduction of O 2 as a rapid response to excessive reduction of PSI, and consequently, for cell survival under (a) WT, (b) Dflv3, (c) Dd1d2, and (d) Dflv3 d1d2 cells were dark-adapted for 10 min, after which absorbance difference changes at 780-820 nm, 820-870 nm, 840-965 nm and 870-965 nm were measured with the DUAL-KLAS-NIR spectrometer during a 30-sec illumination at 503 lmol photons m À2 sec À1 of red actinic light. PC, P700 and Fd signals were deconvoluted from the absorbance difference changes using differential model plots determined for this study ( Figure S3a). Traces were then normalized according to the maximal redox changes determined with the NIRMAX script ( Figure S3b) to obtain relative redox changes of PC, P700 and Fd. Upper panels in a-d show magnifications of the first 1.5 sec of illumination from the lower panels. The experiment was repeated with three independent biological replicates, of which representative traces are shown. (e) Build-up of pmf during transitions from dark to 500 lmol photons m À2 sec À1 of green actinic light. Absorbance difference between 500 and 480 nm (electrochromic shift, ECS) was measured with a JTS-10 spectrometer, and light-induced pmf was determined from the dark interval relaxation kinetics of the ECS signal. Means AE SEM from three to four independent experiments are shown. Small inset shows pmf values after 1 sec of illumination for a clearer view. (f) Conductivity of the thylakoid membrane (gH+) after 1 and 30 sec of illumination as in (e). gH+ was determined as the inverse of the time constant of firstorder post-illumination decay kinetics of the ECS signal. (g) Thylakoid proton flux (vH+), calculated as pmf 9 gH+. Individual data points are shown as colored circles in e-g. (h) Representative traces of 500-480 nm absorbance differences used to determine the values in (e) and post-illumination ECS decay kinetics during a 500 msec dark interval after 30 sec in light (lower panels). First-order fits are shown in green. Cells, after growth in air [CO 2 ]/50 lmol photons m À2 sec À1 for 4 days, were harvested and Chl a concentration adjusted to 10 lg ml À1 with fresh BG-11 for a-d and to 7.5 lg ml À1 for e-h.
© 2020 The Authors. Another alternative electron transport component, the NDH-1 complex, in turn, functions in CET around PSI, using Fd as the electron donor (Saura and Kaila, 2019;Schuller et al., 2019). Besides CET, the NDH-1 1,2 complexes, incorporating the NdhD1 or NdhD2 subunit, are involved in respiration, whereas NDH-1 3,4 complexes, incorporating the NdhD3 or NdhD4 subunit, function in CO 2 uptake. In the current study, we have used Synechocystis mutant strains with different combinations of deficiencies in Flv1/3 and NDH-1 to examine the  (Figure 1d,e). In addition to the absence of these two important pathways, lethality is due to an inability to accumulate low C i -inducible photoprotective CCM proteins (Figure 6c). Combined deficiency of Flv1/3 and NDH-1 3,4 did not result in a lethal phenotype upon similar shifts, indicating that functional redundancy exists specifically between the Mehler-like reaction and NDH-1 1,2 Coordinated functions of Flv1/3 and NDH-1 1,2 protect PSI by maintaining redox poise between the PETC and carbon fixation Cells lacking functional Flv1/3 hetero-oligomers suffer from transient over-reduction of PSI during sudden increases in light intensity (Figures 3 and 5) due to the impairment of the Mehler-like reaction (Figure 2; Allahverdiyeva et al., 2013). Whilst NAD(P)H has been proposed as the electron donor to Flv3 (Vicente et al., 2002;Brown et al., 2019) and Flv1 homo-oligomers (Brown et al., 2019), in vivo experiments have been unsupportive (Mustila et al., 2016), with exact electron donor(s) to FDP hetero-oligomers yet to be proven. In vivo experiments performed in this study demonstrate that the quick reoxidation of Fd after 0.5 sec (Figure 5a) in light is absent in the Dflv3 deletion strains, whereby Fd remains strongly reduced (Figure 5b). This