H⁺ Transport by K⁺ EXCHANGE ANTIPORTER3 Promotes Photosynthesis and Growth in Chloroplast ATP Synthase Mutants^,

Proton antiport across the thylakoid membrane up-regulates photosynthesis and growth in an Arabidopsis mutant with low chloroplast ATP synthase levels and high proton motive force.

Abstract

The composition of the thylakoid proton motive force (pmf) is regulated by thylakoid ion transport. Passive ion channels in the thylakoid membrane dissipate the membrane potential (Δψ) component to allow for a higher fraction of pmf stored as a proton concentration gradient (ΔpH). K⁺/H⁺ antiport across the thylakoid membrane via K+ EXCHANGE ANTIPORTER3 (KEA3) instead reduces the ΔpH fraction of the pmf. Thereby, KEA3 decreases nonphotochemical quenching (NPQ), thus allowing for higher light use efficiency, which is particularly important during transitions from high to low light. Here, we show that in the background of the Arabidopsis (Arabidopsis thaliana) chloroplast (cp)ATP synthase assembly mutant cgl160, with decreased cpATP synthase activity and increased pmf amplitude, KEA3 plays an important role for photosynthesis and plant growth under steady-state conditions. By comparing cgl160 single with cgl160 kea3 double mutants, we demonstrate that in the cgl160 background loss of KEA3 causes a strong growth penalty. This is due to a reduced photosynthetic capacity of cgl160 kea3 mutants, as these plants have a lower lumenal pH than cgl160 mutants, and thus show substantially increased pH-dependent NPQ and decreased electron transport through the cytochrome b₆f complex. Overexpression of KEA3 in the cgl160 background reduces pH-dependent NPQ and increases photosystem II efficiency. Taken together, our data provide evidence that under conditions where cpATP synthase activity is low, a KEA3-dependent reduction of ΔpH benefits photosynthesis and growth.

In plants, photosynthesis occurs in specialized organelles, the chloroplasts. Light energy is collected by light harvesting complexes in the thylakoid membrane, and the resulting excitation energy is transferred to two photosystems, PSI and PSII, where it induces charge separations. These drive electron transfer reactions from water to NADP⁺ along the linear electron transport chain, including plastoquinone (PQ), the cytochrome b₆f (Cyt b₆f) complex, and the soluble, lumen-localized electron carrier plastocyanin (PC). Water oxidation at PSII releases protons into the thylakoid lumen. Additionally, the electron transfer reactions from PSII to Cyt b₆f are coupled to the translocation of protons from the stroma into the thylakoid lumen via the PQ pool. The accumulation of protons in the thylakoid lumen results in a charge difference, leading to a membrane potential (ΔΨ), and a proton concentration gradient (ΔpH) across the thylakoid membrane (reviewed by Armbruster et al., 2017). Both ΔΨ and ΔpH contribute to the proton motive force (pmf) that drives protons through the chloroplast (cp)ATP synthase, providing the energy required for ATP synthesis (reviewed by Allen, 2002).

While both components of the pmf are energetically equivalent, the ΔpH plays an additional role in the feedback regulation of photosynthetic light capture and electron transport in response to light stress or other environmental factors that influence excitation pressure. With rising excitation levels and electron transport rates, downstream carbon fixation and other metabolic processes become limiting, which results in an increased acidification of the thylakoid lumen. Above certain thresholds, the proton concentration induces two processes that lead to the feedback down-regulation of light harvesting and electron transport, respectively. First, protonation of the thylakoid PsbS protein at the lumenal side occurs below a lumen pH of 6.8 (Takizawa et al., 2007). This in turn leads to the rearrangement of PSII supercomplexes and an increase in the thermal dissipation of absorbed light energy (Li et al., 2004; Correa-Galvis et al., 2016; Sacharz et al., 2017). This so-called energy-dependent quenching (qE) component of nonphotochemical quenching (NPQ) is further sustained by zeaxanthin production via the low pH-activated violaxanthin de-epoxidase (VDE) localized in the lumen of land plants (Hager, 1969; Niyogi et al., 1998). Second, when lumen pH decreases below 6.5, plastoquinol (PQH₂) oxidation at Cyt b₆f is slowed down, a process referred to as “photosynthetic control” (Stiehl and Witt, 1969; Tikhonov et al., 1981; Nishio and Whitmarsh, 1993). At a lumen pH of 5.7, Cyt b₆f turnover is decreased 5- to 10-fold compared with its maximum rate (Nishio and Whitmarsh, 1993; Takizawa et al., 2007). This is because PQH₂ oxidation, which releases protons into the lumen, becomes thermodynamically less favorable. Additionally, amino acid residues at the lumenal side of Cyt b₆f, which receive the protons from PQH₂, are partially protonated at low lumenal pH (reviewed by Tikhonov, 2014).

The proton conductivity of the cpATP synthase is reduced in response to environmental factors that limit downstream metabolism, such as low CO₂ and drought stress (Kanazawa and Kramer, 2002; Kohzuma et al., 2009). Low conductivity results in the build-up of high pmf and thereby activates feedback down-regulation of photosynthesis. For wild watermelon (Citrullus lanatus) plants under drought stress conditions, it has been shown that the decreased cpATP synthase activity correlates with a strong down-regulation of its content and that a large fraction of the resulting high pmf is stored as ΔΨ (Kohzuma et al., 2009). The same has been reported for tobacco (Nicotiana benthamiana) and Arabidopsis (Arabidopsis thaliana) mutants with constitutively lower cpATP synthase conductivity, due to decreased levels or activities, which have high pmf and a large fraction of it stored as ΔΨ (Rott et al., 2011; Fristedt et al., 2015; Davis et al., 2016). Whether and how this adjusted pmf composition affects photosynthesis and growth remains to be determined.

Thylakoid ion transport proteins influence the composition of the pmf that is stored as ΔΨ and ΔpH and thus the capacity of the pmf to regulate light harvesting and electron transport. Passive ion channels allow for the dissipation of the ΔΨ component of the pmf. Because the proton buffering capacity of the lumen is high, ΔΨ dissipation by these ion channels has been proposed to be required for storage of pmf as ΔpH (Kramer et al., 2003). In early experiments on isolated thylakoids, it was shown that proton translocation into the lumen is accompanied by counter-ion fluxes (Hind et al., 1974). These mainly consist of Cl⁻, Mg²⁺, and potentially K⁺. Only the VOLTAGE-DEPENDENT CL− CHANNEL1 (VCCN1) protein localized in the thylakoids has been identified so far (Duan et al., 2016; Herdean et al., 2016). Lack of this protein in the vccn1 mutant causes increased pmf, a larger fraction of pmf stored as ΔΨ, and slower induction of NPQ. The opposite behavior was observed when VCCN1 was overexpressed (Herdean et al., 2016). These data suggest that Cl⁻ plays a role in dissipating the membrane potential component of the pmf via VCCN1 in order to allow for a faster induction of thermal dissipation.

While counter ion movement is likely not restricted to Cl⁻, the molecular nature of other counter-ion channels remains unknown: for example, the TWO-PORE K⁺ CHANNEL 3 (TPK3) was found to localize to the thylakoid membrane and have a function comparable with VCCN1 in pmf modulation by mediating K⁺ flux (Carraretto et al., 2013). However, a recent study (Höhner et al., 2019) supports earlier localization experiments, which showed TPK3 to exclusively reside in the tonoplast (Voelker et al., 2006; Dunkel et al., 2008). Additionally, a line with a transfer-DNA insertion in the TPK3 locus did not show any differences in pmf composition and NPQ induction, strongly suggesting that TPK3 does not mediate transthylakoid K⁺ flux (Höhner et al., 2019). Thus, the molecular nature of other ΔΨ-dissipating ion channels besides VCCN1 remains to be elucidated. Recently, it was shown that high ΔΨ favors charge recombination in PSII (Davis et al., 2016). This suggests that, besides the induction of feedback regulation, thylakoid ΔΨ-dissipating channels have an important function for the avoidance of PSII damage.

