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

Lauri Nikkanen, Anita Santana Sánchez, Maria Ermakova, Matthias Rögner,

Laurent Cournac, Yagut Allahverdiyeva

► To cite this version:

Lauri Nikkanen, Anita Santana Sánchez, Maria Ermakova, Matthias Rögner, Laurent Cournac, et al. Functional redundancy between flavodiiron proteins and NDH-1 in Synechocystis sp. PCC 6803. Plant Journal, 2020, 103 (4), pp.1460-1476. 10.1111/tpj.14812. hal-02887161

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The Plant Journal (2020)

doi: 10.1111/tpj.14812

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

Lauri Nikkanen<fnref n="1" /> ID, Anita Santana Sánchez<fnref n="1" />, Maria Ermakova1,†, Matthias Rögner<fnref n="2" />, Laurent Cournac<fnref n="3" /> and Yagut Allahverdiyeva<fnref n="1" />* ID

Received 15 January 2020; revised 30 April 2020; accepted 5 May 2020.

## SUMMARY

In oxygenic photosynthetic organisms, excluding angiosperms, flavodiiron proteins (FDPs) catalyze light-dependent reduction of O₂ to H₂O. 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 (Δflv1 and Δflv3) and the double NDH-1 mutants (Δd1d2, which is deficient in NDH-1₁₂ and Δd3d4, which is deficient in NDH-1₃₄), we studied triple mutants lacking one of Flv1 or Flv3, and NDH-1₁₂ or NDH-1₃₄. We show that the presence of either Flv1/3 or NDH-1₁₂, but not NDH-1₃₄, is indispensable for survival during changes in growth conditions from high CO₂/moderate light to low CO₂/high light. Our results show functional redundancy between FDPs and NDH-1₁₂ under the studied conditions. We suggest that ferredoxin probably functions as a primary electron donor to both Flv1/3 and NDH-1₁₂, allowing their functions to be dynamically coordinated for efficient oxidation of photosystem I and for photoprotection under variable CO₂ and light availability.

**Keywords:** Flavodiiron proteins, Flv, NDH-1, photosynthesis, *Synechocystis* sp. PCC 6803, cyanobacteria, Mehler-like reaction, alternative electron transfer, photoprotection.

## 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 O₂ (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 β-cyanobacterium *Synechocystis* sp. PCC 6803 (hereafter *Synechocystis*) encodes four isoforms of FDPs, namely Flv1-4, which function in O₂ 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 [CO₂], Flv1/3 and Flv2/4 hetero-oligomers function in a coordinated and interdependent manner: Flv1/3 stimulates strong but transient O₂ photoreduction upon the onset of illumination or during increases in light intensity, while Flv2/4 hetero-oligomers catalyze a slower and

<footnote n="1">¹Molecular Plant Biology, Department of Biochemistry, University of Turku, Turku, Finland,</footnote>

<footnote n="2">²Plant Biochemistry, Faculty of Biology & Biotechnology, Ruhr-University Bochum, Bochum, Germany, and</footnote>

<footnote n="3">³Eco&Sols, University of Montpellier, IRD, CIRAD, INRAE, Institut Agro, Montpellier, France</footnote>

<footnote>*For correspondence (e-mail allahve@utu.fi).</footnote>

<footnote>†Present address: Australian Research Council Centre of Excellence for Translational Photosynthesis, Division of Plant Science, Research School of Biology, The Australian National University, Acton, Australia</footnote>
limited steady-state reduction of O₂, also using electrons downstream of PSI (Santana-Sanchez *et al.*, 2019). In elevated [CO₂] and in air [CO₂] 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 [CO₂] stimulates strong steady-state O₂ photoreduction, whereas in air [CO₂] at pH 9, Flv1/3 is solely responsible for strong but transient O₂ photoreduction (Santana-Sanchez *et al.*, 2019). Whilst *in vitro* assays showed that homo-oligomers of recombinant Flv1, Flv3 and Flv4 can reduce O₂ 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 O₂ photoreduction *in vivo* (Mustila *et al.*, 2016). Thus, the electron donor to FDPs remains to be elucidated *in vivo*.