result, together with the lack of impairment in NADP+ reduction and oxidation kinetics of Dflv3 deletion strains (similar conditions, Figure 4f) provide strong support for Fd, rather than NAD(P)H, being the primary electron donor to the Flv1/3 hetero-oligomer in vivo. Accordingly, Fd has been shown to interact with Flv1 and Flv3 by Fd chromatography (Hanke et al., 2011) and with Flv3 using a twohybrid assay (Cassier-Chauvat and Chauvat, 2014). The deficiency of NDH-1 1,2 , in turn, reduced the ability of cells to maintain oxidized P700 and Fd under HL soon after (approximately 50-200 msec) a dark-to-light transition (Figure 5c). This brief time scale may be due to PSI-NDH-1 supercomplexes (Gao et al., 2016) where P700 can be oxidized rapidly upon the onset of illumination. It has been shown that formation of the NDH-1-PSI supercomplex is important to keep PSI functional under various stress conditions (Zhao et al., 2017). NDH-1 1,2 deficiency also caused slightly elevated acceptor side limitation of PSI under LL (Figure 3a), which was probably due to a lack of NADPH oxidation during the low light phases of fluctuating light (Figure 4c). Thus, NDH-1 1,2 appears to play an important role under low light, as has been previously suggested for chloroplastic NDH in angiosperms (Yamori et al., 2011;Yamori et al., 2015) and bryophytes (Ueda et al., 2012). The delayed activation of CBB in Dd1d2 did not result in an inability to oxidize PSI (Figures 3b and 5c), probably due to significant enhancement of FDP-mediated O 2 photoreduction (Figure 2c) providing an enlarged electron sink for the PETC. An opposite order of causation is also possible, whereby the excessive funneling of photosynthetic electrons to O 2 would cause the delay in induction of CO 2 fixation in Dd1d2. However, the observation that there is no NADPH shortage at the onset of illumination in Dd1d2, and rather the consumption of NADPH during transitions from HL to LL is impaired (Figure 4c), suggests that the availability of reductant for the CBB cycle is not the limiting factor. Nor is it probably CO 2 , as CCM is functioning as in WT (Figure 2c), or ATP, as ATP synthase activity was even higher than in WT in Dd1d2 (Figure 5f). One possibility is that the increased funneling of electrons from Fd to the Mehler-like reaction in Dd1d2 (Figure 2c), while not impairing FNR function (and thus NADPH production), might cause a shortage of electrons for the ferredoxin-thioredoxin reductase. This would impair light-dependent activation of the CBB cycle by the thioredoxin system (Michelet et al., 2013;Guo et al., 2014). In addition, it might explain the delay observed in CO 2 fixation in Dd1d2. Nevertheless, simultaneous deficiency of FDPs in addition to NDH-1 1,2 (in Dflv3 d1d2 triple mutant), with the effect of diminishing the flow of photosynthetic electrons to O 2 photoreduction, mostly rescued the delay in CO 2 fixation (Figure 2d) as well as NADPH consumption ( Figure 4d) seen in Dd1d2. Transient O 2 photoreduction at a low rate was still observed in Dflv3 d1d2 during dark-to-HL transitions (Figure 2d), possibly mediated by the thylakoid terminal oxidases (Ermakova et al., 2016) or by photorespiration (Allahverdiyeva et al., 2011). However, the triple mutants have more severe inability to oxidize PSI than Dflv3 during sudden increases in irradiance (Figure 3). These observations suggest that, in addition, Flv1/3 and NDH-1 1,2 have a role in contributing to oxidation of PSI during changes in light conditions or carbon availability. Upon deficiency of NDH-1 1,2 (in Dd1d2), cells prioritize the protection of PSI over efficient CO 2 fixation by upregulating the Mehler-like reaction via an unknown mechanism. The triple mutants cannot do this, leading to the timely induction of CO 2 fixation at the high cost of inability to oxidize PSI (Figure 3a) or Fd (Figure 5d). This results in loss of PSI (Figure 6), possibly due to photodamage to its FeS clusters (Tiwari et al., 2016;Shimakawa et al., 2016).