The fraction of pmf stored as ΔΨ can be increased by another thylakoid ion transport mechanism, via the K⁺ EXCHANGE ANTIPORTER 3 (KEA3; Armbruster et al., 2014; Kunz et al., 2014; Armbruster et al., 2016; Dukic et al., 2019; Wang and Shikanai, 2019). KEA3 exports protons from the lumen in exchange for another cation, most likely K⁺ (Armbruster et al., 2014; Tsujii et al., 2019) and thereby decreases the ΔpH fraction of the pmf. Loss of KEA3 strongly delays NPQ relaxation and thus negatively affects CO₂ assimilation after a transition from high to low light (Armbruster et al., 2014). Plants overexpressing KEA3 instead exhibit faster NPQ relaxation (Armbruster et al., 2016). Additionally, kea3 mutants were reported to have higher NPQ in steady state at low and medium light intensities, whereas KEA3 overexpressors had lower NPQ (Wang and Shikanai, 2019). In high light, there was little difference in pmf composition and NPQ between plants with different KEA3 levels (Armbruster et al., 2014; Armbruster et al., 2016; Wang and Shikanai, 2019). The overexpression of a KEA3 version lacking its C terminus, which contains a predicted regulatory domain, however, resulted in plants with decreased NPQ in high light. This finding strongly suggested that KEA3 activity is inhibited in high light via a mechanism involving its C terminus (Armbruster et al., 2016). Upon transition from high to low light, KEA3 becomes readily activated as seen by the clear effect that different KEA3 levels have on NPQ relaxation. The identification of KEA3 as an important factor for rapid NPQ relaxation suggested that cpATP synthase activity must be insufficient to quickly down-regulate the lumenal proton concentration and thus NPQ during a high to low light transition (Armbruster et al., 2014). Here, activation of KEA3 and thereby increased partitioning of pmf in ΔΨ is required for optimal CO₂ fixation. In the current work, we set out to determine the stoichiometry of KEA3 and cpATP synthase in the thylakoid membrane in Arabidopsis. In addition, we altered KEA3 and cpATP synthase levels by knocking out or overexpressing KEA3 in the conserved only in the green lineage160 (cgl160) mutant, which lacks a cpATP synthase assembly factor and thus accumulates only 20% of wild-type cpATP synthase (Rühle et al., 2014; Fristedt et al., 2015). This allowed us to address whether the reduced ΔpH component of the pmf previously reported for multiple cpATP synthase limited mutants (Rott et al., 2011; Fristedt et al., 2015; Davis et al., 2016) is KEA3-dependent and beneficial for their photosynthesis and growth.

RESULTS

KEA3 Is Much Less Abundant in Thylakoid Membranes than the ATP Synthase

While photosynthetic electron transport reactions generate pmf by depositing protons in the lumen, the majority of protons leave the lumen via the cpATP synthase (Fig. 1A). Both the protein complexes belonging to the pmf-generating electron transport chain, and the pmf-utilizing cpATP synthase are highly abundant and make up a large fraction of the protein content in the thylakoid membrane (Kirchhoff et al., 2002). Proton export from the lumen via KEA3 markedly contributes to increases in the lumenal pH right after high to low light transitions and in steady state at low and medium light intensities (Armbruster et al., 2014; Armbruster et al., 2016; Wang and Shikanai, 2019). KEA3 probably replaces protons with another cation, likely K⁺, and thereby decreases the ΔpH component of the pmf (see scheme in Fig. 1A).

Figure 1.

KEA3 occurs substoichiometrically to the cpATP synthase in the thylakoid membrane. A, Scheme depicting the thylakoid membrane with the main proton transport pathways (i.e. the electron transport chain and the ATP synthase, as well as KEA3). B and C, Representative blots of KEA3 (B) and AtpF (C) quantifications obtained by titrating known amounts of purified recombinant proteins, i.e. the C terminus of KEA3 (which binds the specific epitope antibody, Armbruster et al., 2014) and full-length AtpF, against known amounts of thylakoid membranes (quantified by Chl content). Values derived from the quantifications are given below the blots as average of n = 5 ± sd. D, Ratio of spectral counts identified by MS analysis for the CF_o cpATP b/b` subunits and the CF₁ cpATP synthase subcomplex with KEA3. Average of n = 5 ± sd is displayed.

To obtain information about the stoichiometric relationship of the proton-transporting mechanisms mediated by the cpATP synthase and KEA3, we titrated thylakoid membranes from the wild type against heterologously expressed and purified KEA3 soluble C terminus and ATP synthase CF_o subunit b (AtpF) with C-terminal His tags (Fig. 1, B and C). We chose AtpF as a membrane integral subunit (b-subunit of CF_o) of the thylakoid membrane, unlikely to be lost during thylakoid isolation. From the quantifications obtained by protein blot analyses, we calculated an average abundance in thylakoid membranes of about 4.15 µmol/mol chlorophyll (Chl) for KEA3 (Fig. 1B), and 413 µmol/mol Chl for AtpF (Fig. 1C). This makes about 100 cpATP synthase complexes per KEA3 monomer. In an orthogonal approach, thylakoid membranes from the wild type were subjected to mass spectrometry (MS) analysis and spectral count ratios were calculated for each replicate (Fig. 1D). By this method, we obtained an average CF_o-ATP synthase subcomplex to KEA3 ratio of 120 to 1 and a CF₁-ATP synthase subcomplex to KEA3 ratio of 60:1. The lower amount of CF₁ as compared with CF_o suggests that some of this membrane associated subcomplex was lost during thylakoid isolation. Together, these analyses demonstrate that KEA3 occurs substoichiometrically to the photosynthetic complexes in the thylakoid membrane and that the cpATP synthase is about 100-fold more abundant than KEA3.

The cgl160 Mutant Shows Decreased Membrane Conductivity and ATP Content, But a Strong Increase in Light-Induced pmf

We set out to analyze the effects that loss of KEA3 has on photosynthesis and growth in a mutant with low cpATP synthase content. Here, we chose the cgl160 mutant, which accumulates 20% of wild-type cpATP synthase levels due to a deficiency in cpATP synthase assembly (Rühle et al., 2014; Fristedt et al., 2015). The proton conductivity of the thylakoid membrane (g_H⁺), which serves as a proxy for the capacity of the cpATP synthase for proton efflux from the lumen, and the ECS_t parameter, which is a measure of the light induced total pmf, had been determined by monitoring the decay of the electrochromic shift (ECS) during a short dark interval (Fristedt et al., 2015). This analysis revealed that cgl160 plants have slightly increased light-induced total pmf and decreased g_H⁺, compared with the corresponding wild type (Col-0). However, the previous experiments had only been performed at saturating light intensities (Fristedt et al., 2015).

To understand how different light intensities affect thylakoid proton conductivity and pmf in the cgl160 mutant, we performed ECS measurements of the wild type and the two previously published cgl160 mutants at different actinic light intensities. Plants were grown in a 16-h light/8-h dark photoperiod at 150 µmol photons m⁻² s⁻¹ and showed the previously reported reduction in plant size (Fig. 2A; Rühle et al., 2014; Fristedt et al., 2015). Analysis of the ECS decay kinetics revealed that both cgl160 mutants had a significantly lower g_H⁺ at growth light intensity, which was reduced by 12% to 14% compared with Col-0 (Fig. 2B). The decrease in g_H⁺ as compared with Col-0 was stronger when actinic light intensities were increased (Fig. 2B) and was accompanied by a strong increase in light-induced pmf as expressed by the ECS_t parameter (Fig. 2C). Under actinic light intensities resembling growth light, ECS_t was increased 2.5-fold in both cgl160 mutants compared with the wild type. Particularly, under intermediate light intensities (150-300 µmol photons m⁻² s⁻¹), ECS_t of the cgl160 mutants was more than double the size of Col-0 (Fig. 2C). While in Col-0 light-induced ECS_t increased with actinic light intensities up to 1000 µmol photons m⁻² s⁻¹, ECS_t in cgl160 reached its peak at 600 µmol photons m⁻² s⁻¹. Additionally, we determined whole leaf ATP content in Col-0 and cgl160 plants. In this analysis, both cgl160 lines showed reduced levels of ATP as compared with Col-0 (Fig. 2D; Supplemental Table S1). Taken together, our analyses demonstrate that cgl160 mutants have lower ATP synthase conductivity at growth light. This results in higher pmf and lower ATP levels as compared with the wild type.

Figure 2.

The low cpATP synthase mutant cgl160 has decreased ATP synthase activity, lower ATP levels, and high pmf. A, Picture of 5-week-old Col-0, cgl160-1, and cgl160-2. B and C, Membrane proton conductivity (g_H⁺) as proxy for ATP synthase activity (B) and light-induced thylakoid pmf estimated by ECS_t (C) were calculated from changes in the ECS signal during a short dark interval following 10 min of actinic light illumination at the indicated light intensities. Averages of n = 6 are shown and error bars indicate ± se. ANOVA and a Holm-Sidak pairwise multiple comparison describe g_H⁺ of both mutants to be significantly lower than that of Col-0 (P < 0.05), except where stated ns for nonsignificant. For ECS_t, the tests describe mutants to have significantly higher values at all light intensities as compared with Col-0. D, ATP levels of Col-0 and cgl160-1 or cgl160-2 were determined from leaf discs in two independent experiments as described in Supplemental Table S1, and ratios between Col-0, cgl160-1 or cgl160-2 were calculated.

In the cgl160 Background, KEA3 Is Important for Plant Growth by Increasing the Capacity for Photosynthesis

In order to analyze the role of KEA3 in a mutant with low cpATP synthase levels, we generated multiple cgl160 kea3 double mutants, cgl160-1 kea3-1, cgl160-1 kea3-2, and cgl160-2 kea3-1. Two previously published kea3 mutants (Armbruster et al., 2014; Kunz et al., 2014; Armbruster et al., 2016; Wang et al., 2017; Dukic et al., 2019; Wang and Shikanai, 2019) showed wild-type–like appearance under our standard growth condition of a 16-h light/8-h dark photoperiod with 150 µmol photons m⁻² s⁻¹ (Fig. 3A). In the cgl160 background, lack of KEA3 as in the three cgl160 kea3 double mutants, caused a much stronger growth defect than lack of CGL160 alone (Fig. 3A). While 5-week-old Col-0 and kea3 plants accumulated 12–22 mg of biomass and the cgl160 mutants accumulated 9–15 mg of biomass, the cgl160 kea3 double mutants showed a further reduction in growth capacity and accumulated only 5–11 mg of biomass (Fig. 3B).

