The thylakoid proton motive force (pmf), the transmembrane electrochemical gradient of protons generated during the light reactions of photosynthesis, is a fundamental entity of bioenergetics, coupling light-driven electron transfer reactions to the phosphorylation of ADP via the ATP synthase (Avenson et al., 2004; Kramer and Evans, 2011). In oxygenic photosynthesis, light energy is captured by pigments in light-harvesting complexes and transferred to a subset of chlorophylls in photosystem I (PSI) and photosystem II (PSII), where it drives the extraction of electrons from water and their transfer through redox cofactors to ultimately reduce NADP⁺. The vectorial transfer of electrons across the membrane is tightly coupled with the generation of the pmf, composed of both electric field (Δψ) and pH (ΔpH) gradients.

In addition to its role in energy conservation, the pmf is also critical for feedback regulation of photosynthesis (Cruz et al., 2005). Acidification of the thylakoid lumen activates the photoprotective energy-dependent exciton quenching (q_E) process, which dissipates excess absorbed light energy in the photosynthetic antenna complexes by the activation of violaxanthin deepoxidase (Demmig-Adams and Adams, 1992) and protonation of PsbS (Li et al., 2004). Lumen acidification also regulates the oxidation of plastoquinol by the cytochrome b₆f complex, slowing electron transfer from PSII and preventing the accumulation of electrons on PSI, which can otherwise lead to photodamage (Nishio and Whitmarsh, 1993; Hope et al., 1994). In vitro work has shown that excessive lumen acidification can inactivate PSII (Krieger and Weis, 1993), decrease the stability of plastocyanin (Gross et al., 1994) and severely restrict electron flow through the cytochrome b₆f complex (Kramer et al., 1999). Taken together, the in vivo and in vitro evidence of the susceptibility of photosynthetic components to acidification has led to the proposal that the extent of pmf and its partitioning into Δψ and ΔpH components is regulated to maintain the lumen pH above about 5.8, where it can regulate photoprotection. However, under environmental stresses the regulation of photosynthesis may become overwhelmed, leading to PSII damage, or photoinhibition, from lumen over-acidification, although this has not been shown to occur in vivo (Kramer et al., 1999).

PSII photoinhibition can be a major contributor to loss of photosynthetic productivity (Raven, 2011), particularly under rapid fluctuations in environmental conditions experienced in the field (Kulheim et al., 2002). However, the mechanisms and regulation of photoinhibition remain highly debated (Keren and Krieger-Liszkay, 2011; Tyystjärvi, 2013). Though several mechanisms have been proposed for the photodamage process, it is not known which of these operate in vivo under diverse environmental conditions. In addition, there are differing views on whether the extent of photoinhibition is governed by the rate of photodamage to PSII or by regulation of PSII repair (Takahashi et al., 2007). Answering these questions is essential to understanding how plants respond to rapidly changing conditions and thus of critical importance to improving plant productivity.

The extent of the pmf can be modulated by altering the light-driven influx of protons into the lumen, i.e. by changing the rates of linear electron flow (LEF) or cyclic electron flow, or the efflux of protons through the ATP synthase (reviewed in Kramer et al., 2004). This latter mode of regulation is important for co-regulation of the light reactions with downstream metabolic processes. For example, under CO₂ limitations (Kanazawa and Kramer, 2002) as well as during stromal P_i limited conditions (Takizawa et al., 2008) the chloroplast ATP synthase activity is strongly down-regulated, decreasing the proton conductivity (g_H⁺) of the thylakoid, leading to buildup of pmf and activation of q_E. Similar decreases in g_H⁺ and increases in pmf are seen when ATP synthase protein content is decreased (Rott et al., 2011) or in mutants with an altered ATP synthase γ-subunit (Kohzuma et al., 2012).