On the mechanism of NDH-1-mediated oxidation of PSI
The contribution of NDH-1 to oxidation of PSI during sudden increases in light intensity is not unprecedented. In angiosperms, where FDPs have been lost during evolution (Ilik et al., 2017), Arabidopsis mutants lacking the NDH complex show a delay in oxidation of PSI during increases in light intensity in comparison with Arabidopsis WT (Nikkanen et al., 2018;Shimakawa and Miyake, 2018a). By definition, canonical CET cannot directly increase the relative proportion of oxidized P700, as electrons from the acceptor side of PSI are shunted back to the intersystem electron transfer chain. However, NDH-1 may enhance PSI oxidation by at least three alternative, but mutually non-exclusive mechanisms.
i NDH-1-mediated CET is coupled to the translocation of protons from cytosol to the thylakoid lumen with a 2H + /e À stoichiometry (Strand et al., 2017;Saura and Kaila, 2019). Therefore, it will contribute to build-up of ΔpH, which limits electron transfer to PSI by inhibiting PQH 2 oxidation at Cyt b6f (Shimakawa and Miyake, 2018b) and drives ATP synthesis to accommodate the needs of the CBB, increasing its electron sink capacity. As the Mehler-like reaction also contributes to build-up of ΔpH by consuming H + on the cytosolic side of the thylakoid membrane and by supporting linear electron flow ( Figure 5), (Allahverdiyeva et al., 2013), enhanced FDP activity in Δd1d2 (Figure 2c) partly compensates for the lack of NDH-1 1,2 in respect to the generation of proton flux (Figure 5g). However, this fails to explain the exacerbated impairment of P700 and Fd oxidation in Δflv3 d1d2 in comparison with Δflv3 (Figures 3 and  5) because in the triple mutant pmf was not lower than in Δflv3 (Figure 5e). However, adequate pmf is maintained at the expense of ATP synthase activity (Figure 5f), and it is likely that diminished ATP production contributes to the increased acceptor side limitation of PSI in Δflv3 d1d2 (Figure 3a). Therefore, NDH-1 1,2 could contribute to P700 oxidation by enhancing cytosolic sink capacity by providing a more suitable ATP/NADPH ratio for the CBB. Adjustment of the ATP/ NADPH ratio closer to the theoretically optimal 3:2 has long been considered a fundamental reason for the existence of CET (Kramer et al., 2004;Yamori and Shikanai, 2016). ii NDH-1-mediated respiratory electron transfer, that is, coupling of PQ reduction by NDH-1 to O 2 reduction by thylakoid terminal oxidases (i.e. Cyd and Cox), would also contribute to oxidation of PSI by relieving electron pressure in the intersystem chain during illumination (Ermakova et al., 2016), as well as contribute further to ΔpH by consuming H + on the cytosolic side of thylakoids (Cyd) and pumping protons to the lumen (Cox) (Br€ and en et al., 2006). Interestingly, Liu et al. (2012) have shown that the subcellular localization of NDH-1 complexes is dependent on the redox state of the PQ pool. An oxidized PQ pool causes NDH-1 to accumulate at specific clusters in thylakoid membranes where it would probably transfer electrons (via the PQ pool) to a terminal oxidase. A reduced PQ pool, in turn, results in a more even distribution of NDH-1 within thylakoids (Liu et al., 2012). However, it is important to note that terminal oxidases do not have a high electron sink capacity (Ermakova et al., 2016). iii NDH-1 has been predicted, albeit not yet experimentally shown in photosynthetic organisms, to be able to function in reverse: to oxidize PQH 2 driven by concomitant release of protons from the thylakoid lumen (Strand et al., 2017). Such reverse activity would constitute a "pseudo-linear" electron transfer pathway that would bypass PSI and thereby prevent its over-reduction. This could occur in conditions where the PQ pool is reduced, pmf is high and the Fd pool is oxidized (Strand et al., 2017). Such conditions probably exist transiently during dark-to-light and LL-to-HL transitions (Figures 4 and 5) (Strand et al., 2019). Accordingly, fast re-reduction of P700+ already after 50 msec was observed in Dd1d2 and Dflv3 d1d2 during dark-to-HL transitions ( Figure 5c). As supported by the impaired ability to oxidize Fd in the absence of Flv1/3 ( Figure 5), the presence of the Flv1/3-catalyzed Mehler-like reaction would probably be essential in this model to maintain the Fd pool in a sufficiently oxidized state to provide electron acceptors for reverse-functioning NDH-1. However, it is noteworthy that NDH-1 reverse activity would also have the effect of lowering DpH, thereby relieving photosynthetic control at Cyt b6f. This could counteract the effect of any reverse NDH-1 activity by increasing electron flow to PSI. Hypothetical mechanisms for the coordination of Flv1/3 and NDH-1 1,2 activities are shown in Figure 7.
