Wild type Arabidopsis (Arabidopsis thaliana), Columbia-0 (Col-0) and cfq were grown in a growth chamber in a 16:8 photoperiod with an average of 80 μmol photons⋅m^-2⋅s^-1 light at 23°C. This rather low light was maintained to prevent the accumulation of photodamage in the mutant lines. Plants between 17 and 23 days old were used for the experiments. Seeds for cfq were obtained from Prof. Donald Ort (Departments of Plant Biology and Crop Science, University of Illinois at Urbana-Champaign), and npq4 were from Prof. Krishna Niyogi (Department of Plant and Microbial Biology, University of California at Berkeley). The M₃ generation of the mutant cfq was back-crossed with Col-0 WT for three times, and the homozygous line was chosen from the F₃ generation (Wu et al., 2007). A complementation line expressing wild type atpC1 behind a 35S promoter (35S::atpC1, or comp) was generated as described in Clough and Bent (1998) and Dal Bosco et al. (2004). This strategy for complementing point mutations was based on that presented in Wu et al. (2007) who observed ATP synthase phenotypes upon over-expressing mutant atpC1 variants in a wild type background, thus showing that the native γ-subunit can be outcompeted by other forms.

We used two complementary methods to confirm the expression of wild type atpC1 in comp, first by expression profiling with the insertion-confirmed lines, which showed the q_E value equivalent to the wild type in the Dynamic Environmental Photosynthetic Imaging (DEPI) system (Cruz et al., 2016), and second using a mass spectrometry approach. Briefly for this approach, chloroplasts from a single leaf from either Col-0, cfq, or complemented lines were isolated using Minute^TM Chloroplast Isolation Kit (Invent Biotechnologies, Inc., Eden, MN, USA), lysed and thylakoids were subsequently recovered by centrifugation. Chlorophyll content of thylakoids was determined by the method of Arnon (1949) to give a final concentration of 1 mg chlorophyll/ml for all samples. All thylakoids (5 μg chlorophyll total) samples were solubilized in Laemmeli buffer (Laemmli, 1970) and resolved by SDS-PAGE and stained with Coomassie Blue. Bands in the 37 kDa region (corresponding to the ATP-C protein) were excised and digested according to Shevchenko et al. (1996) and then subjected to mass spectrometry according to protocols established by the MSU Proteomics Facility. Eluted fragments were analyzed by Scaffold^TM 4.0 program. To monitor the rescue of cfq, we compared the recovery of certain fragments specific to either Col-0, cfq or rescued lines. A summary of this analysis is given in Supplementary Figure S7.

Other chlorophyll fluorescence-derived photosynthetic parameters were obtained using the equations and methods described in Kramer et al. (2004b). The F₀ point was measured in extensively (at least 30 min) dark adapted plants. The F_M″ parameter, used to distinguish between q_E and q_I, was measured after 3 min of dark adaptation following steady-state illumination. The dark interval relaxation kinetics (DIRK) of the electrochromic signal at 520 nm was for ECSt (pmf) and thylakoid proton conductivity (g_H⁺) measurement (Avenson et al., 2005b; Baker et al., 2007). ECSt was calculated from the amplitude of the ECS signal during dark intervals of approximately 300 ms, and thylakoid proton conductivity (g_H⁺) was estimated by fitting the ECS decay curve to a first-order exponential as previously described (Kanazawa and Kramer, 2002). The redox states of PSI were estimated from the difference in absorbance changes at 810 and 940 nm (Klughammer and Schreiber, 1994) with modifications described in the text and Figure 4.

Two methods were used for protein quantification. For the result presented in Supplementary Figure S2, leaf samples were rapidly frozen in liquid nitrogen and ground in at 77 K with mortar and pestle, and suspended in an extraction buffer [100 mM Tricine-KOH, pH 7.5, 2 mM MgCl₂, 10 mM NaCl, 1 mM ethylene-diamine-tetra-acetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), and β-mercaptoethanol]. The suspension was centrifuged at 13,000 rpm for 5 min, and the pellet was resuspended in a sample buffer [50 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 10 mM β-mercaptoethanol, 10% glycerol, and bromophenol blue]. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE), and blotted onto polyvinylidene difluoride (PVDF) membranes (Invitrogen, USA). Blots were probed with polyclonal antibodies raised against β, γ, and 𝜀 subunits of ATP synthase, and cytochrome f (cyt f) of b₆f complex. The western blots were exposed to films (Denville Scientific, Inc., Holliston, MA, USA) using ECL⁺ chemiluminescence kit (GE Healthcare, USA) with a peroxidase-conjugated anti-rabbit secondary antibody.