In cyanobacteria, photosynthetic and respiratory electron transfer reactions coexist on thylakoid membranes and are intricately linked, necessitating strict regulation (Mullineaux, 2014). The NAD(P)H dehydrogenase-like complex 1 (NDH-1, photosynthetic complex I) is mainly localized in thylakoid membranes and involved in several bioenergetic reactions, including cyclic electron transfer (CET) around PSI, respiration and CO₂ acquisition via the carbon concentrating mechanism (CCM) (for reviews see Battchikova *et al.*, 2011; Ma and Ogawa, 2015; Burnap *et al.*, 2015; Peltier *et al.*, 2016). The NDH-1 complex is present as several forms with distinct physiological roles: the NDH-1₁ type features the subunits NdhD1 and NdhF1 and functions as complex I of the respiratory electron transfer chain (Zhang *et al.*, 2004; He *et al.*, 2015; Saura and Kaila, 2019); the NDH-1₂ form features the NdhD2 subunit instead of NdhD1, but it remains unclear whether NDH-1₁ and NDH-1₂ serve different physiological functions (Peltier *et al.*, 2016). The NDH-1₃ and NDH-1₄ forms that comprise low-Cᵢ-inducible, high-affinity CO₂ acquisition subunits NdhD3, NdhF3, CupA and CupS, or constitutive, low-affinity CO₂ acquisition subunits NdhD4, NdhF4 and CupB, respectively, function in conversion of CO₂ to HCO₃⁻ (Ohkawa *et al.*, 2000a,b; Shibata *et al.*, 2001; Zhang *et al.*, 2004; Burnap *et al.*, 2013; Han *et al.*, 2017; Schuller *et al.*, 2019). This process is possibly enabled by alkaline pockets at the thylakoid membrane created by NDH-1 proton pumping (Kaplan and Reinhold, 1999). All four NDH-1 types catalyze CET from Fd to plastoquinone (PQ), which is coupled to the pumping of 4H⁺/2e⁻ into the thylakoid lumen (He *et al.*, 2015; Laughlin *et al.*, 2019; Schuller *et al.*, 2019; Saura and Kaila, 2019). However, CET mediated by NDH-1₃,₄ is minor in comparison to CET mediated by NDH-1₁,₂ (Bernat *et al.*, 2011).

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 CO₂ fixation in the Calvin-Benson-Bassham cycle (CBB) under variable light and Cᵢ 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-1₁,₂, but not NDH-1₃,₄, being indispensable for the survival of *Synechocystis* cells under transitions from high [CO₂] conditions to the combined stress conditions of air [CO₂] and high light. We show that the dynamically coordinated and cooperative function of Flv1/3 and NDH-1₁,₂ 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-1₁,₂, although the possible contribution of NADPH as a donor cannot be fully excluded.

## RESULTS

### Simultaneous inactivation of Flv1/3 and NDH-1₁,₂ is lethal upon shift from high to air [CO₂] 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 (Δ*flv1* and Δ*flv3*, respectively; Helman *et al.*, 2003), (ii) both NdhD1 and NdhD2 (Δ*d1d2*, deficient in NDH-1₁ and NDH-1₂; Ohkawa *et al.*, 2000b), (iii) both NdhD3 and NdhD4 (Δ*d3d4*, deficient in NDH-1₃ and NDH-1₄; Ohkawa *et al.*, 2000b), as well as triple mutants with combined deficiencies of both pathways (Δ*flv1* *d1d2*, Δ*flv3* *d1d2*, Δ*flv1* *d3d4*, Δ*flv3* *d3d4*) in conditions of differing CO₂ availability and light intensity. The triple mutants exhibited similar or slightly slower growth compared with the WT and other mutant strains under high [CO₂] (3% CO₂) and moderate light (ML, intensity of 50 µmol photons m⁻² sec⁻¹ of photosynthetically active radiance) (Figure 1a). We also detected no substantial difference in growth when the 3% CO₂/ML pre-grown cells were diluted [optical density (OD)₇₅₀ = 0.1] and shifted to air [CO₂] conditions under constant light intensity (Figure 1b; Figure S1a,c). In contrast, when cultures pre-grown in 3% [CO₂]/ML were diluted (OD₇₅₀ = 0.1) and subjected simultaneously to air [CO₂] and high light stress conditions (HL, intensity of 220 µmol photons m⁻² sec⁻¹), the growth of both Δ*flv3* *d1d2* and Δ*flv1* *d1d2* strains was completely inhibited (Figure 1d). Importantly, Δ*flv1*, Δ*flv3* and Δ*d1d2* strains demonstrated growth similar to WT cells under air [CO₂]/HL conditions.
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Growth of wild-type (WT) and mutant strains under different experimental conditions. All experimental cultures were started from pre-cultures adjusted to OD<sub>750</sub> = 0.1.</figcaption></figure>

(a) Cells were pre-cultivated under 3% [CO₂]/moderate light (ML) (50 µmol photons m⁻² sec⁻¹), adjusted to OD₇₅₀ = 0.1 and grown in the same conditions.

(b) Cells were pre-cultivated under 3% [CO₂]/ML and shifted to air [CO₂]/ML.

(c) Cells were pre-cultivated under air [CO₂]/ML and shifted to air [CO₂]/high light (HL) 220 µmol photons m⁻² sec⁻¹).

(d) Cells were pre-cultivated under 3% [CO₂]/ML and shifted to air [CO₂]/HL. Values presented in a-d are means of three independent biological replicates ± SE.

(e) Growth of WT and mutant cells on agar plates containing BG-11. Cells grown under 3% [CO₂]/ML were harvested and spotted on BG-11 agar plates in 10-fold dilution series starting at OD₇₅₀ = 1, incubation was under air [CO₂]/ML and air [CO₂]/HL for 7 days. See also Figure S1.