Inability to induce a strong CCM network contributes to lethality upon shifts to air [CO 2 ]/HL in cells deficient in both Flv1/3 and NDH-1 1,2 Exposure of Synechocystis cells to low [CO 2 ] induces expression of high-affinity CCM-related genes such as SbtA, a HCO 3 À /Na + symporter on the plasma membrane, and NdhD3, which is part of the NDH-1 3 complex on thylakoids specializing in C i uptake (Ohkawa et al., 2000b;Shibata et al., 2001;Shibata et al., 2002;Zhang et al., 2004). CCM is energetically expensive, but the large C i flux involved in the operation of CCM contributes to dissipation of excess light energy under stress conditions (Xu et al., 2008;Burnap et al., 2015). NDH-1 3,4 in particular has a photoprotective role, using reduced Fd to drive CO 2 conversion to HCO 3 with concomitant translocation of protons into the thylakoid lumen. Despite the low induction level of the CCM proteins, NdhD3 and SbtA, the triple mutants ( Figure 6) survive under standard growth conditions of air [CO 2 ]/ML (Figure 1b,c), but their growth was retarded on solid media (Figure 1e) The difference may be due to the differential diffusivity of CO 2 in solid media in comparison with suspension cultures (Ohkawa et al., 2000a); that is, the impairment of CCM in the triple mutants becomes more limiting on solid media. However, when a decrease in [CO 2 ] is coupled with an increase in irradiance a low amount of CCM, reflected by the low accumulation level of NdhD3 and SbtA in the Dflv1d1d2 and Dflv3d1d2 triple mutants (Figure 6c) fails to dissipate excess energy, thus resulting in photodamage and lethality of those conditions. The regulatory mechanisms controlling the inability of the triple mutants to accumulate low Ci-inducible proteins are unclear and remain to be elucidated. Accumulation of NADP+ and a-ketoglutarate inhibits the induction of CCM gene expression via interaction with the transcription factor NdhR (CcmR) (Daley et al., 2012). NdhR also controls expression of the Flv4-2 operon encoding Flv2, Flv4 and Sll0218 (Eisenhut et al., 2012), all of which also showed decreased accumulation levels in the triple mutants (Figure 6). However, at least in the tested conditions, no substantial increase in the relative amount of NADP+ was detected in Dflv3d1d2 cells (Figure 4). Nevertheless, it is important to note that Flv1/3 and NDH-1 3,4 cooperation alone is not crucial for cell metabolism, as the Dflv1 d3d4 and Dflv3 d3d4 mutant cells do not show clear growth phenotypes ( Figure S1).

Conclusions
In the current study, we have shown that FDPs and NDH-1 function cooperatively to maintain redox balance between the PETC and cytosolic carbon assimilation upon sudden changes in light intensity and/or carbon availability. It is probable that both pathways receive electrons primarily from Fd, which enables coordinated regulation of their activities. Cooperation of FDPs and NDH-1 has an essential photoprotective role during shifts to air [CO 2 ]/HL, contributing to efficient oxidation of PSI, build-up of pmf and induction of expression of low C i -specific CCM-related genes. A key issue yet to be elucidated is the regulatory mechanism(s) by which the dynamic coordination of FDP and NDH-1 activities is achieved. One potential regulatory mechanism could function through the light-dependent Fd-thioredoxin system, which by competing with FDPs and NDH-1 for electrons from Fd, could constitute an effective regulatory feedback-loop. Indeed, light-dependent redox-sensitive cysteine residues have been identified in Flv1 and Flv3 as well as in several subunits of NDH-1 (Guo et al., 2014), and activity of chloroplastic NDH-1 in plants has been reported to be regulated by thioredoxins (Courteille et al., 2013;Nikkanen et al., 2018).
Finally, it is worth noting that simultaneous removal of 2019). Furthermore, our findings about the functional interplay between FDPs and NDH-1 will be highly relevant in projects aiming to enhance crop productivity via introduction of exogenous FDPs to higher plants (Yamamoto et al., 2016;G omez et al., 2018).

Gas exchange measurements
The exchange of 16 O 2 (m/z 32), 18 O 2 (m/z 36) and CO 2 (m/z 44) was measured in vivo with MIMS as described in Mustila et al. (2016). Harvested cells were resuspended in fresh BG-11 pH 7.5 and adjusted to 10 µg Chl a ml À1 , and kept for 1 h in air [CO 2 ]/50 µmol photons m À2 sec À1 . Before measurements, cells were supplemented with 18 O 2 at an equivalent concentration to 16 O 2 and with 1.5 mM NaHCO 3 . Cells were dark-adapted for 15 min, after which gas exchange was monitored over a 5-min illumination period of 500 lmol photons m À2 sec À1 of white actinic light (AL). The gas exchange rates were calculated according to Beckmann et al. (2009).