Figure 3.

KEA3 optimizes growth in the cgl160 mutant by increasing the capacity for photosynthesis. A, Picture of 5-week-old Col-0, kea3-1, kea3-2, cgl160-1, cgl160-2, cgl160-1 kea3-1, cgl160-1 kea3-2, and cgl160-2 kea3-1 plants. B, Dry weight of 5-week-old plants represented as box plots, n = 13. C, Increases in leaf area during 5 weeks for Col-0, kea3-1, cgl160-1, and cgl160-1 kea3-1; averages of n = 6–8 are shown, and error bars indicate ± se. The inset shows daily growth rate ± sd (D) Chl a fluorescence was measured of dark acclimated plants at increasing actinic light intensities, and the electron transport rate through PSII (ETRII) was calculated; averages of n = 18 are displayed and error bars indicate ± se. E, Whole plant assimilation was determined in response to increasing light intensities and recalculated on a leaf area basis; averages of n = 7–13 plus error bars for ± se are shown.

To further characterize the growth of the mutants, we measured increases in leaf area over time for Col-0, kea3-1, cgl160-1, and the corresponding kea3-1 cgl160-1 double mutant and calculated the growth rate. Again, this analysis showed that the kea3 mutant grows similarly to the wild-type Col-0 (Fig. 3C). The growth rate of the cgl160 mutant was lower and after 5 weeks cgl160 mutants showed a reduction in leaf area by ∼30% as compared with Col-0 (Fig. 3C). The growth rate and leaf area of cgl160-1 kea3-1 double mutants were even further reduced with the leaf area only reaching ∼50% of that of the cgl160 mutants (Fig. 3C).

To characterize the photosynthetic performance of the plants, we measured Chl a fluorescence light response curves and calculated the capacity for linear electron transport. Again, this analysis showed that kea3 mutants behaved like Col-0 (Fig. 3D). As previously described, cgl160 mutants (Rühle et al., 2014; Fristedt et al., 2015) exhibited reduced capacity for linear electron transport. The electron transport rate (ETR) was even further decreased in the absence of KEA3 in both independent cgl160 kea3 mutants (Fig. 3D). Particularly, at light intensities >100 and <1000 µmol photons m⁻² s⁻¹, cgl160 kea3 double mutants displayed a much stronger reduction in ETR as compared with cgl160 alone. Light response curves of whole plant CO₂ assimilation were performed with Col-0, kea3-1, cgl160-1, and cgl160-1 kea3-1 and confirmed the reduced capacity of the cgl160 mutants for photosynthesis, which was even further decreased in the absence of KEA3 in the cgl160-1 kea3-1 mutant (Fig. 3E). Taken together, these data show that loss of KEA3 alone does not have a strong effect on growth and photosynthesis in the steady state, when plants are grown under low light conditions. cgl160 mutants, as previously reported, have a lower capacity for photosynthesis and growth (Rühle et al., 2014; Fristedt et al., 2015). Lack of KEA3 in the cgl160 background further decreases the capacity of these plants to perform steady-state photosynthesis and thus negatively affects growth. This shows that cgl160 plants require KEA3 for optimal photosynthesis and thereby biomass accumulation.

cgl160 Mutants Store a Large Fraction of pmf as ΔΨ in a KEA3-Dependent Manner

Proton antiport by KEA3 shifts the pmf composition toward higher ΔΨ and lower ΔpH, while not markedly altering the overall size of the light-induced pmf (Armbruster et al., 2014; Kunz et al., 2014; Dukic et al., 2019; Wang and Shikanai, 2019). Additionally, plants with a low cpATP synthase content and/or activity, including the cgl160 mutant, store a larger fraction of pmf as ΔΨ (Kohzuma et al., 2009; Rott et al., 2011; Fristedt et al., 2015; Davis et al., 2016). To analyze the size of light-induced pmf and its composition in ΔpH and ΔΨ of Col-0, kea3, cgl160, and cgl160 kea3 plants under our growth conditions, we monitored the ECS signal at growth light (150 µmol photons m⁻² s⁻¹) and during relaxation in a prolonged dark period (Fig. 4A; Sacksteder et al., 2000; Cruz et al., 2001). The ECS signal rapidly decays after transition to dark and the extent of this decay gives information about the total size of the light induced pmf (ECS_t). The ECS then recovers to a steady-state signal in the dark. The difference between the ECS signal in the light and steady state in darkness is referred to as ECS_ss and corresponds to the fraction of pmf stored as ΔΨ, whereas the difference between the minimum ECS signal and steady-state dark, which is referred to as ECS_inv, corresponds to the pmf fraction stored as ΔpH (Fig. 4A; Cruz et al., 2001). For Col-0 and kea3 plants at growth light, an ECS_t transmittance signal of around 1.5 * 10⁻³ was measured (Fig. 4B) to which ECS_ss and ECS_inv contributed equally (Fig. 4, A and B). The cgl160 and cgl160 kea3 mutants, at growth light, had much higher ECS_t values of around 4 * 10⁻³, with little difference between the two genotypes (Fig. 4, A and B). In the cgl160 mutant, only 32% of the ECS signal corresponded to ECS_inv, supporting the previous finding that cgl160 mutants store a larger fraction of their pmf as ΔΨ and less as ΔpH (Fristedt et al., 2015). Instead, the cgl160 kea3 double mutants had a significantly larger ECS_inv amplitude (i.e. ΔpH), making up about 45% of the ECS_t. This result strongly suggests that at least part of the high ΔΨ fraction of pmf in the cgl160 mutants is KEA3 dependent. cpATP synthase conductivity (g_H⁺) was not affected by loss of KEA3, only by loss of CGL160 (Fig. 4C). Additionally, we calculated the halftime (t_1/2) of the recovery of the ECS_inv phase. This parameter was recently shown to correlate negatively with KEA3 levels at light intensities where KEA3 is active (Wang and Shikanai, 2019). No differences were observed between Col-0, kea3, and cgl160; however, t_1/2 was slightly, but not significantly, increased in the double mutants (Supplemental Fig. S1A). Taken together, the ECS results demonstrate that cgl160 kea3 mutants, with their increased storage of pmf as ΔpH, have a much lower lumenal pH than cgl160 mutants, suggesting that this may cause increased feedback down-regulation of photosynthesis and thus a reduced capacity to photosynthesize and grow. In order to investigate this hypothesis in detail, we performed multiple analyses that monitor lumen-pH related characteristics of the photosynthetic electron transport chain (Fig. 4D). We performed these analyses by using actinic light that simulated the growth light intensity.

Figure 4.

In the cgl160 mutant, KEA3 decreases the ΔpH component of the pmf and thereby increases photosynthetic electron transport. A and B, At growth light, the cgl160 mutant stores a high fraction of pmf as ΔΨ, which is KEA3-dependent. Representative ECS traces of Col-0, kea3-1, cgl160-1, and kea3-1 cgl160-1 plants during a transition from light (150 µmol photons m⁻² s⁻¹) to dark (A). The difference in the ECS signal between light and the transient minimum in dark is ECS_t, between light and steady-state dark ECS_ss and between transient dark minimum and steady-state dark ECS_inv. ECS_t is a measure for light induced pmf, ECS_ss for ΔΨ and ECS_inv for ΔpH. B, ECS and its composition measured for Col-0, kea3-1, kea3-2, cgl160-1, cgl160-2, cgl160-1 kea3-1, cgl160-1 kea3-2, and cgl160-2 kea3-1. Averages are shown for n = 10, and error bars indicate ± se. Different letters above and in graph indicate significant differences between genotypes with P < 0.05 as determined by ANOVA and a Holm-Sidak pairwise multiple comparison for ECS_t and ECS_inv, respectively. C, Membrane conductivity for protons (g_H⁺). D, Scheme of the thylakoid membrane depicting the composition of the pmf, the regulatory role of the lumenal proton concentration, and parameters that were derived in this work. VDE, violaxanthin de-epoxidase; V, violaxanthin; Z, zeaxanthin; LHCII, light harvesting complex II; OEC, oxygen evolving complex; PQ, plastoquinone; bf, cytochrome b₆f complex; PC, plastocyanin. E to l, photoinhibitory quenching (qI, E), pH-dependent quenching (qE, F and indicated as curvy black arrow in D), xanthophyll deepoxidation state (DEPS, G), PSII quantum efficiency (ΦII, H), PSII acceptor availability (qL, I), cyt-f oxidation state (J), PSI donor side limitation (Y[ND]; K), and PSI acceptor side limitation (Y[NA] L). Average is shown for n = 10 (C, E, F, H) and n = 6–8 (G and I–L), and error bars indicate ± se. A–C and E–L, Measurements were performed on 5-week-old plants at actinic light intensities resembling growth light as described in “Materials and Methods.” Different letters above bars indicate significant differences between genotypes with P < 0.05 as calculated by ANOVA and Holm-Sidak pairwise multiple comparison (C and E–L).