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

We next examined whether pre-adaptation to air [CO₂] would rescue the growth arrest. When cells previously adapted to air [CO₂]/ML were diluted (OD₇₅₀ = 0.1) and shifted to air [CO₂]/HL, no differences in growth patterns between the strains were detected (Figure 1c). Importantly, the triple mutants lacking either Flv1 or Flv3 and NDH-1₃,₄ (Δflv1 d3d4 and Δflv3 d3d4 strains) did not show a lethal phenotype after a shift from 3% [CO₂]/ML to air [CO₂]/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-1₃,₄ complexes is not as crucial as with NDH-1₁,₂. 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% CO₂/ML-grown cells were diluted and grown on plates under either air [CO₂]/ML or air [CO₂]/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 [CO₂]/ML, while no growth was detected under air [CO₂]/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-1₁,₂ is required for the oxidation of PSI during sudden increases in light intensity**

The lethal phenotype observed upon the shift to air [CO₂]/HL, which was caused by the simultaneous impairment of
the Mehler-like reaction catalyzed by Flv1/3 (Helman et al., 2003; Allahverdiyeva et al., 2011, 2013) and by NDH-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 O₂ and CO₂ exchange upon HL illumination of dark-adapted WT and mutant cells grown for 4 days in air [CO₂]/ML. To distinguish O₂ uptake from photosynthetic gross O₂ evolution under illumination, we enriched cell suspensions with ¹⁸O₂ before the measurements. In dark-adapted WT cells, a transient peak in O₂ 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 O₂ reduction in an interdependent manner (Santana-Sanchez et al., 2019). In Δflv3 cells, light-induced O₂ reduction was almost abolished and only a slight impairment of the rate of photosynthetic gross O₂ 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 O₂ uptake during the first minute of illumination, demonstrating sustained O₂ photoreduction at high levels [approximately 75–100 µmol O₂ mg chlorophyll (Chl)⁻¹ h⁻¹] throughout the illumination period. Meanwhile, gross O₂ evolution was diminished nearly two-fold compared with the WT (Figure 2c). Thus, sustained O₂ 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 O₂ uptake transiently during the first minute

of illumination (Figure 2d). The Δflv3 d1d2 cells demonstrated slower induction of photosynthetic O₂ evolution following two-component kinetics, and slightly impaired steady-state gross O₂ evolution compared with WT (Figure 2d).

The initial peak in CO₂ 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-1,4 in addition to NDH-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 [CO₂]/ML, with CO₂ uptake rates peaking at approximately 1.5 mM CO₂ mg Chl⁻¹ h⁻¹. In WT as well as in Δflv3, CO₂ fixation is then induced and the respiratory compensation point surpassed after approximately 1 min of illumination (Figure 2a,b). However, in Δd1d2, induction of CO₂ 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 CO₂ fixation was largely recovered in the triple mutant (Figure 2d).

These MIMS results indicated that combined deficiency of Flv1/3 and NDH-1,2 under standard growth conditions, air [CO₂]/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 µmol photons m⁻² sec⁻¹, LL) and high irradiance (530 µmol photons m⁻² sec⁻¹, 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><img src="image_5.png" /></figure>

**Figure 2.** O₂ and CO₂ exchange rates in wild-type and mutant strains. Cells were grown in air [CO₂]/ moderate light for 4 days, after which the cells were harvested and chlorophyll (Chl) a concentration adjusted to 10 µg ml⁻¹ 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 µmol photons m⁻² sec⁻¹ of white actinic light. Before the measurements, samples were supplemented with ¹⁸O₂ at an equivalent concentration to ¹⁸O₂ to distinguish O₂ uptake from O₂ evolution, and with 1.5 mM NaHCO₃. Dashed line in each panel indicates the compensation point of CO₂ fixation (uptake rate equals respiratory rate). The experiment was repeated with three independent biological replicates, of which representative measurements are shown.
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Photosynthetic parameters in wild-type (WT) and mutants strains in fluctuating light intensities.</figcaption></figure>

(a) Acceptor side limitation of photosystem (PS) I, Y (NA), (b) donor side limitation of PSI, Y(ND), as well as (c) effective yield of PSI, Y(II) were calculated from absorbance difference changes at 875-830 nm according to 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 [CO₂]/moderate light for 4 days, after which the cells were harvested and chlorophyll a concentration adjusted to 10 µg ml⁻¹ with fresh BG-11. Cells were dark-adapted for 10 min, and then illuminated with red actinic light intensity alternating between 25 and 530 µmol photons m⁻² sec⁻¹ in 1 min periods. Saturating pulses (500 msec of 5000 µmol photons m⁻² sec⁻¹) were provided every 15 sec. Values shown are averages from 3-4 independent biological replicates ± SE.

