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. 2019 Aug 19;181(2):762–773. doi: 10.1104/pp.19.00766

Modification of Activity of the Thylakoid H+/K+ Antiporter KEA3 Disturbs ∆pH-Dependent Regulation of Photosynthesis1,[OPEN]

Caijuan Wang 1,2, Toshiharu Shikanai 1,3,4
PMCID: PMC6776848  PMID: 31427465

Overexpression of two versions of KEA3 differentially affects the proton motive force and thereby the regulation of photosynthetic electron transport under stressful light conditions in Arabidopsis.

Abstract

The thylakoid K+ efflux antiporter 3 (KEA3) is required for regulating components of the proton motive force (pmf), proton concentration gradient (ΔpH), and membrane potential (Δψ). The Arabidopsis (Arabidopsis thaliana) disturbed proton gradient regulation mutant (dpgr) is a dominant allele of KEA3, conferring disturbed transport activity. Here, we show that overexpressing the DPGR-type KEA3 (DPGRox) retarded plant growth, whereas overexpressing the wild-type KEA3 (KEA3ox) did not. In KEA3ox lines, the contribution of Δψ to pmf was enhanced, but in DPGRox lines, the size of pmf was reduced. In DPGRox plants, proton conductivity of the thylakoid membrane (gH+) was elevated under high light, implying disturbed stoichiometry of H+/K+ antiport through DPGR-type KEA3 rather than simply enhanced activity. The ΔpH-dependent regulation consisting of thermal dissipation of excessively absorbed light energy and downregulation of cytochrome b6f complex activity was severely and mildly affected in DPGRox and KEA3ox plants, respectively. Consequently, photosystem I was sensitive to fluctuating light in both transgenic plants. Both photosystems were sensitive to constant high light and were slightly photodamaged even at standard growth light intensity in DPGRox plants. KEA3 regulates the components of pmf and optimizes the operation of ∆pH-dependent regulation of electron transport.

The chemiosmotic hypothesis was proposed by Mitchell (1961) and is universally accepted for explaining ATP synthesis both in respiration and photosynthesis (Bernardi, 1999; Allen, 2002). Energy for driving FO/F1 ATP synthase (ATPase) is stored as a proton (H+) motive force (pmf) across the membrane, consisting of a H+ concentration gradient (∆pH) and a membrane potential (Δψ). In mitochondria, H+ is translocated across the inner membrane from the matrix to the intermembrane space and Δψ predominately contributes to pmf (Hoek et al., 1980), though the mitochondrial inner membrane also harbors numerous ion channels (Szabó and Zoratti, 2014). In contrast, H+ is translocated across the thylakoid membrane from the stroma to the thylakoid lumen in chloroplasts. Unlike mitochondria, chloroplasts can store pmf largely in the form of ∆pH (Shikanai and Yamamoto, 2017). In photosynthesis, light energy absorbed by pigments associated with PSII and PSI drives electron transport from water to NADP+ to ultimately generate NADPH. In this linear electron transport pathway, water splitting by PSII and plastoquinol oxidation by the cytochrome (Cyt) b6f complex contributes to the formation of a ∆pH across the thylakoid membrane (Fig. 1; Allen, 2002). Additionally, cyclic electron transport around PSI also contributes to the ∆pH formation without accumulating NADPH (Shikanai and Yamamoto, 2017). The lack of the main PROTON GRADIENT REGULATION 5 (PGR5)/PGR5-like Photosynthetic Phenotype 1 (PGRL1)-dependent cyclic electron transport pathway in the Arabidopsis (Arabidopsis thaliana) pgr5 mutant resulted in severely reduced pmf (Wang et al., 2015).

Figure 1.

Figure 1.