We took advantage of the effects of the ATP synthase on the photosynthetic proton circuit to probe how the pmf influences photoinhibition in vivo. We constructed a series of Arabidopsis thaliana mutants, which we termed minira (minimum recapitulation of ATPC2), in which we complemented a γ₁-subunit (ATPC1) T-DNA knockout line (Dal Bosco et al., 2004) with γ-coding sequences containing site-directed mutations to specifically incorporate amino acid changes around the redox regulatory cysteines present in ATPC2 into ATPC1 (Figure 1A) (Inohara et al., 1991). Each minira line is designated numerically based on amino acid position and independent transformation events; for example minira 4–1, minira 4–2, and minira 4–3 represent three independent transformation events of the same I201V mutation. The minira library was originally developed as part of an on-going 'domain swapping' approach to assess functional differences in the two ATPC paralogs in Arabidopsis, ATPC1 and ATPC2, but the minira mutants also shows a range of ATP synthase activities useful to the present work. As described below, we also confirmed key aspects of the work using previously characterized ATPC1 tobacco antisense lines (Rott et al., 2011). However, variations in ATP synthase suppression in the antisense lines between leaves and plant generations limited their experimental utility. The current mutagenesis approach was preferable as it allowed repeatable analyses of multiple photosynthetic parameters in stable, identical genetic backgrounds within each mutant line.

Figure 1

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We assessed the effects of minira modifications on g_H⁺ and the extent of the light-driven pmf in vivo based on the decay kinetics of the electrochromic shift (ECS), which reports changes in the thylakoid electric field (Sacksteder and Kramer, 2000). The minira lines displayed a range of ATP synthase activities (Figure 1C), from about 30–120% that of wild type (Wassilewskija-2, Ws-2), resulting in similar variations in light-driven pmf (Figure 1D). Multiple independent transformations were utilized for the same minira mutation, as the g_H⁺ changes likely reflect both intrinsic ATP synthase activity changes due to the mutations, as well as changes due to protein expression level or stability of the mutated subunit within the complex (Figure 1B). While some minira mutants display an increase in g_H⁺ relative to the wild type and will acidify the lumen at a slower rate, others have a large decrease in total ATP synthase content. For the purpose of understanding how a high pmf impacts photosynthesis we have primarily focused on those mutants that modified g_H⁺ while maintaining an ATP synthase content similar to wild type levels.

The minira library was then screened for photosynthetic phenotypes using whole plant chlorophyll fluorescence imaging (Cruz et al., 2016) over a consecutive three-day photoperiod (Figure 2A). Photosynthetic parameters were calculated from these images (videos are shown in videos 1–9) and shown as kinetic traces (Figure 2—figure supplements 1–15) or as log-fold changes compared to wild type (Figure 2B–D). Under 'standard' laboratory growth chamber lighting on day one, most minira lines showed relatively small differences from wild type in LEF (Figure 2B), q_E (Figure 2C), and photoinhibitory quenching (q_I) (Figure 2D), with the exceptions of minira 3–1 and 11–1, which also showed the most severe decreases in g_H⁺ (Figure 1C). Stronger photosynthetic phenotypes appeared on days two and three, implying that the decreased g_H⁺ and pmf effects were enhanced by intense or fluctuating illumination.

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In general, LEF decreased with decreasing g_H⁺ (Figure 2B, rows are ordered by increasing g_H+) while q_E increased (Figure 2C). The increases in q_E were especially pronounced at higher light intensities on days two and three. These effects can be explained by slowing of proton efflux through the ATP synthase in the mutants that results in increased pmf for a given LEF. This is reflected in the higher lumen pH-sensitive q_E response for a given LEF (Figure 2E), which is strikingly similar to that attributed to ATP synthase regulation in wild type plants during limitations in carbon fixation (Kanazawa and Kramer, 2002) or decreases in ATP synthase content (Rott et al., 2011). The q_E sensitivities for the mutants remained similar throughout the experiments, i.e. the data for each mutant followed similar curves, implying that the ATP synthase activities were relatively constant within a particular line, consistent with a lack of light-dependent modulation of g_H⁺ that has previously been observed (Avenson et al., 2005). The extents of q_E did not exceed about 3.5 units in any of the lines, suggesting that either the lumen pH was restricted to a moderate acidity or that the capacity of the q_E response was saturated as light intensities increased.

We also observed a strong correlation between increased q_E and q_I, (for quantitative comparisons, see Figure 2—figure supplement 2), implying that decreases in ATP synthase activity in the mutants led to not only higher photoprotection but also higher rates of PSII photoinhibition. This result appears counterintuitive, in that we would expect the photoprotective q_E response to prevent photoinhibition. Particularly striking was the relative loss in q_E near the peak light intensities on days two and three in the most strongly affected minira lines (Figure 2C). We attribute this effect to strong accumulation of PSII photoinhibition (Figure 2D) leading to the loss of photosynthetic capacity (see Figure 2B) that limited acidification of the thylakoid lumen.