previous studies (Helman *et al.*, 2003; Allahverdiyeva *et al.*, 2013). However, during subsequent 1 min cycles of LL/HL, the ability of Δflv3 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 Δd1d2 (Figure 3a) as PSI was oxidized similarly to the WT (Figure 3b). However, interestingly, Δd1d2 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 Δflv3 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 Δflv3 mutant, Δflv3 d1d2 did not show improvement in the PSI acceptor side limitation during subsequent cycles. This implies a compensating activity of NDH-1 in Δflv3 mutant under the studied conditions. Interestingly, diminished donor side limitation in Δflv3 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 b₆f, 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 Δflv3 and Δd1d2, but not in Δflv3 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 µmol photons m⁻² sec⁻¹ (HL) similarly to Figure 3. Upon dark-to-LL transitions, NADPH rapidly accumulated close to a maximal amount in WT, Δflv3 and Δd1d2 cells, while in Δflv3d1d2 the NADP pool was highly reduced already in darkness. Reoxidation of NADPH then occurred in WT and Δflv3d1d2 cells, while in Δflv3 a slower oxidation phase preceded a transient re-reduction phase during the first minute of illumination (Figure 4a,b). Strong reduction of the NADP+ pool was also detected in Δflv3 during the second HL phase of the experiment. In Δd1d2 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 CO₂ fixation in the CBB cycle (Figure 2c), and impaired CET or dark respiration. In contrast, NADPH was strongly oxidized in Δflv3 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 Δd1d2 (Figure 4c). Therefore, the PIFR peaks
<figure><img src="image_7.png" /><figcaption>**Figure 4.** NADPH and chlorophyll (Chl) a fluorescence under fluctuating light in wild-type (WT) and mutant strains.</figcaption></figure>

(a) WT, (b) Δ*flv3*, (c) Δ*d1d2*, and (d) Δ*flv3 d1d2* cells were grown in air [CO₂]/moderate light for 4 days, after which the cells were harvested and Chl *a* concentration adjusted to 5 µg ml⁻¹ 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 µmol photons m⁻² sec⁻¹ 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 µmol photons m⁻² sec⁻¹. 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.
probably originate from cyclic and respiratory electron
transport via NDH-1,2 (Holland *et al.*, 2015). However, both
Chl PIFR peaks were observed in Δ*flv3 d1d2* cells, indicat-
ing involvement of a complementary pathway, possibly via
succinate dehydrogenase (Cooley and Vermaas, 2001), or a
CET pathway dependent on PGR5 and PGRL1-like (Yere-
menko *et al.*, 2005; Dann and Leister, 2019). Finally, we
monitored NADPH redox kinetics under conditions mimick-
ing the MIMS experiments, where strong O₂ photoreduc-
tion occurs in WT and Δ*dcd2* during the first minute of
dark-to-high light transition (Figure 2). No substantial dif-
ferences were observed between the strains, as close to
maximal reduction of NADP+ was obtained within approxi-
mately 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-1,2, 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-1,2

To elucidate the molecular mechanism behind the photo-
synthetic phenotypes of the studied mutant strains, we
next utilized a DUAL-KLAS-NIR spectrophotometer to dis-
tinguish 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 µmol photons m⁻² sec⁻¹). Upon illumination of
WT cells, rapid oxidation of P700 and PC occurred, fol-
lowed 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 Δflv3 cells, P700 and PC remained mostly
reduced after the initial oxidation transient until approxi-
mately 3 sec, when another transient and partial reoxida-
tion 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 oxi-
dized. 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 O₂,
accepting electrons presumably from reduced Fd (after
approximately 0.5 sec in light). In Δd1d2, redox changes of
PC, P700 and Fd were similar to WT, except that the reduc-
tion of P700+ following initial oxidation as well as reduc-
tion of Fd occurred already after approximately 50 msec
(Figure 5c). In Δflv3 d1d2, re-reduction of P700+ and reduc-
tion of Fd also occurred already after approximately
50 msec (Figure 5d), suggesting involvement of NDH-1,2

as an acceptor of electrons from Fd at that stage. Fd was
reduced more quickly in Δflv3 d1d2 than in any other
strains and was slowly reoxidized over 30 sec (Figure 5d),
probably due to the shortage of electron acceptors (Fig-
ure 3a). In summary, these NIR-spectroscopic measure-
ments revealed that Flv1/3 and NDH-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-1,2 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 Syne-
chocystis (Viola et al., 2019). In vivo measurement of light-
induced ECS revealed that after 1 sec of illumination of
dark-adapted WT cells with 500 µmol photons m⁻² sec⁻¹,
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 Δflv3 and
Δflv3 d1d2, pmf and thylakoid proton flux (vH+) were lower
than in WT after 1 sec (Figure 5g). In Δflv3, pmf remained
drastically lower than in WT during the first seconds of illu-
mination, but differed only slightly from WT thereafter due
to diminished conductivity of the thylakoid membrane
(gH+) (Figure 5f), which is mainly determined by the activ-
ity of the adenosine triphosphate (ATP) synthase (Cruz
et al., 2005; Viola et al., 2019). In Δd1d2, the thylakoid pro-
ton 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 (Fig-
ure 5e) because of elevated conductivity (Figure 5f). In con-
trast, 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 dimin-
ished 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 kinet-
ics in single, double and triple mutant cells studied here,
we conclude that both Flv1/3 and NDH-1,2 contribute to
proton flux during transitions from dark to HL. However,
deficiency in NDH-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
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>
<caption>**Figure 5.** Redox changes of plastocyanin (PC), P700 and ferredoxin (Fd), and build-up of pmf during dark-to-high irradiance transitions.</caption>

(a) WT, (b) Δflv3, (c) Δd1d2, and (d) Δflv3 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 µmol photons m⁻² sec⁻¹ 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 µmol photons m⁻² sec⁻¹ 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 ± 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 first-order post-illumination decay kinetics of the ECS signal.