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The proposed regulatory function of KEA3 in modulating the partitioning of pmf between two components, ∆pH and ∆ψ. In the photosynthetic linear electron transport chain (yellow arrows), water splitting by PSII and the quinone cycle at the Cyt b6f complex contribute to H+ translocation across the thylakoid membrane. Cyclic electron transport around PSI (blue arrow) also contributes to pmf formation. Because of the large buffering capacity of the thylakoid lumen, the initially transported H+ is absorbed by some anions. During this process, the energy of H+ translocation is stored in the form of ∆ψ. With the aid of thylakoid ion channels decreasing the contribution of ∆ψ, H+ accumulates in the thylakoid lumen. Both ∆ψ and ∆pH contribute to the pmf, which is ultimately used to drive ATPase activity. By antiporting H+ and K+, KEA3 likely affects the partitioning of the pmf. The activity of ATPase could be estimated by H+ conductivity through the thylakoid membrane (gH+) in the ECS decay kinetics during a dark pulse. In addition to ATP synthesis, ∆pH-dependent lumenal acidification also plays a role in regulating photosynthetic electron transport by inducing thermal dissipation at PSII, monitored as nonphotochemical quenching (NPQ) of chlorophyll fluorescence, and by downregulating the activity of the Cyt b6f complex, monitored as Y(ND) (donor-side regulation of PSI). It is important to induce donor-side regulation to avoid the acceptor-side limitation from PSI, monitored as Y(NA). Y(NA) is due to unbalanced electron input to PSI and electron uptake from PSI and may result in PSI photodamage. Fd, ferredoxin; FNR, NADP+ oxidoreductase; PQ, plastoquinone; PC, plastocyanin.

Although both components of pmf equally contribute to ATP synthesis (Soga et al., 2017), the formation of a ∆pH downregulates electron transport via acidification of the thylakoid lumen (Kanazawa and Kramer, 2002; Cruz et al., 2005b). Lumenal acidification triggers the thermal dissipation of excessively absorbed light energy from PSII antennae, a process that is monitored as an energy-dependent (qE) component of NPQ of chlorophyll fluorescence (Fig. 1; Niyogi, 1999; Li et al., 2009; Ruban, 2016). Low lumenal pH also downregulates the rate of electron transport through the Cyt b6f complex to slow down the electron transport toward PSI, monitored as the Y(ND) in Figure 1 (Tikhonov, 2013). This process is called photosynthetic control, and it is essential for preventing overreduction of the reaction center chlorophyll pair of PSI (P700) and, consequently, PSI photodamage, monitored as the acceptor-side limitation of PSI Y(NA) in Figure 1 (Suorsa et al., 2013). To maintain optimal photosynthetic performance under fluctuating environments, particularly changes in light intensity, it is necessary to properly adjust the partitioning of pmf components, as well as the total size of pmf.

If the ion permeability of the thylakoid membrane were extremely low, H+ translocated into the thylakoid lumen would be absorbed by buffering negative charges, although most H+s are exported from the thylakoid lumen via ATP synthase with the aid of the ∆ψ (Fig. 1). In this case, energy for H+ translocation is stored in the form of ∆ψ. However, earlier studies in electrode-impaled giant chloroplasts indicated that virtually all of the ∆ψ component of pmf was rapidly dissipated under continuous illumination (Bulychev et al., 1972; Vredenberg and Bulychev, 1976; Van Kooten et al., 1986), probably owing to diverse ion channels and transporters localized in the thylakoid membrane. Regulation of ion translocation across the thylakoid membrane plays a crucial role by making space for ∆pH in pmf (Pottosin and Shabala, 2016; Szabó and Spetea, 2017). To adapt to harsh light environments, terrestrial plants have developed a ∆pH-dependent regulatory system of photosynthetic electron transport (Tikhonov, 2013).

As elucidated in Figure 1, it has been proposed that KEA3 substitutes Δψ for ∆pH via antiporting H+ with K+, which is required for optimization of photosynthesis during the induction of photosynthesis and in fluctuating light environments via rapidly relaxing ∆pH-dependent downregulation (Armbruster et al., 2014; Wang et al., 2017). We have identified a dominant mutant allele of KEA3, disturbed proton gradient regulation (dpgr), which harbors a point mutation in the 11th transmembrane domain and shows a phenotype opposite the knockout (KO) allele (kea3-1) in terms of NPQ induction, suggesting that the repression of KEA3 activity may be disturbed (Wang et al., 2017). Recently, the dpgr mutation in KEA3 was shown to enhance K+ transporting activity in Escherichia coli, although H+ antiport activity was not detected (Tsujii et al., 2019). Taking this information into account, we overexpressed this dominant mutant allele of KEA3, as well as wild-type KEA3, to further obtain information about the regulation of KEA3 activity and its regulatory function during steady-state photosynthesis.