The observed increases in photoinhibition at high pmf could be caused by several mechanisms. It has been proposed that photoinhibition is primarily controlled by modulating the rate of PSII repair, i.e. the rate of damage is dependent solely on light intensity but repair being inhibited by stress-induced ROS production (Nishiyama and Murata, 2014). However, blocking PSII repair with the chloroplast translation inhibitor lincomycin (Tyystjarvi and Aro, 1996) revealed that, when compared to wild type or minira lines with wild type like g_H⁺ (e.g. minira 6–1), minira lines with low g_H⁺ and high pmf (e.g. minira 3–1, Figure 1C, Figure 3) had higher rates of photodamage as reflected in both decreased maximal PSII quantum efficiency and loss of t capacity to perform charge separation in PSII (Joliot and Delosme, 1974; Joliot et al., 1977; Bailleul et al., 2010) and significantly decreased levels of D1 protein (Figure 3C–E). Thus, decreasing ATP synthase activity led to increased PSII photodamage rather than decreased rates of repair, in contradiction with strict control of photoinhibition by repair (Takahashi et al., 2007).

Figure 3

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Photodamage can be induced in vitro by excitation of PSII centers with previously reduced primary quinone acceptor (Q_A) leading to the formation of the doubly-reduced Q_AH₂ state (Keren and Krieger-Liszkay, 2011). This situation might be expected if a high pmf slowed electron transfer through the b₆f complex, resulting in the accumulation of electrons on PSII acceptors. When data at a range of light intensities are compared, the relationship between the Q_A redox state (q_L) (Kramer et al., 2004) and q_I is statistically significant (ANOVA, p=2 × 10⁻¹⁶) (Figure 4). However, both the light intensity (p=3 × 10⁻⁵) and q_E (p=7 × 10⁻³) are significant interacting factors. At any one light intensity, q_L was relatively stable, whereas q_I was strongly dependent upon the mutant background and underlining pmf changes, indicating that while Q_A reduction may be a contributing factor, it cannot by itself explain the observed extents of photoinhibition in the minira lines. On the other hand, this dependence is also consistent with an alternative model, proposed below, that involves effects on PSII recombination rates.

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Changes in chlorophyll content have also been correlated with increases in PSII photoinhibition (Pätsikkä et al., 2002), likely due to less light being absorbed at the leaf surface and the subsequent increased light penetration into the leaf reaching more PSII centers. While the leaf chlorophyll content was altered in the minira mutants from wild type levels (Supplementary file 3), the leaf chlorophyll content does not fall below where (Pätsikkä et al., 2002) observed correlations between a lack of chlorophyll content and photoinhibition.

We next hypothesized that the most probable explanation for the increased photoinhibition is direct sensitization of PSII to photodamage by pmf. In the 'acid-damage' model (Kramer et al., 1999), it was proposed that excessive lumen acidification at high ΔpH (i.e. low lumen pH) could sensitize PSII centers to photodamage. To test this possibility, we compared the rates of photoinhibition with the extents of the ΔpH and Δψ components of the pmf by measuring the relaxation kinetics of the ECS signal (Takizawa et al., 2007). Surprisingly, increased extents of photoinhibition in low g_H⁺ lines were not correlated with ΔpH, but were with Δψ (Figure 5), contrary to what was expected with the acid-damage model. Consistent with this result, the rates of P₇₀₀⁺ reduction, which reflect the lumen pH-sensitive turnover of the cytochrome b₆f complex remained in a range consistent with a lumen pH above or near the pK_a for b₆f down-regulation, i.e. above about 6.0 (Figure 5—figure supplement 1).

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These results are consistent with an increase in the partitioning of pmf into Δψ as the total pmf increased (Figure 5—figure supplement 2), as have been observed previously (Avenson et al., 2004), and are likely caused by alterations in ion movements across both the thylakoid and chloroplast envelope membranes (Avenson et al., 2004; Armbruster et al., 2014; Kunz et al., 2014). Our results suggest that pmf partitioning acts to maintain a permissible lumen pH during large pmf increases.

We observed similar results throughout the range of minira mutants with low g_H⁺ as well as with tobacco γ-subunit antisense plants (Rott et al., 2011) with reduced ATP synthase complexes, finding that a large increase in total pmf was accompanied by increased partitioning of pmf into Δψ (Figure 5—figure supplement 3), and increased rates of photodamage in the presence and absence of lincomycin (Figure 5—figure supplement 3). These results suggest that low g_H⁺ or high pmf-related PSII damage is a more general phenomenon, which is related to excess Δψ and likely to be independent of such factors as changes in the protein or supercomplex content (see also discussion in Rott et al., 2011).