(g) Thylakoid proton flux (vH+), calculated as pmf × 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 [CO₂]/50 µmol photons m⁻² sec⁻¹ for 4 days, were harvested and Chl a concentration adjusted to 10 µg ml⁻¹ with fresh BG-11 for a–d and to 7.5 µg ml⁻¹ for e–h.

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-1,2 shifted to air [CO₂] and HL

The results from the growth assays, along with the real-time gas exchange, P700 redox change and ECS measurements suggested that in Δd1d2 cells, an increase in the activity of FDPs compensates for the absence of a functional NDH-1,2 complex, allowing efficient oxidation of PSI under HL. Conversely, in Δflv1 and Δflv3 mutants, over-reduction of the electron transport chain occurs initially upon exposure to HL, but another mechanism(s) eventually allow acclimation and survival in high [CO₂] and high light (Helman et al., 2003). Under air [CO₂] Flv1/3, hetero-oligomers catalyze transient O₂ photoreduction, which is why no strong growth phenotype is observed in Δflv1 or Δflv3 under constant HL (Allahverdiyeva et al., 2013; Santana-Sanchez et al., 2019). However, when both Flv1/3 and NDH-1,2 are absent and the electron sink capacity of the cytosol is not elevated by high [CO₂], 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 Δflv1 or Δflv3 cells grown at 3% [CO₂]/ML and shifted to air [CO₂]/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 Δflv1 d1d2 and Δflv3 d1d2 triple mutants, while an increased amount of both proteins was detected in Δd1d2 (Figure 6a). After 24 h

exposure to air [CO₂]/HL, PsaB amount had decreased even further in Δflv1 d1d2 and Δflv3 d1d2 (Figure 6c). Furthermore, 77K fluorescence spectra revealed that while Δflv3 d1d2 and WT cells grown under air [CO₂]/ML had similar PSI/PSII ratios (Figure 6d), the 24-h exposure to air [CO₂]/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 [CO₂]/HL. Interestingly, a substantial decrease in D1 content in air [CO₂]/HL was also observed in the triple mutants (Figure 6b,c).

Importantly, the Δflv1 d1d2 and Δflv3 d1d2 strains were unable to induce substantial accumulation of the proteins encoded by the flv4-2 operon after a shift from 3% to air [CO₂] (Figure 6a,c). Only when pre-grown for 4 days in air [CO₂]/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 [CO₂]/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% [CO₂] to air [CO₂]/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 [CO₂]/HL (shifted from air [CO₂]/ML) (Figure 6b). In the Δd1d2 mutant, the NdhD3 content was similar to WT, but Flv3 as well as the flv4-2 operon proteins (Flv2, Flv4 and SII0218) were upregulated in all conditions tested (Figure 6), which probably contributes to the increased rate of O₂ 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 O₂ as a rapid response to excessive reduction of PSI, and consequently, for cell survival under
<figure><img src="image_11.png" /><figcaption>**Figure 6.** Immunodetection of proteins related to photosynthesis and carbon concentrating mechanism and 77K fluorescence emission spectra in wild-type (WT) and mutant strains.</figcaption></figure>

Pre-cultures were grown for 3 days at 3% [CO₂]/moderate light (ML) (50 µmol photons m⁻² sec⁻¹) and then pelleted, resuspended in fresh BG-11 at OD₇₅₀ = 0.15 and (a) incubated for 24 h in air [CO₂]/ML, (b) grown for 4 days in air [CO₂]/ML, harvested, resuspended in BG-11 at OD₇₅₀ = 0.15 and then incubated for 24 h in air [CO₂]/HL (220 µmol photons m⁻² sec⁻¹), (c) incubated for 24 h in air [CO₂]/high light (HL). Total protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with specific antibodies. Immunoblots shown are representative of two to three independent biological replicates.

(d) 77K fluorescence emission spectra from intact WT and Δflv3 d1d2 cells grown for 4 days in air [CO₂]/ML.

(e) After pre-growth at 3% [CO₂]/ML cells were pelleted, resuspended in fresh BG-11 at OD₇₅₀ = 0.15 and incubated for 24 h in air [CO₂]/HL. Cells (d and e) were pelleted, adjusted to [chlorophyll *a*] of 7.5 µg ml⁻¹, and excited with 440 nm light at 77K. Spectra were normalized to the PSII fluorescence peak at 685 nm.

fluctuating light conditions (Allahverdiyeva *et al.*, 2013; Santana-Sanchez *et al.*, 2019). Moreover, we recently showed that in air [CO₂], Flv2/4 hetero-oligomers also catalyze low-level light-induced steady-state reduction of O₂ on the acceptor side of PSI in a coordinated manner with Flv1/3 (Santana-Sanchez *et al.*, 2019).