Loss of KEA3 in the cgl160 Background Exacerbates Low pH-Dependent Quenching and Photosynthetic Control

As discussed before, a high proton concentration in the lumen triggers feedback down-regulation of photosynthesis by increasing qE and the de-epoxidation state of xanthophylls (DEPS), and slowing down electron transfer via the Cyt b₆f complex (Hager, 1969; Nishio and Whitmarsh, 1993; Niyogi et al., 1998; Takizawa et al., 2007). Changes in the rate of PQH₂ oxidation can be estimated from the redox states of both the PSII acceptor side (qL, corresponding to PSII acceptor availability) and cytochrome f of the Cyt b₆f complex (cyt-f), as well as the PSI donor side (Y[ND]; Fig. 4D). Alterations in pmf composition may also affect PSII function, as it has been shown that a high membrane potential increases PSII charge recombinations and thus photoinhibitory quenching (qI; Davis et al., 2016).

Our analyses performed at growth light intensities showed that none of the genotypes differed with respect to photoinhibitory quenching (qI; Fig. 4E). Also, the wild-type and both kea3 mutants did not differ significantly in any of the other measured parameters (Fig. 4, F–L). For cgl160 mutants, qE and DEPS were significantly increased as compared with Col-0 and kea3 (Fig. 4, F and G). This finding is in line with the larger ECS_inv signal, suggesting lower lumenal pH in the cgl160 mutants as compared with Col-0 (Fig. 4B). Whereas PSII efficiency (ΦII) was decreased in the cgl160 mutants (Fig. 4H), qL was comparable with Col-0 (Fig. 4I). However, cyt-f oxidation was significantly increased by about 30%, and PSI donor side limitation was approximately six times higher in both cgl160 mutants as compared with Col-0 (Fig. 4, J and K). Additionally, PSI acceptor side limitation was decreased by ∼50% (Fig. 4L). In nearly all cases, alterations in photosynthetic parameters measured for the cgl160 mutants as compared with Col-0 and kea3 were more strongly affected in the cgl160 kea3 mutants (Fig. 4, F–L ). Both independent cgl160 kea3 double mutants showed a two-fold increase in qE as compared with their cgl160 parents (Fig. 4F), and DEPS was increased by ∼20% (Fig. 4G), which again is in line with the larger ECS_inv signal in the cgl160 kea3 mutants as compared with the cgl160 mutants (Fig. 4B). ΦII was also significantly reduced (Fig. 4H). qL was only lower in cgl160-2 kea3-1, but not in cgl160-1 kea3-2 as compared with the other genotypes (Fig. 4I).

A stronger inhibition of PQ oxidation was evident from the cyt-f oxidation measurement, which showed that the cgl160 kea3 mutants had a 25% higher cyt-f oxidation as compared with the cgl160 mutants (Fig. 4J). The amplitude changes of the cyt-f transmission signal, when measured at saturating light, were indistinguishable between genotypes (Supplemental S1B), supporting that the difference at growth light intensity stemmed from a higher relative oxidation level of cyt-f. PSI donor side limitation Y(ND) was increased by ∼50% in the cgl160 kea3 mutants as compared with the cgl160 mutants (Fig. 4K), whereas PSI acceptor side limitation Y(NA) was not significantly different from the single mutants (Fig. 4L). We also analyzed leaf adenylates and showed that lack of KEA3 in the cgl160 background did not change leaf ATP levels (Supplemental S1, C and D).

Loss of KEA3 in the cgl160 Mutant Is Associated with a Decrease in Plastocyanin Content

Except for the cpATP synthase, thylakoid complexes in the cgl160 mutant accumulate at wild-type levels (Rühle et al., 2014; Fristedt et al., 2015). Also, lack of KEA3 does not affect thylakoid complex content (Armbruster et al., 2014). To investigate whether the thylakoid composition was different in cgl160 kea3 mutants as compared with the other genotypes, we analyzed pigment levels, quantified PSII, Cyt b₆f, plastocyanin (PC), and PSI spectroscopically and performed immunoblot analyses (Fig. 5). No clear difference in Chl per leaf area could be demonstrated between genotypes except for kea3-1 and cgl160-2 kea3-1 (Fig. 5A). Additionally, no strong differences between genotypes were observed for PSII, Cyt b₆f, and PSI content, when normalized to Chl and leaf area (Fig. 5, B–D; Supplemental Table S2). Interestingly, the cgl160-1 kea3-2 double mutant showed significantly lower PC content as compared with its cgl160-1 parent, when levels were normalized to Chl (Fig. 5E). When calculated on a leaf area basis, PC was reduced in both cgl160 kea3 lines as compared with each of their two parents by ∼20% (Fig. 5F).

Figure 5.

In the cgl160 mutant, lack of KEA3 decreases plastocyanin content. A, Chl content per leaf area is very similar between 5-week-old Col-0, kea3-1, kea3-2, cgl160-1, cgl160-2, cgl160-1kea3-2, and cgl160-2kea3-1 plants. B to F, PSII (B), Cytb₆f (C), PSI (D), and PC (E) were quantified spectroscopically with respect to Chl in isolated thylakoids as described in the methods. F, PC per leaf area was calculated from data in (A) and (E). Averages are shown for n = 6, and error bars indicate ± se. Different letters above bars indicate significant differences between genotypes with P < 0.05 as calculated by ANOVA and post hoc Tukey’s test. Note that PC per leaf area for cgl160-1kea3-2 and cgl160-2kea3-1 is significantly different from both parents, cgl160-1, kea3-2 and cgl160-2, kea3-1, respectively. G, Western blot analysis of total leaf protein from Col-0 (dilution series), kea3-1, cgl160-1, and cgl160-1kea3-1 with antibodies as indicated on the right. Before immune detection, membranes were stained with Ponceau Red.

To verify these findings, we performed protein blot analyses on total protein extracts by using specific antibodies. We probed total protein extracts from Col-0, kea3-1, cgl160-1 and the double mutant with antibodies specific for KEA3 and CGL160 (Fig. 5G). The results clearly showed that CGL160 and KEA3 were present at wild-type levels in the kea3 and cgl160 mutants, respectively, but lacking in the corresponding mutants. Additionally, we probed total leaf protein with the AtpB antibody that recognizes the CF₁ β-subunit of the cpATP synthase. This analysis confirmed the decrease in cpATP synthase in both the cgl160 and cgl160 kea3 mutants to levels < 25% of the wild type, as shown previously (Rühle et al., 2014; Fristedt et al., 2015). Levels of PsbS, which is known to determine the amplitude of energy pH-dependent quenching (Niyogi et al., 1998; Li et al., 2002), were not changed between genotypes. Protein blot analyses corroborated our spectroscopic quantification results: Levels of complexes from the photosynthetic electron transport chain were determined by using specific antibodies for PsbA (D1-subunit of PSII), PetA (cyt-f subunit of Cyt b₆f) and PsaB (B subunit of PSI) and showed few differences between genotypes (Fig. 5G). Only PC content was again measurably reduced in the cgl160 kea3 mutants to about 75% of the other genotypes.

Additional pigment analyses suggested some minor remodeling of the protein complex composition in the thylakoid membrane in the cgl160 kea3 mutants as compared with the wild type. The Chl a/b ratio was slightly, but significantly lower in the cgl160 kea3 mutants as compared with Col-0 and the two kea3 mutants, while the Chl a/b ratio for the cgl160 mutant was between that of wild type and the cgl160 kea3 mutants, but not significantly altered as compared with either (Table 1). However, no changes were found in the PSI/PSII ratio as calculated from the spectroscopic quantification of each complex (Supplemental Table S2). This was corroborated by unaltered levels of β-carotene, a pigment more abundant in PSI, between cgl160 kea3 and Col-0 (Table 1). Also, levels of neoxanthin, which only binds to the antenna of PSII, were similar between genotypes. Together with western blot results from the light harvesting complex b1 (Lhcb1) antibody showing no difference between genotypes, this strongly suggests that the size of the PSII antenna remained unchanged (Table 1).

Table 1. Pigments from Col-0, kea3, cgl160, and cgl160kea3 Plants.

Pigments were extracted and quantified as described in “Materials and Methods.” Values for neoxanthin, lutein, carotene, violaxanthin + antheraxanthin + zeaxanthin are given in millimole per mole Chl. The Chl a/b ratio was calculated, and PSI/PSII is derived from the spectroscopic quantification shown in Figure 5. Average values (n = 5) and standard deviations are provided. The letters indicate the results from ANOVA and post hoc Tukey’s test.