As described in Figure 8, we hypothesize that high Δψ accelerates photodamage by favoring recombination reactions within PSII (de Grooth and van Gorkom, 1981; Diner and Joliot, 1976; Satoh and Katoh, 1983; Rappaport et al., 1999) that lead to the formation of chlorophyll triplet states that in turn generate ¹O₂ (Johnson et al., 1995).

To test this model, we studied the relationship between elevated Δψ and PSII charge recombination in isolated spinach thylakoids (Figure 6), which unlike Arabidopsis can be isolated as highly intact chloroplasts and tightly coupled thylakoids. Consistent with our model, we found that elevated Δψ, produced by artificial decyl-ubiquinol mediated cyclic electron flow through PSI, increased the rate of charge recombination from the S₂Q_A⁻ state compared to samples treated with gramicidin to dissipate Δψ (Figure 6A). This recombination reaction can also occur in vivo during normal turnover when the S₂Q_A⁻ state is formed given that the equilibrium constant for sharing of electrons between Q_A and Q_B is small (Robinson and Crofts, 1983). The extent of Δψ generated in these experiments was similar to that observed in minira leaves under photoinhibitory conditions (Figure 6B), suggesting similar increases in recombination rates should occur in vivo.

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We next tested the predicted connection between elevated Δψ and singlet oxygen (¹O₂) generation (Figure 7D). In wild type leaves, moderate illumination (30 min of 300 μmol photons m⁻²s⁻¹) resulted in no detectable light dependent changes in ¹O₂ (Figure 7D) using Singlet Oxygen Sensor Green (SOSG) dye fluorescence (Dall'Osto et al., 2012; Ramel et al., 2013; Shumbe et al., 2016). In contrast, the low g_H⁺minira 3–1 line showed a strong induction of SOSG fluorescence within the first 10 min of illumination, which saturated by about 30 min. Infiltration of leaves with valinomycin, a potassium ionophore that decreases the Δψ component of pmf (Satoh and Katoh, 1983), partially inhibited the rise in in SOSG fluorescence (Figure 7—figure supplement 1). While care must be taken making quantitative estimates of ¹O₂ from SOSG fluorescence (Koh and Fluhr, 2016), within the limits of these experiments the Δψ-dependence of the SOSG fluorescence increases strongly support a role for Δψ-induced ¹O₂ production from photosynthesis.

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Singlet oxygen is produced by the interaction of O₂ with triplet excited states of pigments, most likely from the ³P₆₈₀ chlorophyll within the PSII reaction centers generated by recombination reactions (Krieger-Liszkay et al., 2008; Telfer, 2014), as further discussed below. It is unlikely that such triplets could be generated in the bulk light harvesting pigments because high NPQ in the mutants will decrease the lifetime of antenna excited states, and chlorophyll triplets generated in light harvesting complexes are efficiently quenched by carotenoids (Telfer et al., 2008). We thus propose that elevated Δψ induces ¹O₂ production by accelerating PSII recombination in low g_H⁺ mutants when pmf is large. Keren et al. (Keren et al., 1997) suggested that recombination-induced triplet formation could explain the photoinhibitory effects of very low light, when PSII charge recombination is preferred over the forward electron transfer reactions. Our work implies that this type of phenomenon is greatly accelerated by high Δψ, potentially making it relevant to photosynthesis under growth light conditions.

The effects of high Δψ would be expected to alter the rates of PSII recombination through P⁺Pheo⁻ (where P⁺ is the oxidized primary chlorophyll donor and Pheo the D1 subunit pheophytin) in an increasing dependence upon the fraction of Q_A⁻, consistent with the observed correlation between q_I and q_L as the light intensity increased (Figure 4). To test this relationship, we estimated the recombination rates from S₂Q_A⁻ through the P⁺Pheo⁻ pathway, considering Q_A redox state (estimated by 1-q_L) and the expected impact of Δψ on the equilibrium constant for sharing electrons between Pheo and Q_A (based on ECS_ss and the position of Q_A in the structure relative to the membrane dielectric). The basis of this estimate is described in more detail in Materials and methods. As shown in Figure 5C, we see a positive correlation between q_I and estimated recombination through P⁺Pheo⁻ over both mutant variants and light intensities, indicating that the combined effects of Δψ and Q_A redox state can explain a large fraction of the observed extents of photoinhibition. While it is likely that multiple mechanisms of photoinhibition exist, which may also explain some of the q_I variation, overall the greatest impact upon photoinhibition under these conditions can be explained by Δψ-mediated changes in PSII electron recombination.