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 CO₂ 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
physiological significance of these alternative electron
transport pathways and the possibility of functional inter-
dependence between them. Our results provide compelling
evidence for the concomitant physiological functions of
Flv1/3 and NDH-1,2 and their dynamic coordination for the
efficient oxidation of PSI (thus protecting it from photo-
damage) under variable light conditions, and when low
[CO2] limits the consumption of reductants in the CBB
cycle. The two pathways can compensate for each other's
absence to some extent, but absence of both the Flv1/3
hetero-oligomer and NDH-1,2 is lethal when cells are
transferred from elevated [CO2] to the combined condition
of air level [CO2] and HL (Figure 1d,e). In addition to the
absence of these two important pathways, lethality is due
to an inability to accumulate low C1-inducible photoprotec-
tive 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-1,2

Coordinated functions of Flv1/3 and NDH-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 experi-
ments 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 Δflv3 deletion strains,
whereby Fd remains strongly reduced (Figure 5b). This
result, together with the lack of impairment in NADP+
reduction and oxidation kinetics of Δflv3 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 chrom-
matography (Hanke et al., 2011) and with Flv3 using a two-
hybrid assay (Cassier-Chauvat and Chauvat, 2014).

The deficiency of NDH-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 (Fig-
ure 5c). This brief time scale may be due to PSI-NDH-1
supercomplexes (Gao et al., 2016) where P700 can be oxi-
dized 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-1,2 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-1,2 appears to play an impor-
tant 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 Δd1d2 did not result in an
inability to oxidize PSI (Figures 3b and 5c), probably due to
significant enhancement of FDP-mediated O2 photoreduc-
tion (Figure 2c) providing an enlarged electron sink for the
PETC. An opposite order of causation is also possible,
whereby the excessive funneling of photosynthetic elec-
trons to O2 would cause the delay in induction of CO2 fixa-
tion in Δd1d2. However, the observation that there is no
NADPH shortage at the onset of illumination in Δd1d2, and
rather the consumption of NADPH during transitions from
HL to LL is impaired (Figure 4c), suggests that the availabil-
ity of reductant for the CBB cycle is not the limiting factor.
Nor is it probably CO2, as CCM is functioning as in WT (Fig-
ure 2c), or ATP, as ATP synthase activity was even higher
than in WT in Δd1d2 (Figure 5f). One possibility is that the
increased funneling of electrons from Fd to the Mehler-like
reaction in Δd1d2 (Figure 2c), while not impairing FNR func-
tion (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
CO2 fixation in Δd1d2. Nevertheless, simultaneous defi-
ciency of FDPs in addition to NDH-1,2 (in Δflv3 d1d2 triple
mutant), with the effect of diminishing the flow of photo-
synthetic electrons to O2 photoreduction, mostly rescued
the delay in CO2 fixation (Figure 2d) as well as NADPH con-
sumption (Figure 4d) seen in Δd1d2. Transient O2 photore-
duction at a low rate was still observed in Δflv3 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
Δflv3 during sudden increases in irradiance (Figure 3).
These observations suggest that, in addition, Flv1/3 and
NDH-1,2 have a role in contributing to oxidation of PSI dur-
ing changes in light conditions or carbon availability. Upon
deficiency of NDH-1,2 (in Δd1d2), cells prioritize the protec-
tion of PSI over efficient CO2 fixation by upregulating the
Mehler-like reaction via an unknown mechanism. The triple
mutants cannot do this, leading to the timely induction of
CO2 fixation at the high cost of inability to oxidize PSI (Fig-
ure 3a) or Fd (Figure 5d). This results in loss of PSI (Fig-
ure 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 $2H^+/e^-$ stoichiometry (Strand *et al.*, 2017; Saura and Kaila, 2019). Therefore, it will contribute to build-up of $\Delta pH$, 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 $\Delta pH$ 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 $\Delta d1d2$ (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 $\Delta flv3 d1d2$ in comparison with $\Delta flv3$ (Figures 3 and 5) because in the triple mutant pmf was not lower than in $\Delta flv3$ (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 $\Delta flv3 d1d2$ (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 $\Delta pH$ by consuming $H^+$ on the cytosolic side of thylakoids (Cyd) and pumping protons to the lumen (Cox) (Brändén *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 $\Delta d1d2$ and $\Delta flv3 d1d2$ 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 $\Delta pH$, 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 [CO₂]/HL in cells deficient in both Flv1/3 and NDH-$1_{1,2}$