Genotype	Chl a/b	Carotene	Neoxanthin	Lutein	Violaxanthin
Col-0	2.96 ± 0.17 a,b	63.2 ± 1.4 a,b	26.4 ± 0.9 a	88.9 ± 4.2 a	28.3 ± 2.1 a
kea3-1	3.03 ± 0.07 a	63.5 ± 2.6 a,b	27.9 ± 1.3 a	101.4 ± 7.5 a,b	41.9 ± 4.3 a,b
kea3-2	3.06 ± 0.02 a	64.0 ± 3.1 a	27.1 ± 1.5 a	96.8 ± 8.2 a,b	39.5 ± 5.5 a,b
cgl160-1	2.83 ± 0.05 b	58.7 ± 3.8 b	27.3 ± 0.9 a	102.4 ± 7.2 a,b	51.3 ± 9.8 b
cgl160-2	2.83 ± 0.03 b,c	62.6 ± 2.5 a,b	25.6 ± 1.0 a	95.1 ± 6.9 a,b	49.8 ± 7.5 b
cgl160-1kea3-2	2.70 ± 0.02 b,c	58.4 ± 1.8 b	27.6 ± 0.9 a	107.5 ± 7.2 b	60.9 ± 7.8 b,c
cgl160-2kea3-1	2.65 ± 0.03 c	59.0 ± 2.5 a,b	25.4 ± 1.3 a	109.2 ± 7.0 b	69.1 ± 9.8 c

Only lutein, which binds all antenna proteins, was significantly higher in the cgl160 kea3 mutants as compared with Col-0 (Table 1). The VAZ pool, which is the sum of the xanthophylls violaxanthin, antheraxanthin and zeaxanthin, was significantly increased in cgl160 mutants compared with Col-0, as shown previously (Rühle et al., 2014), and was even further, but not significantly increased in both cgl160 kea3 mutants. Additionally, the relative antenna cross sections of the photosystems were determined from 77K Chl a fluorescence emission spectra (Supplemental Fig. S2). No changes in the ratio of the emission signals of PSII-LHCII peaking at 687 nm wavelength and PSI-LHCI peaking at 733 nm wavelength were observed, supporting that antenna cross sections and excitation distribution between the photosystems were unchanged between genotypes.

Loss of cgl160 Increases High Light Sensitivity

Previously, it was shown that high ΔΨ favors charge recombination within PSII (Davis et al., 2016; Davis et al., 2017) and that low lumenal pH below 5.5 triggers the release of calcium from the oxygen evolving complex (Krieger and Weis, 1993). Both high ΔΨ and low lumen pH can thus increase the slowly relaxing photoinhibitory quenching component qI that accompanies PSII damage (Krause and Weis, 1991). However, when we analyzed Col-0, kea3, cgl160, and cgl160 kea3 for qI under actinic light intensities resembling growth light, no differences were observed (Fig. 4E). These results suggested that if PSII damage was increased in cgl160 and cgl160 kea3 mutants as compared with the other genotypes under growth light, it did not exceed the capacity of the plants for PSII repair. This finding was corroborated by measurements of the maximum quantum yield of PSII, Fv/Fm, which was also unchanged between genotypes (Fig. 6A).

Figure 6.

cgl160 plants are sensitive to high light independent of KEA3. A, Maximum quantum yield of 5-week-old Col-0, kea3-1, kea3-2, cgl160-1, cgl160-2, cgl160-1 kea3-2, and cgl160-2 kea3-1 was measured before and 3 h after a shift to 900 µmol photons m⁻² s⁻¹. Averages are shown for n = 10, and error bars indicate ± se. Different letters above bars indicate significant differences between genotypes with P < 0.05 as calculated by ANOVA and post hoc Tukey’s test. B, Light-induced pmf (ECS_t) and fraction stored as ΔΨ (ECS_ss) and ΔpH (ECS_inv) was measured at 1000 µmol photons m⁻² s⁻¹ actinic light for Col-0, kea3-1, cgl160-1, and cgl160-1 kea3-1. Average is shown for n = 5, and error bars indicate ± se. Different letters above graph indicate significant differences between genotypes for ECS_t, in top bar for ECS_ss and in bottom bar for ECS_inv with P < 0.05 as calculated by ANOVA and post hoc Tukey’s test.

Plants were then challenged with light stress by exposing them to six times the growth light intensity (900 µmol photons m⁻² s⁻¹) for 3 h: all genotypes showed a significant decrease in Fv/Fm after the high light treatment, but it was more severe in cgl160 plants independent of whether they were additionally lacking KEA3 (Fig. 6A). In order to analyze the link between pmf composition and PSII photoinhibition, we determined light induced pmf (ECS_t) and its composition, ΔpH (ECS_inv), and ΔΨ (ECS_ss) of plants after 15 min of high light. This analysis showed that all genotypes had a very similar ECS_ss independent of the size of ECS_t, which in turn was higher in plants lacking cgl160 (Fig. 6B). This could indicate that the size of ΔΨ is regulated to remain below a certain threshold, potentially to avoid ΔΨ-induced PSII charge recombination. The results also suggest that at high light KEA3 does not influence the composition of pmf, which is in line with previous publications (Armbruster et al., 2016; Wang and Shikanai, 2019), not even in the cgl160 background.

Overexpression of KEA3 in the cgl160 Background Further Decreases qE But Not Photosynthetic Control

Because we observed that cgl160 mutants require KEA3 for efficient photosynthesis and growth, we investigated whether we could improve photosynthesis and growth by overexpressing the major KEA3 isoform KEA3.2 in the cgl160 background. Therefore, we transformed cgl160-1 plants with a construct to overexpress a KEA3.2-GFP fusion, which was previously shown to complement kea3 mutant phenotypes, when expressed at wild-type level (Armbruster et al., 2014). We isolated two lines, which showed a more than 4-fold overexpression of KEA3-GFP compared with cgl160-1, which will be referred to as oeKEA3_cgl160 (Supplemental Fig. S3). The oeKEA3_cgl160 plants had strongly decreased cpATP synthase levels similar to those of the cgl160 mutants, which was determined by Western blot analysis of total protein by using the AtpB antibody (Supplemental Fig. S3). oeKEA3_cgl160 plants looked slightly larger than cgl160-1 plants, but still much smaller than Col-0 (Fig. 7A). While cgl160-1 mutants accumulated significantly less dry weight than Col-0, the dry weight of the two KEA3 overexpression lines was between that of Col-0 and cgl160-1, and not significantly different from either (Fig. 7B).

Figure 7.

Overexpression of KEA3 in the cgl160 background promotes photosynthesis by down-regulating qE. A, Picture of 5-week-old Col-0, cgl160-1 and two independent lines overexpressing KEA3-GFP in the cgl160-1 background. B, Dry weight of 5-week-old plants; n = 13–15. Different letters above graph indicate significant differences between genotypes with P < 0.05 as calculated by ANOVA on ranks and Dunn’s test (C and D) pH-dependent quenching (qE; C) and PSII quantum efficiency (ΦII; D) were measured of Col-0, cgl160-1, and oeKEA3 lines 1 and 2 (L1 and L2) in the cgl160-1 background. Averages are shown for n = 6, and error bars indicate ± se. E to H, Light-induced pmf (ECS_t) and fraction stored as ΔΨ (ECS_ss) and ΔpH (ECS_inv; E), cpATP synthase conductivity (g_H⁺; F), halftime (t_1/2) of ECS_inv recovery (G), and cyt-f oxidation state (H) were measured for Col-0, cgl160-1, oeKEA3 in cgl160-1 L1 and L2. Data for both overexpression lines were pooled for representation and statistical analysis. Average is shown for n = 3–10, and error bars indicate ± se. C to H, Measurements were performed at actinic light intensities resembling growth light as detailed in “Materials and Methods.” Different letters above graph indicate significant differences between genotypes with P < 0.05 as calculated by ANOVA and Holm-Sidak pairwise multiple comparison.

Then we measured Chl a fluorescence and ECS based parameters at actinic light intensities resembling growth light (Fig. 7, C–G). Chl a fluorescence analysis demonstrated that KEA3 overexpression in the cgl160-1 background lowers pH-dependent quenching (qE) to levels comparable with Col-0 (Fig. 7C). This decrease in qE was accompanied by a significant increase in PSII efficiency (ΦII) in both oeKEA3_cgl160 lines as compared with cgl160-1 (Fig. 7D). We determined ECS_t, ECS_ss, and ECS_inv to analyze the effect of high KEA3 levels on light induced pmf and its composition in the cgl160-1 background (Fig. 7E). This analysis showed that oeKEA3_cgl160 had more light-induced pmf stored as ΔΨ compared with Col-0 and cgl160-1. However, differences were not significant. Interestingly, while ECS_t was much higher in cgl160-1 as compared with Col-0 (as was also shown in Figs. 2C and 4B), ECS_t was reduced in the overexpressors to levels that were not significantly different from the wild type (Fig. 7E). The membrane conductivity for protons (g_H⁺) behaved in the opposite way, that is, while g_H⁺ in cgl160-1 was significantly decreased as compared with Col-0, overexpression of KEA3 in oeKEA3_cgl160 resulted in g_H⁺ values that were in between Col-0 and cgl160-1, not significantly different to either (Fig. 7F). The halftime of ECS_inv recovery (t_1/2) tended to be higher in cgl160-1 than in the wild type, but this difference was not significant. However, overexpression of KEA3 further reduced t_1/2 to levels significantly lower than those in cgl160-1 (Fig. 7G).