RESULTS

Overexpression of DPGR-Type KEA3 Resulted in Retarded Plant Growth

Although the KO allele of kea3-1 induces high NPQ after transition from dark to low light (110 μmol photons m−2 s−1), the amino acid substitution (Gly-422Arg) in the dpgr mutant causes a dominant mutant phenotype of reduced NPQ, suggesting elevated transport activity in the DPGR-type KEA3 (Wang et al., 2017). This dpgr phenotype is not obvious during steady-state photosynthesis, suggesting that the activity of the DPGR-type KEA3 is still regulated. To study the impact of the substitution of ∆ψ for ∆pH during steady-state photosynthesis, we overexpressed the DPGR-type KEA3, as well as wild-type KEA3, under the control of the cauliflower mosaic virus 35S promoter in the kea3-1 mutant background. Four lines accumulating high levels of KEA3 protein were selected from 42 independent DPGRox (lines 18 and 36) and 35 KEA3ox T1 plants (lines 5 and 32). Protein blot analysis in the T2 generation indicated that KEA3ox lines 5 and 32 accumulated ∼19 and ∼24 times more KEA3 protein, respectively, than in the wild type, and DPGRox lines 18 and 36 accumulated 18 and 11 times more mutant KEA3, respectively (Fig. 2B). Although overexpression of wild-type KEA3 did not affect plant growth, cotyledons of some DPGRox seedlings were yellow, and some seedlings died by 2 weeks after germination, especially in line 18 (Supplemental Fig. S1). The initial growth of DPGRox seedlings was retarded, and consequently, the seedling size was smaller after growing for 5 weeks (Fig. 2A). Blue Native-PAGE (Supplemental Fig. S2) and immunoblot analysis of thylakoid proteins (Fig. 2B) did not detect any defects in the accumulation of major protein complexes including PSII, PSI, Cyt b6f, and ATPase in any lines.

Figure 2.

Figure 2.

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Growth and protein accumulation of plants overaccumulating wild-type (WT; KEA3ox lines 5 and 32) or DPGR-type KEA3 (DPGRox lines 18 and 36). A, Plants were cultured in a growth chamber at 50 μmol photons m−2 s−1 under long-day conditions (16-h light/8-h dark) for 5 weeks. B, Immunoblot analysis of thylakoid membrane proteins. Thylakoid membrane proteins corresponding to 2 µg chlorophyll (×1) were loaded with a dilution series of wild-type proteins. For each genotype, three independent seedlings used for the physiological analysis were pooled. The antibodies used are indicated on the right. Lhca1, a subunit of the PSI light-harvesting complex; Lhcb1, a subunit of the PSII light-harvesting complex; PsaA, subunit A of the PSI reaction center; PsbA, D1 protein of PSII; PsbD, D2 protein of PSII; PetA, cytochrome f; AtpB, ß subunit of ATP synthase.

Transient NPQ Induction Was Mildly Reduced in KEA3ox and Severely Reduced in DPGRox Plants

The phenotypes of kea3-1 and dpgr mutants were most evident during the induction and relaxation of NPQ by relatively low light intensity of 110 µmol photons m−2 s−1 (Wang et al., 2017). The transient induction of NPQ observed in the wild type reflects acidification of the thylakoid lumen, followed by its relaxation by ATPase and KEA3 activity. After 30 min of dark adaptation, photosynthesis was induced by low light of 100 μmol photons m−2 s−1 using a MINI-PAM system (Fig. 3). Consistent with the previous result (Wang et al., 2017), kea3-1 induced higher NPQ than did the wild type, and this NPQ was not relaxed during 5 min of illumination (Fig. 3A). In two KEA3ox lines, NPQ was transiently induced, as in the wild type, after 1-min illumination, but the peaks were slightly lower than in the wild type. In contrast, transient NPQ induction was almost completely abolished in two DPGRox lines (Fig. 3A). In particular, in DPGRox line 36, the low level of NPQ was not relaxed in the dark after 3 min, suggesting that this line could not induce ∆pH-dependent NPQ (qE) entirely. In addition to the defect in qE induction, the quantum yield of PSII [Y(II)] was slightly reduced in DPGRox lines, but this was not the case in the KEA3ox lines (Fig. 3B).