For the results presented in Supplementary Figure S5, total leaf tissue from cfq and Col-0 plants under control or fluctuating light were collected and homogenized using Minute-Chloroplast Isolation Kit (Invent Biotechnology, Inc. Plymouth, MN, USA) according to manufacturer protocol. Total leaf homogenates were quantitated for chlorophyll content and 5 μg chlorophyll/lane for each sample were analyzed by either SDS-PAGE or Western blot analysis. For western blots, proteins corresponding to 5 μg chlorophyll/lane were transferred onto PVDF membrane (Invitrogen) and probed with antibodies purchased from Agrisera (Vännäs, Sweden) according to manufacturer specifications [PsaB(AS10 695), CytF(AS10 695), PsaD(AS09 461), PsaF(AS06 104), PsbA(AS05 084A), and PC(AS06 141)] or produced in house (Tic110 and Toc75 using 1:5,000 dilutions). The detection method employed used a secondary anti-rabbit conjugated to alkaline phosphatase (KLP, Inc. Gaithersburg, MD, USA) at 1:5,000 dilution for 1 h in 5% DM/TBST. The blots were developed using a standard AP detection system with BCIP/NBT as substrates (Sigma-Aldrich, St. Louis, MO, USA). Bands were imaged and quantitated digitally using ImageJ (Rasband, 2008).

Under typical low light used for Arabidopsis growth (to avoid photodamage), 16 h light: 8 h dark photoperiod with an average of 80 μmol photons⋅m^-2⋅s^-1 light at 23°C (for more details, see Materials and Methods), the cfq mutant grew with leaf area and thickness values that were indistinguishable from Col-0 (see Supplementary Figure S1). The chlorophyll content per leaf area was also indistinguishable (17.2 ± 0.5 μg Chl⋅cm^-2 for Col-0, and 16.8 ± 0.7 μg Chl⋅cm^-2 for cfq). Larger differences in growth phenotypes were observed when plants were grown for longer times under fluctuating illumination, as will be discussed in a forthcoming paper.

In order to understand the impact of variations in ATP synthase properties on the regulation of photosynthesis, we compared the photosynthetic response of Col-0, cfq and cfq 35S::atpC1 (comp) expressing wild type atpC1 in the cfq background. It is important to note that all experiments shown here were performed on plants that had been grown under non-stressed conditions. As will be discussed in detail in a forthcoming paper, exposing cfq plants to environmental stresses such as high light, drought, etc. results in long-term changes in the photosynthetic responses. In this work we focus on the short-term effects (from minutes to hours). The maximal PSII quantum efficiencies, measured by the fluorescence parameter F_V/F_M taken just prior to our experiments were indistinguishable among cfq, Col-0, and comp (0.81 ± 0.2, 0.82 ± 0.2, and 0.80 ± 0.3), indicating that PSII was not damaged in the mutant prior to the experiments. Figure 1 compares the responses of photosynthetic parameters of intact, attached leaves to a range of steady-state intensities of photosynthetically active radiation (PAR) under ambient (∼400 ppm) CO₂. Photosynthesis was allowed to acclimate 30 min at each PAR to ensure steady-state conditions. In Col-0, LEF (Figure 1A) increased continuously with PAR, reaching half-saturation at approximately 100 μmol photons⋅m^-2⋅s^-1, and a light-saturated LEF of approximately 35 μmol electrons⋅m^-2⋅s^-1, consistent with previous results on Arabidopsis grown under these conditions (e.g., Livingston et al., 2010). There were no statistically significant differences in LEF among Col-0, cfq and comp at 300 μmol photons⋅m^-2⋅s^-1 (Figure 1A) but there were substantial differences in the q_E responses (Figure 1B). In Col-0 and comp, q_E increased continuously with PAR, reaching a value of about 0.6–0.8 at 300 μmol photons⋅m^-2⋅s^-1, comparable to previous results for plants grown under these conditions (Livingston et al., 2010).