Interestingly, cyt-f oxidation, which was increased significantly in cgl160-1 compared with Col-0 (as was also shown for cgl160-2 in Fig. 4J) was not further changed by overexpression of KEA3-GFP (Fig. 7H; Supplemental Table S3). Additionally, the thylakoid complex composition was analyzed spectroscopically, and no significant differences were found between Col-0, cgl160-1, and oeKEA3_cgl160 (Supplemental Table S4). Together, the data indicate that overexpression of KEA3 in the cgl160 background may pose an advantage for photosynthesis and growth. The results clearly demonstrate that higher KEA3 levels decrease pH-dependent quenching, increase PSII efficiency, and accelerate ECS_inv recovery in the cgl160-1 background.

DISCUSSION

Photosynthesis and Growth of the cgl160 Plants, with Low cpATP Synthase Content, Benefit from KEA3 Activity

Previously, photosynthetic phenotypes of kea3 mutants and KEA3 overexpressors were described. It was shown that KEA3 decreases the ΔpH component of the pmf and thereby downregulates NPQ (Armbruster et al., 2014; Kunz et al., 2014; Armbruster et al., 2016; Wang et al., 2017; Dukic et al., 2019; Wang and Shikanai, 2019). The main function of KEA3 is in intermediate light conditions and during transition from high to low light (Armbruster et al., 2014; Dukic et al., 2019; Wang and Shikanai, 2019). However, so far lack of KEA3 or overexpression of naturally occurring KEA3 isoforms has not been associated with a strong effect on growth.

In the current work, we set out to analyze the influence that the absence or overexpression of KEA3 has in the cgl160 mutant, which has strongly reduced cpATP synthase levels (<25% of wild type (Rühle et al., 2014; Fristedt et al., 2015; Fig. 5G). We could show that in this background, KEA3-mediated proton export from the lumen promotes growth, that is, cgl160 kea3 mutants accumulate less biomass than cgl160 single mutants (Fig. 3, A–C). In the cgl160 mutant, with high pmf, KEA3 enables higher photosynthesis rates by down-regulating energy-dependent quenching (qE) and up-regulating the rate of plastoquinol oxidation at the Cyt b₆f complex (i.e. decreasing photosynthetic control; Fig. 4). Together, the data demonstrate that photosynthesis in cgl160 kea3 mutants is limited by low electron transport rates as a result of a high ΔpH.

In cgl160 single mutants instead, KEA3 counteracts the high lumenal proton concentration by converting ΔpH into ΔΨ and thereby upregulates photosynthesis. Interestingly, while levels of all other protein components of the electron transport chain remained unchanged between genotypes, PC content was reduced in mutants that lacked both CGL160 and KEA3. In the cgl160 kea3 mutants, PC may either be directly destabilized by low lumenal pH, as previously suggested (Kramer et al., 1999), or decreased by mechanisms at the transcriptional or posttranscriptional level. Many mutants affected in photosynthesis instead up-regulate PC content, starting at the transcriptional level in response to starvation signals (i.e. low sugar; Oswald et al., 2001; Schöttler et al., 2004; Rott et al., 2011). Hence, the reduction in PC content in the cgl160 kea3 mutants is unlikely to be a response to the decrease in assimilation capacity we observed in this double mutant, because in this case we would expect the opposite, that is, an increase in PC content. Thus, it appears plausible that PC content may be indeed sensitive to lumenal pH.

Regardless of the mechanism by which PC content is decreased, the question remains of whether the low PC content contributes to the decrease in photosynthesis observed in the cgl160 kea3 mutants. A substantial decrease in PC content can result in PC being rate-limiting for photosynthesis (Schöttler et al., 2004). At such low PC levels, PSI would be strongly donor side limited and cyt-f of Cyt b₆f more reduced (Schöttler et al., 2004). However, cyt-f is more oxidized in the cgl160 kea3 mutants (Fig. 4J), which points to PQ oxidation and not PC reduction being the rate-limiting step at Cyt b₆f in this background.

The Regulation of pmf Composition by KEA3 Affects the Rate of Plastoquinol Oxidation at Cyt b₆f

The increased oxidation level of cyt-f and PSI (Fig. 4) in the cgl160 mutants and even more strongly in the cgl160 kea3 double mutants suggest that electron transport from PQ via cyt-f to PSI (i.e. the high potential chain) is slowed down at growth light intensity due to lower Cyt b₆f turnover. An alternative explanation for increased oxidation of cyt-f and PSI could be imbalanced excitation rates of the photosystems, so that PSI is excited in excess and thereby oxidizes the high potential chain. This alternative explanation is unlikely, because neither photosynthetic complex contents nor antenna cross sections of the photosystems were altered (Fig. 5; Supplemental Fig. S2). Thus, the higher cyt-f oxidation in the cgl160 kea3 mutants compared with the cgl160 mutants strongly supports a role for KEA3-dependent proton antiport in alleviating photosynthetic control, at least in the cgl160 background.

Recent work by Wang and Shikanai (Wang and Shikanai, 2019) interpreted a decrease in PSI donor side limitation (Y[ND]) in KEA3 overexpressors as compared with wild type as a result of increased Cyt b₆f turnover. In the present work, we show by measurement of the cyt-f redox state that overexpression of KEA3 does not increase Cyt b₆f turnover in the cgl160 background (Fig. 7). Thus, while lack of KEA3 in the cgl160 mutants increases both qE and photosynthetic control (Fig. 4), KEA3 overexpression in the cgl160 mutants only downregulates qE (Fig. 7H). Together, these data support the notion that qE and Cyt b₆f turnover are affected differently by high pmf.

While the main determinant of qE seems to be the net proton concentration in the lumen, which behaves inversely to KEA3 levels in the cgl160 background (Figs. 4B and 7E), photosynthetic control appears not only to be dependent on ΔpH, but also on ΔΨ. This is in line with thermodynamic considerations and results obtained with the homologous mitochondrial bc1 complex, which show that collapsing the membrane potential increases the rate of electron transport through this complex (Rich and Clarke 1982). However, the strong increase in cyt-f oxidation seen in the cgl160 kea3 mutants as compared with the cgl160 mutants supports an additional kinetic down-regulation of Cyt b₆f turnover by low lumenal pH, possibly due to the protonation of amino acids involved in proton transfer from the plastoquinol binding site to the lumen (Tikhonov, 2014).

KEA3 Activity Is Tightly Regulated

KEA3 activity changes the composition of the pmf toward higher ΔΨ. KEA3 may achieve this either directly by increasing the membrane potential through electrogenic antiport, for example, if it transports multiple K⁺ per proton, and/or indirectly by dissipating the ΔpH component, thus forcing a larger fraction of pmf to be stored as ΔΨ. The exact transport mechanism of KEA3 has yet to be determined. Our results demonstrate KEA3 to be present in thylakoid membranes at much lower levels than the cpATP synthase and, therefore, at first sight may question a direct competition of KEA3 with the cpATP synthase for proton export from the lumen (Fig. 1). However, maximum proton transport rates for antiporters can be much higher than those for the cpATP synthase: The Escherichia coli Na+/H+ antiporter A (NhaA), which belongs to the same class of cation proton antiporter2 (CPA2) transporters as KEA3 (Masrati et al., 2018) has been shown to have a maximum turnover rate of 9 × 10⁴ s⁻¹ (Taglicht et al., 1991). At high pmf, a maximum ATP synthesis rate of ∼400 ATP s⁻¹ per protein complex has been reported (Gräber et al., 1987). In line with a ∼4.7 H⁺/ 1 ATP stoichiometry (Petersen et al., 2012), one cpATP synthase complex could achieve a maximum proton transport rate of ∼ 1.9 × 10³ protons s⁻¹. Assuming an equally high maximum activity for KEA3 as reported for NhaA, this would imply that one KEA3 has the same maximum proton transport capacity as 50 cpATP synthases. With ∼100 cpATP synthases per KEA3, proton export from the lumen by KEA3 could reach at least 50% of the cpATP synthase, assuming no kinetic or thermodynamic constraints on each of the two transport processes.

Proton transport via KEA3 could be thermodynamically limited by low concentrations of the antiported cation. However, stromal concentrations of free K⁺ are in the millimolar range (discussed in Cruz et al., 2001) and thereby in excess of the lumenal proton concentration. While K⁺ has been shown be transported by KEA3 (Tsujii et al., 2019), a high selectivity for K⁺ has not yet been unequivocally demonstrated. Also, other cations, such as Na⁺ and Mg²⁺, are present at concentrations exceeding the micromolar range (Robinson and Downton, 1984; Ishijima et al., 2003). Thus, exchange of the entire ΔpH with a cation concentration gradient should be feasible through KEA3. This clearly does not occur. Therefore, proton export from the lumen via KEA3 must be kinetically controlled, either by a lower affinity of KEA3 for the antiported ions as compared with NhaA or via regulation of its activity.

KEA3 activity has been shown to be regulated via its C terminus (Armbruster et al., 2016). This C terminus contains a conserved K+ transport nucleotide-binding domain, which in other systems gates K⁺ transport in response to nucleotide binding (Choe, 2002; Roosild et al., 2002; Cao et al., 2013; Kim et al., 2015). The two main products of the light reactions of photosynthesis, ATP and NADPH, have been shown to strongly decrease in response to a high to low light transition (Stitt et al., 1989), the very condition where an activation of KEA3 occurs (Armbruster et al., 2016). Thus, it is tempting to speculate that KEA3 is activated in response to lower levels of one or both of these nucleotides or an increase in ADP or NADP⁺, their low energy counterparts.