A DUAL-KLAS-NIR spectrophotometer (Walz) was used to measure absorbance difference changes at 780-820, 820-870, 840-965 and 870-965 nm, from which PC, P700 and Fd redox changes ( Figure 5) were deconvoluted based on differential model plots (DMPs) for PC, P700 and Fd (Klughammer and Schreiber, 2016;Schreiber, 2017;Setif et al., 2019). The redox changes were then normalized according to the maximal redox changes determined with the NIRMAX script of the instrument software ( Figure S3b), consisting of a 3 sec illumination of red AL (200 lmol photons m À2 sec À1 ), with a saturating pulse after 200 msec of illumination to reduce the Fd pool fully. After 4 sec of darkness thereafter, cells were illuminated under far red light for 10 sec with a saturating pulse administered in the end of the illumination period to oxidize P700 fully. In WT Synechocystis, very fast reoxidation of Fd upon illumination impedes the measurement of model spectra. To circumvent this issue, we measured the DMPs from Dflv3 d1d2 cells where P700 oxidation was severely delayed in comparison with WT ( Figure 3b). The DMPs ( Figure S3a) were measured using scripts provided with the instrument software. Harvested cells were adjusted to 10 µg Chl a ml À1 with fresh BG-11 pH 7.5, and kept for 1 h in air [CO 2 ]/50 µmol photons m À2 sec À1 and dark-adapted for 30 min before illumination for 30 sec with 503 lmol photons m À2 sec À1 of red AL. It is noteworthy that cytochrome c 6 and (F A F B ) redox changes probably contribute to the PC and Fd signals, respectively (Setif et al., 2019).
NADPH fluorescence changes between 420 and 580 nm, induced by excitation at 365 nm, were measured simultaneously with Chl a fluorescence with the Dual-PAM 100 and its 9-AA/ NADPH accessory module (Walz) (Schreiber and Klughammer, 2009;Kauny and Setif, 2014). Experimental samples were prepared as above and [Chl a] was adjusted to 5 µg ml À1 . Cells were dark-adapted for 20 min and subjected to the same fluctuating light regime as in Figure 3, but without saturating pulses.
The ECS was measured as the absorbance difference between 500 and 480 nm (Viola et al., 2019). Absorbance changes were measured from dark-adapted cell suspensions with a JTS-10 spectrophotometer (BioLogic, Seyssinet-Pariset, France) using 500 and 480 nm CWL 50 mm 10 nm FWHM bandpass filters (Edmund Optics, Barrington, NJ, USA) and BG39 filters (Schott, Mainz, Germany) protecting the light detectors from scattering effects. Absorbance changes induced by measuring light only were subtracted from changes under ML + AL. ECS values were normalized to ECS change induced by a single turnover flash provided by an XST-103 xenon lamp (Walz). Experimental samples were prepared as above and [Chl a] was adjusted to 7.5 µg ml À1 , and illuminated with green AL of 500 lmol photons m À2 sec À1 for 1 or 60 sec interspersed with 500 msec dark intervals at 2, 7.5, 18, 30 and 45 sec. Pmf was calculated as the extent of ECS decrease at the dark intervals. Thylakoid conductivity (gH+) was calculated as the inverse of the time constant of a first-order fit to ECS relaxation kinetics during a dark interval, and proton flux (vH+) as pmf 9 gH+ (Cruz et al., 2005). Representative ECS traces of whole 60 sec measurements (upper panels) as well as fast kinetics during dark intervals after 30 sec illumination (lower panels) from WT, Dflv3, Dd1d2 and Dflv3 d1d2 cells are shown in Figure 5h.
77K fluorescence emission spectra were measured with a QE Pro spectrophotometer (Ocean Optics, Dunedin, FL, USA). Cells were harvested and [Chl a] was adjusted to 7.5 µg ml À1 with fresh BG-11 pH 7.5. Cells were frozen in liquid N 2 and excited at 440 nm. Raw spectra were normalized to the PSII-fluorescence peak at 685 nm.