In contrast, cfq showed q_E responses 2–3-fold smaller than in Col, reaching only 0.3 at 400 μmol photons⋅m^-2⋅s^-1, implying a loss of light-induced lumen acidification or q_E-related antenna responses in cfq. The decrease in q_E response was largely reversed in comp and thus we attribute the major effects on photoprotection to the cfq mutation. It may seem surprising that changes in NPQ extent did not strongly affect LEF, but this effect is generally observed with moderate levels of NPQ seen in higher plants (e.g., Li et al., 2002; Brooks and Niyogi, 2011), because the NPQ dissipation of light energy competes with a very rapid capture of excitation energy by photochemistry; NPQ will, however, effectively decrease the quantum efficiency when photosynthesis is strongly limited by light intensity. In effect, the rate-limiting step for LEF is not at the level of light capture, but downstream at the level of the cytochrome b₆f complex which in turn is controlled by lumen pH, so, that with moderate changes in NPQ, the PSII quantum efficiency (and thus LEF) is not strongly affected, but how the energy is lost from the photosynthetic apparatus is changed (Strand and Kramer, 2014). Inactivating q_E results in less NPQ, but in more PSII centers becoming closed by reduction of Q_A, implies that primary function of NPQ is not to regulate electron transfer but to prevent the accumulation of reduced Q_A^-.

To distinguish between possible mechanisms for the loss of q_E in cfq, we probed the proton circuit of photosynthesis using in vivo kinetic spectroscopy. The DIRK of the electrochromic shift (ECS) can be used to monitor light-induced electron and proton transfer reactions that affect the thylakoid pmf (Avenson et al., 2005a; Baker et al., 2007). The amplitude of the ECS signal during the dark 0.5 s interval, termed ECSt, is related to the amplitude of light-driven pmf. The dependences of q_E on light-induced pmf (Figure 1D) for cfq and comp were all similar to that in Col-0, suggesting that the mutation did not substantially affect the partitioning of pmf into ΔpH and Δψ or the response of q_E to lumen acidification. If the fraction of pmf partitioning into ΔpH had increased, we would expect to see a higher extent of q_E, which is responsive to lumen acidification, for a given total pmf, as we reported earlier (Avenson et al., 2005a; Baker et al., 2007). We thus conclude that the loss of q_E in cfq was most likely caused by decreased acidification of the lumen rather than altered antenna responses to lumen acidification.

The cfq mutant showed substantially lower amplitudes of light-induced pmf changes (as indicated by the ECSt parameter) (Figure 1C), implying an effect on proton efflux from the lumen rather than on light-driven proton transfer. This interpretation was confirmed by the data in Figure 1E, which shows that the loss of pmf in cfq can be explained by higher conductivity of the thylakoid to proton efflux (g_H⁺), which in turn is attributable to the activity of the chloroplast ATP synthase (Kanazawa and Kramer, 2002; Avenson et al., 2005b). The fact that the decay of the ECS signal in leaves with inactive ATP synthase are similar in cfq and Col-0 (Wu et al., 2007) indicates that the signals we observe here are attributable to the ATP synthase activity and not to membrane leakage. The difference in g_H⁺ was minimal at low light, but increased with light intensity, reaching about threefold higher values at 300–400 μmol photons⋅m^-2⋅s^-1, suggesting that the effect was related to the metabolic state of the chloroplast rather than to any basal increases in the rate of proton leakage. These results support the conclusion that the loss of q_E in cfq was caused by high ATP synthase activity that depletes the thylakoid pmf.