Exposure of *Synechocystis* cells to low [CO₂] induces expression of high-affinity CCM-related genes such as SbtA, a $HCO_3^-/Na^+$ symporter on the plasma membrane, and NdhD3, which is part of the NDH-$1_3$ complex on thylakoids specializing in Cᵢ uptake (Ohkawa *et al.*, 2000b; Shibata *et al.*, 2001; Shibata *et al.*, 2002; Zhang *et al.*, 2004). CCM is energetically expensive, but the large Cᵢ 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 CO₂ conversion to $HCO_3^-$ with concomitant translocation of protons into the thylakoid lumen.
<figure><img src="image_12.png" /><figcaption>**Figure 7.** Schematic models for the coordination of the activities of flavodiiron proteins (FDPs) and NDH-1 in preventing over-reduction of photosystem (PS) I. (a) In conditions where the ferredoxin (Fd) pool is highly reduced and the plastoquinone (PQ) pool is not, NDH-1L transfers electrons from Fd to the PQ pool, coupled to O₂ reduction by thylakoid-localized terminal oxidases (RTOs). NDH-1L pumps 2H⁺/e⁻ into the lumen while FDPs and RTOs consume H⁺ when reducing O₂ to H₂O on the cytosolic side of thylakoids. This increases transthylakoid pmf, which in turn drives adenosine triphosphate (ATP) synthesis as well as inhibits PQH₂ oxidation at Cyt b₆f (depicted by a blunt-ended red curve), thereby preventing excessive electron supply to PSI. Carbonic anhydrase activity of NDH1-MS converts CO₂ to HCO₃⁻, consuming H⁺ in the cytosol, probably also coupled to translocation of H⁺ to the lumen, further increasing pmf. Increased availability of CO₂ and ATP/NADPH ratio enhances the electron sink capacity of the Calvin-Benson-Bassham cycle (CBB) on the acceptor side of the photosynthetic electron transport chain.</figcaption></figure>

(b) If the PQ pool is highly reduced, Fd pool is oxidized, and pmf is high, NDH-1L could hypothetically function in reverse, transferring electrons from PQH₂ to Fd⁺, powered by release of 2H⁺/e⁻ from the lumen. A strong Mehler-like reaction catalyzed by Flv1/3 would be required to maintain a sufficient pool of oxidized Fd, with Flv1/3 probably accepting electrons directly from Fd⁻. Most components depicted in (a) are omitted from (b) to enhance clarity, not to suggest their absence. Extent of the color fill for PQ/PQH₂ and Fd depict level of reduction. FNR, ferredoxin-NADP⁺-reductase; PC, plastocyanin.

Despite the low induction level of the CCM proteins, NdhD3 and SbtA, the triple mutants (Figure 6) survive under standard growth conditions of air [CO₂]/ML (Figure 1b,c), but their growth was retarded on solid media (Figure 1e) The difference may be due to the differential diffusivity of CO₂ 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 [CO₂] is coupled with an increase in irradiance a low amount of CCM, reflected by the low accumulation level of NdhD3 and SbtA in the Δflv3d1d2 and Δflv3d1d2 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 α-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 Δflv3d1d2 cells (Figure 4). Nevertheless, it is important to note that Flv1/3 and NDH-1₃,₄ cooperation alone is not crucial for cell metabolism, as the Δflv1 d3d4 and Δflv3 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 [CO₂]/HL, contributing to efficient oxidation of PSI, build-up of pmf and induction of expression of low Ci-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 Flv1/3 and NDH-1,₂ as competitive electron sinks downstream of PSI may be a useful biotechnological tool for maximizing the direction of photosynthetic electrons to desired pathways under controlled environmental conditions (McCormick et al., 2013; Thiel et al., 2019; Jokel et al.,
2019). Furthermore, our findings about the functional inter-
play between FDPs and NDH-1 will be highly relevant in
projects aiming to enhance crop productivity via introduc-
tion of exogenous FDPs to higher plants (Yamamoto et al.,
2016; Gómez et al., 2018).

EXPERIMENTAL PROCEDURES

Strains and culture conditions

In the current study we used the glucose-tolerant WT strain *Synechocystis* sp. PCC 6803 (Williams, 1988), single mutant strains Δ*flv1* and Δ*flv3* (Helman *et al.*, 2003), M55 mutant (Δ*ndhB*) (Ogawa, 1991), double mutants Δ*d1d2* and Δ*d3d4* (Ohkawa *et al.*, 2000a) and triple mutants Δ*flv1* *d1d2*, Δ*flv3* *d1d2*, Δ*flv1* *d3d4* and Δ*flv3* *d3d4* obtained from the CyanoMutants collection (Nakamura *et al.*, 1999). The triple mutants were constructed by T. Ogawa in Δ*d1d2* and Δ*d3d4* backgrounds. All the mutants demonstrated complete segregation in the presence of antibiotic. Pre-experimen-
tal cultures were always grown in 30 ml batches of BG-11 medium
pH 7.5 (Williams, 1988) under 3% [CO₂] at 30°C under continuous
white light of 50 µmol photons m⁻² sec⁻¹ (ML) with agitation.
Mutant pre-cultures were supplemented with the appropriate
antibiotics. At the logarithmic growth phase, cells were harvested
and resuspended in fresh BG-11 without antibiotics at OD₇₅₀ of
0.1-0.2, as described in the appropriate figure legends. Cells were
then shifted to air [CO₂]/30°C and illuminated continuously with
white light of either 50 or 220 µmol photons m⁻² sec⁻¹, as
described in the figure legends.

Gas exchange measurements

The exchange of ¹⁶O₂ (m/z 32), ¹⁸O₂ (m/z 36) and CO₂ (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⁻¹, and kept for 1 h in air [CO₂]/50 µmol photons m⁻² sec⁻¹. Before measurements, cells were supplemented with ¹⁸O₂ at an equivalent concentration to ¹⁶O₂ and with 1.5 mM NaHCO₃. Cells were dark-adapted for 15 min, after which gas exchange was monitored over a 5-min illumination period of 500 µmol photons m⁻² sec⁻¹ of white actinic light (AL). The gas exchange rates were calculated according to Beckmann *et al.* (2009).