The obvious questions are: does Δψ-induced photoinhibition occur in wild type plants and if so under what conditions? Photosynthesis is known to be particularly sensitive to rapid fluctuations in light intensity (Kulheim et al., 2002), at least some of this sensitivity is associated with photoinhibition of PSI, especially in cyanobacteria (Allahverdiyeva et al., 2015). However, such fluctuations should also result in large transient changes in Δψ, as the thylakoid membrane has a low electrical capacitance and low permeability to counter-ions, while the lumen and stroma have high proton buffering capacity (Cruz et al., 2001). The slow onset of q_E and other down-regulatory processes in photosynthesis should exacerbate these effects and allow for large fluxes of electrons when light levels are rapidly increased, resulting in large, transient Δψ 'pulses'.

We therefore hypothesized that Δψ-induced photodamage may contribute to the increased photodamage seen under fluctuating light. To test this possibility, we measured light-driven pmf (Δψ and ΔpH) and ¹O₂ generation in wild type Arabidopsis under fluctuating light conditions (Figure 7A, replicating the first 8 hr of Figure 2A day three). The initial dark-to-light transition resulted in an immediate, transient 'spike'in Δψ, even though the light intensity was low (39 μmol photons m⁻²s⁻¹, Figure 7B). The spike was transient, and decreased to steady-state levels within tens of seconds, as shown in the blue trace in Figure 7B. Spikes in Δψ of similar amplitudes were also seen upon each increase in light at the onset of each fluctuation, though the recovery kinetics tended to be more rapid than those seen at the first dark-light transient. An example of these transients, taken at the transition between 167 and 333 μmol photons m⁻² s⁻¹ is shown in (Figure 7B). A more complete set of transient kinetics, over the entire course of the experiment is presented in Figure 7—figure supplement 2. The amplitudes of these Δψ transients were similar to or larger than those seen in the minira 3–1 line under constant 300 μmol photons m⁻² s⁻¹ light, which also induced ¹O₂ generation (see red horizontal bars in Figure 7B). These spikes reflect increases in Δψ above that already produced by steady-state photosynthesis, so the true extent of Δψ is likely considerably higher, and based on estimates of the calibration of the ECS signal likely range between 150–260 mV (see Figure 7—figure supplement 2).

By contrast, wild type plants under constant 300 μmol photons m⁻² s⁻¹ produced much lower Δψ extents (Figure 7B green bars) and had no detectible ¹O₂ generation (Figure 7D). These results suggest that even low amplitude light fluctuations are capable of inducing Δψ large enough to produce ¹O₂ and PSII photodamage. Supporting this interpretation, wild type leaves under these fluctuating light conditions produced substantial amounts of ¹O₂ during the first hour of fluctuating conditions compared to higher intensity, but constant illumination (Figure 7D).

The above results lead us to conclude that fluctuating light likely induces strong effects through Δψ-induced recombination reactions in PSII (see below for considerations of potential contributions to PSI). In addition to direct damage to the enzymes of photosynthesis, ¹O₂ can also activate plant light stress-related gene expression and programmed cell death (Zhang et al., 2014), suggesting a possible physiological linkage between pmf-enhanced recombination and plant regulatory pathways that may result in long-term acclimation to fluctuating light.

At a mechanistic level, we propose that imposing a large Δψ across the PSII complex will decrease the standard free energy gap between the vectorial electron transfer steps and thus the back-reaction will be accelerated in competition with forward (energy-storing) reactions (de Grooth and van Gorkom, 1981; Vos et al., 1991; Johnson et al., 1995) (Figure 8). It is known that decreasing the energy gap between Pheo and Q_A favors recombination from P⁺Q_A⁻ via Pheo⁻ (de Grooth and van Gorkom, 1981; Johnson et al., 1995) rather than directly from Q_A⁻ to P⁺ (Johnson et al., 1995; Krieger-Liszkay and Rutherford, 1998; Rutherford et al., 2012). The observed increased recombination rates in vitro (Figure 6) when Δψ is present, combined with ¹O₂ production under high Δψ conditions in vivo suggest that the fraction of electrons recombining to P⁺ through a Pheo⁻ intermediate is greatly increased, as the recombination pathway via the Pheo⁻ has a high yield for formation of the triplet state of P (³P) that in turn can interact with O₂ to produce ¹O₂ (Johnson et al., 1995; Keren and Krieger-Liszkay, 2011; Rutherford et al., 2012).