Activation by low ATP or high ADP may explain why we see such a strong effect of KEA3 on pmf composition specifically in the cgl160 mutant with decreased ATP levels (Fig. 2D). However, the strong effect that loss of KEA3 has in the cgl160 background on pmf composition can be alternatively explained by the following: (1) low proton transport rates through the cpATP synthase, because of its low content, and/or (2) high rates of proton efflux via KEA3 because of high pmf. However, both explanations predict that further increases of pmf, for example, by high light, would exacerbate the effect that KEA3 has on pmf composition in the cgl160 background. This is, however, not the case, as the difference in pmf composition between the cgl160 and cgl160 kea3 mutants collapses in high light (Fig. 6B). This strongly suggests that also in the cgl160 background KEA3 gets inactivated in high light.

Dissipation of pmf via Simultaneous Activity of KEA3 and a Counterion Channel?

One interesting observation is that overexpression of KEA3 in the cgl160 background causes significantly reduced light-induced pmf (as demonstrated by the ECS_t parameter), while absence of KEA3 does not affect the pmf size. One possible explanation for this is the simultaneous activity of KEA3 and the corresponding thylakoid cation (likely K⁺) channel in the cgl160 background, when KEA3 is overexpressed. Together, both would be cycling K⁺ with net efflux of protons from the lumen and thus dissipate pmf. In the cgl160 mutant, K⁺ channel activity must be much lower than that of KEA3, because we do observe a strong effect on pmf composition (i.e. higher ΔΨ in cgl160 than in cgl160 kea3), which should not be present if both antiporter and channel had the same ion transport capacity. However, if we speculate that the K⁺ channel is voltage gated, the membrane potential may reach a critical threshold in the oeKEA3_cgl160 line at which the K⁺ channel is activated and together with KEA3 starts dissipating pmf.

Is there any evidence that the thylakoid K⁺ channel is voltage gated? First of all, the function of thylakoid counter ion channels such as the K⁺ channel is the dissipation of ΔΨ, and thus it is intuitive that these channels should be activated by high ΔΨ. Multiple studies have reported on the presence of a K⁺ channel in the thylakoid membrane by electrophysiological approaches (Tester and Blatt, 1989; Enz et al., 1993; Pottosin and Schönknecht, 1996), and indeed in one study a moderate voltage dependence of the channel was described (Pottosin and Schönknecht, 1996). However, so far, the molecular nature of the thylakoid K⁺ channel and thus its regulation remains unknown (Höhner et al., 2019). The only thylakoid counter-ion channel that has convincingly been shown to reside in the thylakoid membrane is the Cl⁻ channel VCCN1, and this is in fact voltage-gated (Duan et al., 2016; Herdean et al., 2016). Besides lower pmf size, the oeKEA3_cgl160 plants also displayed significantly faster ECS_inv recovery (Fig. 7). The recovery phase of the ECS signal in the dark (ECS_inv) occurs after proton flux from the lumen has caused the generation of an inverse membrane potential.

Two mechanisms can explain the phenomenon of the ECS recovery: slow ion flux that collapses the inverse membrane potential (Cruz et al., 2001) or proton flux from the stroma into the lumen by the cpATP synthase (Szabò and Spetea, 2017). Previously, the acceleration of ECS_inv by KEA3 has been shown to correlate with an increase in the parameter g_H⁺, which reflects the proton conductivity of the thylakoid membrane and has been used to describe cpATP synthase activity (Wang and Shikanai, 2019). This correlation was used to support that KEA3 transport activity causes increases in g_H⁺. We also see a slight increase in g_H⁺ in the oeKEA3_cgl160 line as compared with the cgl160 mutant. However, how and whether these two parameters are directly correlated remains to be determined.

CONCLUSIONS

This study aimed at characterizing the functional interaction of two exit routes for protons from the thylakoid lumen, that is, the cpATP synthase and the proton antiporter KEA3, on the physiological level. Hereby, we could show that in a mutant with low cpATP synthase content (i.e. cgl160), the presence of KEA3 confers a physiological advantage by decreasing the lumenal proton concentration, which in turn increases the capacity of the cgl160 mutant for light harvesting and electron transport. This is of physiological significance, because it suggests that under conditions that limit ATP synthase activity, proton transport via KEA3, which functions to increase the fraction of the pmf as ΔΨ, controls photosynthesis and growth.

MATERIAL AND METHODS

Plant Material, Propagation, and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) kea3-1 (Gabi_170G09), kea3-2 (SAIL_556_E12), cgl160-1 (SALK_057229), and cgl160-2 (WiscDsLoxHs024_02B) T-DNA insertion lines have been described previously (Armbruster et al., 2014; Kunz et al., 2014; Rühle et al., 2014; Fristedt et al., 2015). The cgl160 kea3 plants were generated by crossing and identifying double homozygous mutants in the F2 generation by PCR-based genotyping using primers as detailed in Supplemental Table S5. cgl160-1 plants expressing KEA3.2-GFP (KEA3_cgl160) were generated via Agrobacterium-mediated floral dip (Clough and Bent, 1998) by using the pB7FWG2-KEA3.2 construct described in Armbruster et al., 2014.

Wild-type Arabidopsis accession Col-0 and transgenic seeds were stratified for 2 d at 4°C in 1% agarose with gibberellic acid A4 and A7 (0.2% and 0.1% [v/v] respectively) to synchronize germination. Seeds were sown on soil, grown in long-day conditions (16-h light/8-h darkness at 20°C/ 6°C, 60%/75% relative humidity) at 250 µmol photons m⁻² s⁻¹ for 7 d. Plants were then moved to long-day conditions with 120-150 µmol photons m⁻² s⁻¹ and 16°C night temperature. After 2 weeks, plants were pricked into individual pots. All measurements were performed with plants that were 4 to 5 weeks old.

Growth and Biomass Accumulation Analysis

The leaf area of whole rosettes was determined by detecting the minimal Chl a fluorescence (F₀) under measuring light using the Walz Imaging PAM (Walz GmbH) and quantification using Image J according to Schneider et al., 2012. The increase in leaf area over time was fitted exponentially to derive the daily growth rate. For representation only those plants were selected where the R²-value of the fit was >0.99. For dry weight measurements, 5-week-old rosettes were harvested and dried for 3 d under vacuum at 65°C.

Expression and Purification of Recombinant KEA3-C Terminus and AtpF Protein

The KEA3 C terminus and AtpF were amplified from complementary DNA by using primers described in Supplemental Table S5 and cloned into the pET28a (+) vector (Novogen). His-tagged KEA3 C terminus (KEA3_C-termHis₆) and AtpF (AtpF-His₆) were obtained by using standard protocols for recombinant protein expression and purification using Ni-NTA affinity chromatography. The purity of the recombinant protein was checked on SDS PAGE (SDS-PAGE) stained with Coomassie brilliant blue. The concentrations of the recombinant proteins were determined by Pierce BCA Protein Assay kit (Thermo Scientific).

Mass Spectrometric Identification and Quantification of ATP Synthase and KEA3

Thylakoids resuspended in 8 m urea and 10 mm Tris-HCl (pH 8.0) were reduced with dithiothreitol, modified with iodoacetamide and digested on column with trypsin (Promega) in 100 mm ammonium bicarbonate (ratio trypsin to protein of 1:25 [w/w]). Digest was performed for 14 h at 37°C. Peptides were eluted with 0.5 m NaCl. Trifluoroacetic acid (10% [w/v]) was used for peptide acidification to pH < 3.0. The peptide mixture was purified and desalted on C18 SEP-Pak columns (Tecknokroma). Measurements were performed on a Q Exactive Plus combined with HF mass spectrometer coupled with a nLC1000 nano‐HPLC (both Thermo Scientific). Quantitative analysis of tandem MS measurements was performed with MaxQuant software (Cox and Mann, 2008), and Mascot search engine was used to annotate peptide sequences using the Arabidopsis TAIR10 genome annotation database. The following settings were used for a search: 10 ppm peptide mass tolerance; 0.8 D tandem MS tolerance; maximum of two missed cleavages allowed, threshold for validation of peptides set to 0.01, carbamidomethylation of Cys was set as a fixed modification and oxidation of Met was set as variable modification. The ‘label‐free quantification’ and ‘match between runs’ options were selected. The minimum peptide length of six amino acids was used. The quantification was performed for proteins with minimum of one unique and one razor peptide. Known contaminants such as keratins were removed from further analysis.

Nucleotide Quantification

ATP, ADP, and AMP were quantified fluorometrically after specific derivatization with chloroacetaldehyde as described in Bürstenbinder et al., 2007. ATP, ADP, and AMP were quantified fluorometrically after specific derivatization with chloroacetaldehyde as described in Bürstenbinder et al., 2007. Nucleotides were extracted from frozen ground material (20 mg) in 50 μL of 0.1 m HCl using a mixer mill with a zirconia bead for 1 min at 25 Hz, and centrifuged at 16,000 g at 4°C for 10 min. After centrifugation, 15 µL of the supernatant mixed with 77 µL of CP buffer [62 mm citric acid-1hydrate and 76 mm (Na)₂HPO₄0.2H₂O, pH 4] was derivatized by adding 8 µL of 45% (v/v) chloroacetaldehyde for 10 min at 80°C. Separation of derivatized nucleotides was carried out as in Estavillo et al., 2011 using a HyperClone C18 (ODS) column (Phenomenex) on a Summit HPLC system (Dionex). Nucleotide amounts were determined by measuring the area under the curve and calculated based on a calibration curve of derivatized nucleotide standards.