We next tested if the responses of ATP synthase activity to CO₂ levels were different in cfq by probing photosynthetic parameters during steady-state illumination (100 μmol photons⋅m^-2⋅s^-1) at ambient (400 ppm), low (50 ppm) and very low (less than 10 ppm) CO₂ levels (Figure 2). Lowering CO₂ led to decreases in LEF in both the wild type and mutant (Figure 2A), but the effect was somewhat larger in cfq, likely because of higher levels of photoinhibition (see below). In Col-0, decreasing CO₂ from ambient to 10 ppm resulted in a progressive slowing of g_H⁺, from 60 to about 12 s^-1, likely reflecting decreased ATP synthase activity as previously described (Kanazawa and Kramer, 2002), with increased q_E responses (Figure 2C) resulting from higher pmf (Figure 2D). In contrast, g_H⁺ in cfq was almost twofold higher at 400 ppm (96 s^-1), and increased fractionally as CO₂ was lowered to 50 ppm. Only at the lowest CO₂ level did we observe a decrease in g_H⁺ to about 60 s^-1, close to that seen in Col-0 at 400 ppm. The q_E response of cfq roughly followed that of the pmf, and was largely independent of decreased in CO₂ with a substantial increase at very low CO₂. These results imply that the control or regulation of g_H⁺, and thus likely the activity of the ATP synthase, is substantially altered in cfq.

Supplementary Figure S2 summarizes results of western blot analysis of selected thylakoid membrane proteins in Col-0 and cfq, showing a loss of about 50% of ATP synthase proteins β, γ, and 𝜀 in cfq compared to Col-0. For comparison (load control), the levels of cytochrome f of the cytochrome b₆f complex, and these were similar in Col-0 and cfq, as was consistent with the similar levels of photo-oxidizable cytochrome f measured in intact leaves in Col-0 and cfq (Supplementary Figure S3). Despite the fact that ATP synthase activity in cfq estimated by g_H⁺ was equal to or higher than that in Col-0, the content of the ATP synthase proteins was about 50% lower than that of Col-0, suggesting that the existing ATP synthase had an effectively high specific activity. The reason for this effect is not known, but it suggests that the native ATP synthase operates below its potential maximal activity, perhaps as will be discussed below, as a regulatory mechanism. In this respect it is interesting that Rott et al. (2011) presented evidence that a substantial fraction of ATP synthase can be in inactive forms, giving a low effective specific activity, and one possibility is that the cfq mutation prevents the accumulation of this inactive form leaving to higher overall activity.

Figure 3 shows representative re-reduction kinetics of P₇₀₀⁺ upon rapid light–dark transition from steady-state illumination (at 300 μmol photons⋅m^-2⋅s^-1) at ambient (about 400 ppm) and low (50 ppm) CO₂ in Col-0 and cfq. In Col-0, at ambient CO₂, P₇₀₀ was re-reduced with a half time of about 5–7 ms, similar to previous results (Rott et al., 2011). Decreasing CO₂ led to an increased extent of P₇₀₀⁺ oxidation state and a slower rate of re-reduction (half time of about 25 ms) in the dark, likely reflecting the slowing of PQH₂ oxidation with increased lumen acidity at low CO₂ levels (Kanazawa and Kramer, 2002). In contrast, cfq showed very rapid P₇₀₀⁺ re-reduction kinetics. At ambient CO₂, P₇₀₀ was only marginally oxidized and re-reduced rapidly with a half time of about 5 ms, though the precise time was difficult to quantify because of the small signal size. At 50 ppm CO₂, no obvious P₇₀₀⁺ re-reduction was observed during the dark interval, suggesting that in cfq, the rate of electron delivery to PSI was substantially more rapid than its oxidation by PSI photochemistry. In other words, the cfq mutant appears to have a constitutively rapid ATP synthase so that the normal buildup of ΔpH that initiates “photosynthetic control” of electron transfer at the cytochrome b₆f complex is lost, so that re-reduction of P₇₀₀⁺ is more rapid than its light-driven oxidation.