Figure 8

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This proposed mechanism of Δψ-mediated photoinhibition has broad implications for the energy limitations of photosynthesis. Although the two components of pmf are energetically equivalent for driving ATP synthesis (Hangarter and Good, 1982), they have distinct effects on the regulation of photosynthesis (Kramer et al., 1999; 2004; Finazzi et al., 2015). It has thus been proposed that the partitioning of pmf into Δψ and ΔpH is regulated to maintain a balance between efficient energy storage and regulation of light capture (Cruz et al., 2005). We demonstrate here another important constraint on this balance: the avoidance of photodamage caused by recombination reactions in PSII, and this may explain the need for complex ion balancing systems in chloroplasts (Cruz et al., 2001; Armbruster et al., 2014; Kunz et al., 2014).

The effect of Δψ on recombination and ¹O₂ production may, at least in part, explain the severe effects of fluctuating light on photosynthesis, and thus could constitute a significant limitation to photosynthetic productivity. In the absence of a large Δψ, the energy gap between the P⁺Pheo⁻ and P⁺Q_A⁻ appears to be sufficient to keep detrimental recombination to a manageable level when the usual regulatory mechanisms are functional (Rutherford et al., 2012). During photosynthesis, and especially under fluctuating light, though, the energy gaps between the photo-generated radical pairs vary dynamically under the influence of the pmf, so that high Δψ renders the charge-separated states in the photosystems considerably less stable. The Δψ is also expected to influence the trap depth (i.e. the energy level between P^* and the first radical pair(s), the most relevant probably being P⁺Pheo⁻) and this could potentially affect the quantum yield of charge separation and the yield of radiative recombination (luminescence).

Transthylakoid electric fields also influence recombination reactions in PSI reaction centers (van Gorkom, 1996). It is thought that P₇₀₀ triplet formation is minimized in PSI by the presence of the higher potential quinone in PsaA, making this side of the reaction center the safe charge recombination pathway (Rutherford et al., 2012). In light of the present findings, it is worth considering whether a transiently large Δψ could make this protective mechanism less efficient, though this question has yet to be addressed experimentally.

Over evolutionary time scales, the Δψ-effect may have constrained other bioenergetics features of photosynthesis. It has long been known that oxygenic photosynthesis is limited, in most organisms, to wavelength ranges shorter than about 700 nm (the 'red limit'), resulting in the loss of a large fraction of the light energy hitting the plant (Blankenship et al., 2011). (Gust et al., 2008) proposed that the red limit may be the result of certain limitations imposed in part by key biochemical properties of life (e.g. the properties of energy storage molecules NAD(P)H and ATP, the use of certain biochemical pathways, etc.) that evolved before the advent of photosynthesis. Milo (Milo, 2009) came to a different conclusion based on estimates of the theoretical wavelength dependence of energy conversion efficiency for plant photosynthesis, based on the Shockley and Queisser equation (Shockley and Queisser, 1961). The maximum efficiency was about 700 nm, similar to the red limit of oxygenic photosynthesis, suggesting that evolution has selected for photosynthetic energetics based on this fundamental limit, rather than any biological imperative. However, the predicted wavelength-dependence of the energy efficiency is very broad, with only about a 15% decrease from the peak at wavelengths out to 800 nm, far beyond the red limit, even in organisms with red-shifted reaction centers. Marosvolgyi and van Gorkom (Marosvolgyi and van Gorkom, 2010) drew a similar conclusion but with additional restraints and suggested a narrower maximum more closely overlapping with the red absorption of chlorophyll a.

Rutherford et al. (Rutherford et al., 2012) took a different view based on an analysis of the bioenergetics of reaction centers. They noted that PSII was in a uniquely difficult situation in energy terms: (1) it does multi-electron chemistry (at both sides of the reaction center) and so cannot prevent back reactions by kinetic control, (2) it has a very energy demanding reaction to do: water oxidation and quinone reduction with a ∆E ~920 meV in functional conditions, and requires a significant over-potential not just for attaining a high quantum yield of photochemistry but also for achieving water oxidation and quinol release, and (3) its chlorophyll cation chemistry is uniquely oxidizing and thus it cannot use carotenoids to protect itself from chlorophyll triplet formation at the heart of the reaction center. This situation means that unlike other type-II reaction centers, PSII is unable to prevent electrons from the bound semiquinones getting back to the Pheo and then recombining with P⁺ and forming ³P.