ECS, Chl a Fluorescence, and P700 Measurements

Near simultaneous Chl a fluorescence and ECS measurements were performed on intact leaves of 30-min dark-adapted plants using a custom-made spectrophotometer (Hall et al., 2013). Plants were illuminated for 10 min at various light intensities (Fig. 2, B and C), 150 µmol photons m⁻² s⁻¹ (Figs. 4, A and B, and 7, E–G–G), and 1000 µmol photons m⁻² s⁻¹ (Fig. 7) to reach steady state. The dark interval relaxation kinetics (DIRK) of the 520 nm ECS signal monitored for 300 ms was used to estimate thylakoid proton conductivity (g_H⁺) by fitting the ECS decay curve to a first order exponential, and the amplitude of the dark-light signal to estimate ECS_t (light-induced thylakoid pmf) as previously described (Kanazawa and Kramer, 2002). DIRKs for three wavelengths (505, 520, and 535 nm) were monitored for 40 s, and the ECS signal was deconvolved as described (Cruz et al., 2001) to determine the amplitude and partitioning of the pmf. The half time (t_1/2) of the ECS_inv recovery signal was calculated by fitting data to the equation y = A₁*e^(x/t1) + y₀ using Origin (OriginLab) and settings for orthogonal distance regression. All ECS signals were normalized to the total Chl content of the leaf.

For Chl a fluorescence yield measurements, saturation light pulses as in Strand et al., 2017 were applied after dark acclimation (for Fm), steady-state illumination (for Fm’), and 10 min of dark relaxation (for Fm’’). qE and qI components of NPQ were calculated as [(Fm’’− Fm’)/Fm’] and [(Fm − Fm’’)/ Fm’’], respectively. A Dual-PAM-100 instrument (Walz GmbH) was used to measure PSII acceptor availability (qL) and light-dependent electron transport rate through PSII as in Baker, 2008. For P700 measurements, leaves were illuminated for 12 min at 135 µmol photons m⁻² s⁻¹. Fv/Fm values in Figure 6 were measured using the Imaging PAM (Walz GmbH).

77K Chl a fluorescence emission spectra were measured on freshly isolated thylakoid membranes equivalent to 10 μg chlorophyll mL⁻¹ using a F-6500 fluorometer with a red-sensitive photomultiplier (Jasco Inc.). The sample was excited at 430-nm wavelength with a bandwidth of 10 nm, and the emission spectrum was recorded between 655- and 800-nm in 0.5-nm intervals with a bandwidth of 1 nm. All signals were normalized to the emission maximum of PSII-LHCII at 686.5 nm wavelength.

Cyt-f Oxidation State Measurements

Light-dark difference transmittance signals after 10-min illumination at 111 and 1295 µmol photons m⁻² s⁻¹ followed by 30-s dark in the dark were measured at multiple wavelengths between 505 and 570 nm using a KLAS-100 spectrophotometer (Walz GmbH). Transmittance signals for cyt-f were deconvoluted from the ECS signal, scattering, and other cytochromes as previously described (Klughammer et al., 1990; Rott et al., 2011). The deconvoluted redox kinetics of cyt-f were normalized to the chlorophyll content of the leaf.

Quantification of Photosynthetic Complexes

PSII, Cyt b₆f, PSI, and PC were quantified spectroscopically in isolated thylakoids extracted as in Schöttler et al., 2004. PSII and Cyt b₆f contents were determined from differences in absorption signals of cyt-b₅₅₉ (PSII) and cyt-f and -b₆ (cyt-bf). After complete oxidation of all cytochromes by the addition of 1 mm potassium ferricyanid (+III), cyt-f and the high-potential form of cyt-b₅₅₉ were reduced by the addition of 5 mm sodium ascorbate, followed by the addition of 10 mm sodium dithionite, to reduce the low-potential form of cyt-b₅₅₉ and the cytochromes b₆. For each redox condition, ten absorbance spectra between 580 and 535 nm wavelength were averaged using a Jasco V-550 photometer equipped with a head-on photomultiplier with a scanning speed of 100 nm * min⁻¹ and a spectral bandwidth of 1 nm (Jasco GmbH). Then, differences in absorbance spectra were calculated, baseline-corrected and deconvolved as described (Kirchhoff et al., 2002; Lamkemeyer et al., 2006).

Redox-active PSI content was determined from light-induced absorption changes of P₇₀₀ in the presence of 10 mm sodium ascorbate as the electron donor and methylviologen as the artificial electron acceptor (Schöttler et al., 2007). PC contents, relative to P₇₀₀, were determined in leaves by in vivo difference absorption spectroscopy in the far-red range of the spectrum and then recalculated based on the absolute P₇₀₀ quantification in isolated thylakoids as described in Schöttler et al., 2007 and finally renormalized to a leaf area basis with the known chlorophyll content per leaf area.

CO₂ Fixation Measurements

Light response curves of net photosynthesis rates were determined using the LI-6400 photosynthesis system (Li-Cor). The soil was covered using parafilm M (Brand) to reduce soil-borne evaporation. Plants were analyzed in the 6400-17 whole plant Arabidopsis chamber, which was equipped with the 6400-18A RGB light source. Measurements were performed at 400 ppm CO₂, 21% O₂, and 55% to 65% relative humidity, 20°C cuvette temperature, and a flow rate of 500 µmol air s⁻¹. Plants were exposed to increasing light intensities up to 1500 μmol m⁻² s⁻¹. At each light intensity, after steady state was reached, data were logged 10× over a period of 30 s; these data points were averaged for the representation. After the rosette was cut off, soil respiration was determined to correct values. Projected leaf area was determined using ImageJ.

Western Blot Analyses

Total protein was extracted from liquid nitrogen frozen leaf tissue (1 cm² diameter leaf disc) in 100 µL 4 × Laemmli buffer as described in Armbruster et al., 2016. Total leaf protein was separated by SDS PAGE and blotted onto a nitrocellulose membrane as in Schwarz et al., 2015. After incubation with specific antibodies—Anti-KEA3 (Armbruster et al., 2014), Anti-PsbS (Correa-Galvis et al., 2016; Correa-Galvis et al., 2016), Anti-Actin (Sigma), and Anti-CGL160, AtpB, PsbA, PetA, PC, PsaB, AtpF and Lhcb1 (Agrisera)—signal was detected by using a C-DiGit Blot Scanner (LI-COR Biosciences).

Pigment Analyses

Total chlorophyll from a 5-mm diameter leaf disc was extracted by the addition of 80% (v/v) acetone and measured according to Porra et al., 1989 for the normalization of ECS and cyt-f oxidation signals as well as for data in Figure 5. Additionally, total chlorophyll, xanthophylls, and β-carotene were extracted from 1 cm² leaf disc by 1 mL acetone (100%). The supernatant was filtered through a 0.2 μm membrane filter (GE Healthcare). Pigments were separated and quantified by HPLC analysis as previously described (Färber et al., 1997). The DEPS of xanthophylls was calculated as (zeaxanthin + 0.5 anteraxanthin)/(violaxanthin + anteraxanthin + zeaxanthin).

Statistical Analyses

ANOVAs and Holm-Sidak or Tukey pot hoc tests were performed by using the program SigmaPlot.

Accession Numbers

The following genes and proteins were investigated: KEA3 (At4g04850), CGL160 (At2g31040), AtpB (ATCG00480), PetA (ATCG00540 ), PsaB (ATCG00340), PsbA (ATCG00020), plastocyanin (PC, AT1G20340, AT1G76100), Lhcb1 (AT1G29910, AT1G29920, AT1G29930, AT2G34420, AT2G34430), and PsbS (AT1G44575). Further information on the mutant lines that were used in the study can be found in the “Material and Methods” section under plant material.

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Spectroscopic data and adenylate quantification.

• Supplemental Figure S2. The 77K Chl a fluorescence emission spectra support an unaltered antenna distribution between both photosystems in Col-0, kea3, cgl160 and cgl160 kea3 plants.

• Supplemental Figure S3. Western blot analysis of oeKEA3_cgl160 plants.

• Supplemental Table S1. Leaf ATP, AMP and ATP contents of Col-0, cgl160-1 and cgl160-2.

• Supplemental Table S2. PSII, Cyt b₆f and PSI content per leaf area and PSI/PSII of Col-0, kea3, cgl160 and cgl160kea3 plants.

• Supplemental Table S3. Fv/Fm, qI and Cyt-f oxidation of Col-0, cgl160-1 and oeKEA3_cgl160.

• Supplemental Table S4. Thylakoid complexes are unchanged between Col-0, cgl160-1 and oeKEA3_cgl160.

• Supplemental Table S5. Primer purpose, names, and DNA sequence used in this study.