Figure 4 shows representative kinetics traces reflecting light-induced P₇₀₀/P₇₀₀⁺ absorbance changes using a procedure modified from that introduced by Klughammer and Schreiber (1994) to test for changes in the redox state of PSI centers in Col-0 (Figures 4A,B) and cfq (Figures 4C,D) under ambient (Figures 4A,C) and 50 ppm CO₂ (Figures 4B,D). Several parameters can be inferred from these experiments (Klughammer and Schreiber, 1994). Here, we extend or modify the established terminology (Klughammer and Schreiber, 2008) to avoid potentially confusing interpretations and to emphasize the special features relevant to the current data sets. For example, we observe large changes in the total photo-oxidizable P₇₀₀ signal as the over the course of the experiment and between traces so that it is not possible to use this extent from individual traces as a reference point. We thus define our terms as follows, using parameters starting with P as in an indicator of P₇₀₀⁺ absorbance signals and PSI referencing calculated PSI redox states:

Compared to Col-0, cfq showed strong suppression of both the steady-state P₇₀₀⁺ oxidation state (PSI_ox) and open PSI centers (PSI_o) as well as an increase in the reduction state of the PSI electron acceptors, as indicated by an increase in PSI_or (Figure 4). These distinct responses likely reflect differences in the rate-limitations for electron transfer. In Col-0, electron flow was likely limited at the cytochrome b₆f complex, which is typically controlled by acidification of the lumen, leading to net oxidation of P₇₀₀⁺. In cfq, by contrast, electron flow appeared to be limited on the acceptor side of PSI, resulting in accumulation of electrons on stromal electron carriers and within PSI. Consistent with this view, lowering the CO₂ resulted, in Col-0, in an increased in PSI_ox, indicating a slowing of the b₆f complex upon buildup of pmf. In contrast, in cfq, decreasing CO₂ led to strongly decreased PSI_ox and increased PSI_or, likely indicating a further reduction of the PSI electron acceptor pool. Also noteworthy was the large decrease in P₇₀₀⁺ during the saturation pulse (PSI_s), presumably reflecting the buildup of electrons in the stromal electron acceptor pool and subsequent inhibition of PSI photochemistry. Under our conditions, the extent of PSI_act was about 10 and 20% lower in cfq than in Col-0 under ambient and 50 ppm CO₂, likely indicating a loss of active PSI centers in the mutant (see also below).

To determine the effects of extended exposure to low CO₂, we performed a time course for the PSI and PSII photosynthetic parameters, in Col-0, cfq and npq4 (Figure 5). The npq4 mutant was included to distinguish the effects of loss in cfq of q_E from that of decreased pmf, as discussed below. The extent and rate of PSI photoinhibition (Figure 5A), as reflected in the decrease in PSI_act, were highest in cfq, which lost over 65% of photoactive PSI centers during the experiment; this effect was partially rescued in comp, see Supplementary Figure S4). By contrast, Col-0 lost less than 25% of active PSI. Interestingly, PSI in npq4 appeared to be marginally less sensitive to these conditions, losing activity slightly more slowly. The trends in PSI inhibition were well-correlated with the extents of the PSI_or parameter (Figure 5B) as expected if over-reduction causes PSI photodamage as proposed by several groups (Terashima et al., 1994; Scheller and Haldrup, 2005; Suorsa et al., 2012; Allahverdiyeva et al., 2015). The highest extent of PSI_or was seen in cfq, at early time points accounting for over 80% of the total PSI centers, but decreasing to about 55% by the end of the experiment, possibly reflecting a decrease in delivery of electrons at later time points. In contrast, in Col-0, PSI_or remains at about 30% or lower throughout the experiment. The extent of PSII photoinhibition, estimated by the q_I parameter (Figure 5C), was but substantially higher in npq4 compared to Col-0, as anticipated from previous studies (Brooks et al., 2014). In contrast, cfq showed very similar q_I responses to Col-0, suggesting that the stability of PSI, but not PSII was strongly compromised in cfq by illumination at low CO₂. Interestingly, npq4 showed the lowest extent of PSI_ox, possibly reflecting decreased rates of delivery of electrons to PSI as a result of PSII photoinhibition, possibly reflecting the proposed role of PSII photoinhibition in protecting PSI (Tikkanen et al., 2014). The realized steady-state PSII quantum efficiency was most severely affected in cfq, implying that PSI photoinhibition had a stronger effect on overall photosynthesis.