This lack of energy 'headroom' was seen not only as a major factor in PS II’s susceptibility to photodamage but also as a reason why oxygenic photosynthesis is pinned to chlorophyll a photochemistry at around 680 nm as the red limit. The existence of efficient oxygenic photosynthesis at longer wavelengths seemed to question that view (Miyashita et al., 1996; Kuhl et al., 2005; Gan et al., 2014; Behrendt et al., 2015). However, it was pointed out that these species seem to exist in very stable environments that have very little variation in light conditions (Cotton et al., 2015). Under such a narrow range of illumination conditions it is not unreasonable that less energy 'headroom' is required (Cotton et al., 2015).

Clearly, these specific energy limitations of PSII will be exacerbated by spikes in the Δψ reported here. Indeed, it seems reasonable to suggest that the existing 'energy headroom' postulated in normal chlorophyll a-containing PSII, while too small to avoid photodamage altogether, exists quite specifically to mitigate the extra photodamage from back-reactions enhanced by spikes in Δψ due to variable light intensities. In this way, the extent of the variable light-induced Δψ may be considered to contribute to the position of the red limit of oxygenic photosynthesis.

The need to prevent Δψ-induced recombination may also have guided the evolution of other photosynthetic components. Recent mechanistic models of the ATP synthase suggest that the ratio of protons passed through the ATP synthase per ATP synthesized depends on the number of subunits in the c-ring (Silverstein, 2014). The chloroplasts of green plants and algae thus far studied possess ATP synthase complexes with larger c-ring stoichiometries than their mitochondrial and bacterial homologues (Seelert et al., 2000), imposing higher fluxes of protons to generate ATP and necessitating the engagement of additional bioenergetic processes, including cyclic electron flow, to make up the ATP deficit needed to sustain photosynthesis (Kramer et al., 2004; Avenson et al., 2005). While this increased H⁺ demand is often viewed as a bioenergetic limitation to photosynthetic electron and proton transfer, a high H⁺/ATP ratio decreases the pmf needed to maintain a given ATP free energy state (ΔG_ATP), thus allowing photosynthesis to operate at a decreased steady-state pmf. In this context, the deleterious electron recombination effects of a high pmf may have favored the evolution of ATP synthase complexes with high H⁺/ATP ratios in chloroplasts.

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Author details

1. Geoffry A Davis

   1. Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, United States
   2. Graduate Program of Cell and Molecular Biology, Michigan State University, East Lansing, United States

   Contribution

   GAD, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

2. Atsuko Kanazawa

   1. Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, United States
   2. Department of Chemistry, Michigan State University, East Lansing, United States

   Contribution

   AK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

3. Mark Aurel Schöttler

   Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany

   Contribution

   MAS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

4. Kaori Kohzuma

   Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, United States

   Present address

   Graduate School of Life Sciences, Tohoku University, Sendai, Japan

   Contribution

   KK, Conception and design, Drafting or revising the article

   Competing interests

   No competing interests declared.

5. John E Froehlich

   Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, United States

   Contribution

   JEF, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

6. A William Rutherford

   Department of Life Sciences, Imperial College London, London, United Kingdom

   Contribution

   AWR, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

7. Mio Satoh-Cruz

   Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, United States

   Contribution

   MS-C, Conception and design, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

8. Deepika Minhas

   Department of Horticulture, Washington State University, Pullman, United States

   Contribution

   DM, Conception and design, Acquisition of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

9. Stefanie Tietz

   Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, United States

   Contribution

   ST, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

10. Amit Dhingra

    Department of Horticulture, Washington State University, Pullman, United States

    Contribution

    AD, Conception and design, Drafting or revising the article

    Competing interests

    No competing interests declared.

11. David M Kramer

    1. Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, United States
    2. Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, United States

    Contribution

    DMK, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    kramerd8@msu.edu

    Competing interests

    DMK: Founder and stock holder in Phenometrics, Inc. and a founder of the PhotosynQ.org project, these entities manufacture and distribute instrumentation for plant and algal phenotyping. However, neither of these technologies are used in the current work.

    "This ORCID iD identifies the author of this article:" 0000-0003-2181-6888

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