Measurement of fluorescence and absorbance changes

Experimental cultures for all spectroscopic experiments were grown for 4 days in air [CO₂]/50 µmol photons m⁻² sec⁻¹ at 30°C in BG-11 pH 7.5. Chl *a* fluorescence and P700 oxido-reduction (875-830 nm absorbance difference) were simultaneously recorded (Figure 3) with the Dual-PAM 100 spectrophotometer (Walz, Effeltrich, Germany). Harvested cells were resuspended in fresh BG-11 pH 7.5 and adjusted to 10 µg Chl *a* ml⁻¹, and kept for 1 h in air [CO₂]/ 50 µmol photons m⁻² sec⁻¹ and dark-adapted for 10 min before being subjected to a fluctuating light regime alternating between 1-min periods of 25 and 530 µmol photons m⁻² sec⁻¹ of red AL. Saturating pulses (500 msec of 5000 µmol photons m⁻² sec⁻¹) were administered at 15 sec intervals. Photosynthetic parameters were calculated as follows: Y(NA) = (P<sub>m</sub> - P<sub>m'</sub>)/P<sub>m'</sub>; Y(ND) = P<sub>m'</sub>/P<sub>m</sub>; Y(I) = 1 - Y(ND) - Y(NA) (Klughammer and Schreiber, 2008); Y(II) = (F<sub>m'</sub> - F)/F<sub>m</sub>' (Genty *et al.*, 1989).

A DUAL-KLAS-NIR spectrophotometer (Walz) was used to mea-
sure 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 µmol photons m⁻² sec⁻¹), 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 reoxygenation of Fd upon illumination impedes the measurement of model spectra. To circumvent this issue, we measured the DMPs from Δ*flv3* *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⁻¹ with fresh BG-11 pH 7.5, and kept for 1 h in air [CO₂]/50 µmol photons m⁻² sec⁻¹ and dark-adapted for 30 min before illumination for 30 sec with 503 µmol photons m⁻² sec⁻¹ of red AL. It is noteworthy that cytochrome *c*₆ and (FₐF₇)ₐ 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⁻¹. 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⁻¹, and illuminated with green AL of 500 µmol photons m⁻² sec⁻¹ 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 × 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, Δ*flv3*, Δ*d1d2* and Δ*flv3* *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⁻¹ with fresh
BG-11 pH 7.5. Cells were frozen in liquid N₂ and excited at
440 nm. Raw spectra were normalized to the PSII-fluorescence
peak at 685 nm.

Protein extraction and immunoblotting

Total protein extracts from cultures were isolated as previously
described (Zhang *et al.*, 2009), separated by sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis on 12% gels with 6 *m*urea
and blotted on polyvinylidene fluoride membranes. Membranes were probed with antibodies raised against D1, PsaB (AS10 695; Agrisera, Vännäs, Sweden), PetH (kindly shared by H. Matthijs), OCP (kindly shared by D. Kirilovsky), SbtA (kindly shared by T. Ogawa), NdhD3 (Eurogentec, Liége, Belgium), Flv2, Flv4 and Flv3 (Antiprot, Puchheim, Germany), and Sll0218 (MedProbe, Oslo, Norway). Horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Chicago, IL, USA) and Amersham ECL (GE Healthcare) were used for detection.

## ACKNOWLEDGEMENTS

We thank Prof. Teruo Ogawa for sharing the FDP and NDH-1 mutants and Dr. Henna Mustila for construction of the ΔM55 *flv3* strain. Dr. Duncan Fitzpatrick is thanked for expertise and assistance with MIMS. We also thank Dr. Pierre Setif and Dr. Gert Schansker for their expert advice on measuring the DMPs for the DUAL-KLAS-NIR spectrophotometer, and Prof. Jens Appel for critical reading of the manuscript. This work was supported by the Academy of Finland (project no. 315119 to Y.A. and the Finnish Center of Excellence, project no. 307335), the NordForsk Nordic Center of Excellence 'NordAqua' (no. 82845) and SFB480, Germany.

## AUTHOR CONTRIBUTIONS

LN and YA conceptualized the research; LN, ASS, ME and YA designed the research; ME and ASS conducted growth experiments; LN performed MIMS and spectrophotometric experiments; ASS performed immunoblotting; all authors analyzed the data; LN and YA wrote the article with input from ASS and ME.

## CONFLICT OF INTEREST

The authors declare no conflict of interest.

## DATA AVAILABILITY STATEMENT

All relevant data can be found within the article and its Supporting information.

## SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article.

**Figure S1.** Growth of Δ*flv1* *d3d4* and Δ*flv3* *d3d4* and creation of M55 Δ*flv3* mutant strains.

**Figure S2.** O₂ and CO₂ fluxes in M55 and in WT at pH 6.

**Figure S3.** Near-infrared (NIR) differential model plots (DMPs) for deconvolution of PC, P700 and Fd signals with the DUAL-KLAS-NIR 100 spectrometer.

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