Rapid changes in actinic light intensity are known to severely impact photosynthetic processes and application of fluctuating light can reveal new photosynthesis-related phenotypes in mutants that would otherwise show only weak effects (Suorsa et al., 2012; Allahverdiyeva et al., 2015; Cruz et al., 2016). The data in Figure 6 shows the effects on chlorophyll fluorescence parameters of exposure of previously dark-adapted leaves to fluctuating light alternating between 100 and 1,000 μmol photons⋅m^-2⋅s^-1 at 30 min intervals. The maximal quantum efficiency of PSII photochemistry in dark adapted leaves (F_V/F_M, Figure 6A) was slightly smaller in cfq, likely indicating the presence of a small extent of photoinhibition prior to the start of the experiment. During the first 30 min of 100 μmol photons⋅m^-2⋅s^-1 the steady-state PSII quantum efficiency (ϕ_II) was similar in Col-0 and cfq (as seen in the experiments above), but much more strongly suppressed by exposure to high light (1,000 μmol photons⋅m^-2⋅s^-1). The loss of ϕ_II strongly decreased LEF, especially at higher light intensities (Figure 6B). During subsequent low light cycles, ϕ_II and LEF recovered in Col-0. In contrast, ϕ_II and LEF did not recover at low light in cfq, but rather showed a progressive decrease with each cycle of high and low light exposure. Despite this loss of activity, quantum efficiency in the dark (F_V″/F_M″) recovered to nearly the same extent Col-0 and cfq upon dark adaptation for 20 min, likely indicating that, although the mutant suffered some PSII photoinhibition, it likely did not account for most of the decreased electron transfer capacity during the low light conditions. Instead, it appears the loss of ϕ_II was related to decreased activity of downstream steps in photosynthetic electron transfer.

During the first low and high light cycle, NPQ was smaller in cfq than Col-0, consistent with the results above showing a loss of the q_E response in the mutant (Figure 6C). In subsequent illumination cycles, Col-0 showed a nearly constant pattern of increased NPQ at high light and return to lower values in the weak light. In contrast, cfq showed a gradual increase in NPQ with each cycle, consistent with a moderate increase in the rate of photoinhibition and loss of PSII activity, but not sufficient to account for the decrease in ϕ_II seen at low light. Instead, the strong decreases in the q_L parameter in the mutant (Figure 6D) indicate that PSII became limited by buildup of electrons on Q_A, implying that electron transfer was blocked at a step following plastoquinone reduction, most likely at the acceptor side of PSI. The decrease in the q_L parameter increased throughout the remaining light cycles, suggesting that this blockage became progressively more restrictive with fluctuating light.

Figure 7 shows the effects of fluctuating light on ECS parameters, related to the generation and dissipation of the thylakoid pmf. The g_H⁺ parameter (Figure 7A), was relatively constant for Col-0, increasing only slightly during the high intensity illumination, consistent with previous observations that the activity of the ATP synthase can be dependent on conditions such as CO₂ levels, but relatively stable over this range of light intensity (Kanazawa and Kramer, 2002). During the first low light and high light exposures, g_H⁺ was about 50% higher in cfq compared to Col-0, largely accounting for the small extent of the light-induced pmf, as estimated by the ECSt parameter (Figure 7B). Interestingly, the g_H⁺ values increased in cfq during the higher light treatments, possible reflecting alterations in metabolic regulation. The amplitude of the light-induced pmf (ECSt) in cfq decreased with each successive light cycle, likely reflecting the combination of high g_H⁺ and decreased light-induced electron and proton fluxes. These results suggest that the loss of ATP synthase control in cfq leads to a cascade of events under fluctuating light, starting with the light reactions and leading to secondary effects on downstream metabolic or regulatory processes.

Figure 7 also shows the effects of fluctuating light on the redox state (Figure 7C) and PSI activity (Figure 7D). In Col-0, the extent of the PSI_or parameter was relatively constant at about 0.2 over the course of the experiment, indicating that even at high light electrons do not substantially accumulate on PSI acceptor side. In cfq by contrast, PSI was substantially over-reduced throughout the experiment, with PSI_or starting at 0.6 during the first low light treatment and increasing to about 0.9 during the first high light treatment, with only partial recovery in the following low light treatments. Overall, the results on fluctuating light are consistent with loss of pmf control of electron flow in cfq, leading to over-reduction of PSI and subsequent PSI photoinhibition.

Supplementary Figure S5 shows that the exposure to a 4-h fluctuating light regime (as in Figure 6) induced only small changes in the content of thylakoid proteins related to PSI (PsaD, PsaF, PsbA), PSII (PsbA), cytochrome b₆f complex (cyt f) and plastocyanin (PC). To distinguish between PSI damage and loss of downstream electron acceptors, we infiltrated leaves with DCMU to block PSII and Methyl viologen (MV) as a rapid exogenous PSI electron acceptor and observed the extent and kinetics of P₇₀₀ oxidation upon illumination (Supplementary Figure S6), comparing the effects of pre-exposure of leaves to 20 min of low light (100 μmol photons⋅m^-2⋅s^-1) or a single cycle of high light. The extent of photooxidizable P₇₀₀ was lower in cfq than Col-0 and decreased strongly upon the high light treatment even though the major PSI proteins were unaffected, suggesting that while light treatment caused PSI damage, it did not result in loss of the PSI proteins that were assayed on the time scale of a few hours. This result suggests that the loss of PSI activity results from damage to the core electron transfer components, but not degradation of the PSI proteins themselves.

Interestingly, the photosynthetic phenotypes of cfq were strikingly similar to those of “proton gradient regulation 5” pgr5 mutant. Munekage et al. (2002) ascribed the loss of pmf in pgr5 to a defect in CEF around PSI and argued that this process was essential for maintaining photoprotection of PSII. The fact that both of these mutants show increased ATP synthase activity, leading to decreased lumen acidification leading to loss of q_E response, over-reduction of PSI (Avenson et al., 2005a; Suorsa et al., 2012) and severe PSI photodamage (Suorsa et al., 2012), suggests that the major function of PGR5 may be in adjusting the activity of the ATP synthase rather than regulating or catalyzing CEF.

We describe a mutant, cfq, containing a single point mutation in the γ₁-subunit of the chloroplast ATP synthase that affects the regulation and activity of the chloroplast ATP synthase. Previously, the cfq mutation was reported (Wu et al., 2007) to shift the redox potential of the γ-subunit regulatory thiols, which should make it more sensitive to oxidative down-regulation, i.e., one might expect that cfq-ATP synthase would have slower activity. Instead, we find that the activity of the cfq-ATP synthase as probed by g_H⁺ was considerably faster than wild type-ATP synthase (Figures 1E, 2B) despite having a lower protein content of ATP synthase subunits (Supplementary Figure S2), suggesting that the specific activity of the ATP synthase is increased in cfq. The fact that cfq-ATP synthase activity responds differently to light (Figure 1), altered CO₂ levels (Figure 2) and fluctuating light (Figure 7) suggests that the increased g_H⁺ is caused by altered regulation or control of the ATP synthase. It is important to note that our results are not in contradiction with those of Wu et al. (2007) as the phenotypes we observed occur under higher or fluctuating lighting conditions.

Overall, these results are in broad agreement with lumen acidification being an essential component of the feedback regulatory system of light reactions, activating the photoprotective q_E response and governing the cytochrome b₆f complex. The effects were minimal under permissive “laboratory-like” growth conditions but become particularly severe under low CO₂ or fluctuating light, implying that ATP synthase regulatory control is an essential regulatory response of the light reactions to environmental or metabolic fluctuations. The loss of control of the thylakoid pmf during photosynthesis resulted in changes in the rate-limiting step in electron transfer, from the cytochrome b₆f complex to the acceptor side of PSI, resulting in accumulation of electrons on PSI and subsequent PSI photodamage, possibly via the production of reactive oxygen species (Scheller and Haldrup, 2005; Rutherford et al., 2012; Huang et al., 2016; Takagi et al., 2016) that may result in destruction of the PSI iron sulfur complexes. Finally, the results suggest that engineering efforts to improve photosynthesis by increasing the rates of key rate-limiting steps in photosynthesis (e.g., the ATP synthase) can do more harm than good by short circuiting essential feedback regulatory systems.