Figure 3.

Figure 3.

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Time course of induction of photosynthesis by low light (100 μmol photons m−2 s−1) after 30 min dark adaptation. Induction and relaxation of NPQ (A) and Y(II) (B), were measured by chlorophyll fluorescence in wild-type (WT), kea3-1, KEA3ox, and DPGRox plants. Data represent means ± sd (n = 6–10 biological replicates).

∆pH-Dependent Control of Electron Transport Was Disturbed in KEA3ox and DPGRox Plants

To assess the effect of overaccumulation of wild-type and DPGR-type KEA3 on electron transport during steady-state photosynthesis, we analyzed the light intensity dependence of PSI and PSII photochemistry by measuring absorption changes in P700 and chlorophyll fluorescence, respectively, using a Dual-PAM system (Fig. 4). Consistent with the reduced NPQ induction (Fig. 3A), two DPGRox lines showed lower levels of NPQ than in the wild type at light intensities of >110 µmol photons m−2 s−1 (Fig. 4E). Mild reduction in NPQ was also observed in KEA3ox plants at moderate light intensities from 110 to 558 μmol photons m−2 s−1. The Y(ND) parameter of P700 analysis represents the donor-side regulation of PSI, which is mainly induced by ∆pH-dependent downregulation of Cyt b6f activity and was used to estimate the operation of photosynthetic control (Fig. 1; Yamamoto and Shikanai, 2019). In the wild type and kea3-1, Y(ND) was induced upon increased light intensity, representing induced photosynthetic control at the donor side of PSI (Fig. 4B). However, consistent with the observation in NPQ (Fig. 4E), the induction of Y(ND) was severely suppressed in the DPGRox plants, representing weakened photosynthetic control due to overexpressed DPGR-type KEA3 (Fig. 4B). It was mildly suppressed in the KEA3ox plants. When photosynthetic control is not induced, excess electrons transferred to PSI induce acceptor-side limitation in PSI, a process that is monitored as an increase in Y(NA) (Fig. 1). High Y(NA) was indeed observed in DPGRox plants, but not in KEA3ox plants (Fig. 4C). Probably reflecting the slightly lower Y(ND) in KEA3ox lines, the light energy used for PSI photochemistry [Y(I)] was higher in these lines than in the wild type (Fig. 4A). A similar trend was also observed in Y(II) (Fig. 4D). Taking all these findings together, we consider that excess electrons are transferred to PSI mainly due to the absence of photosynthetic control, resulting in limitation of electron acceptors from PSI in DPGRox plants. In contrast, photosynthetic control was only mildly reduced in KEA3ox plants, resulting in an increase in Y(I) rather than Y(NA).

Figure 4.

Figure 4.

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Impact of the overexpression of wild-type (WT) and DPGR-type KEA3 on steady-state photosynthesis. The light intensity dependence of P700 parameters (A–C) and chlorophyll fluorescence parameters (D and E) was determined in detached leaves from wild-type, kea3-1, KEA3ox (lines 5 and 32), and DPGRox (lines 18 and 36) plants. Parameters were Y(I) (A), Y(ND) (B). C, Acceptor-side limitation of PSI. D, Y(II). E, NPQ. Data represent means ± sd (n = 6 biological replicates). PFD, photon flux density.

Partitioning of pmf Components Was Altered in KEA3ox Plants, But the Size of the pmf Was Reduced in DPGRox Plants

It has been previously assumed that KEA3 increases the contribution of ∆ψ to pmf at the expense of ΔpH (Armbruster et al., 2014). To investigate the impact on pmf of overexpressing two types of KEA3, we performed analysis on the light intensity dependence of electrochromic shift (ECS; Fig. 5). The ECS signal represents the absorption change of photosynthetic pigments (mainly carotenoids) peaking at 515–520 nm, which depends on the presence of an electric field across the thylakoid membrane (Klughammer et al., 2013). The total amplitude of the ECS (ECSt) during the light-to-dark transition is the light-dark difference in the ECS signal, representing the total size of the pmf formed during illumination, as shown in the ECS decay kinetics (Supplemental Fig. S3). Consistent with the pmf phenotype in the KEA3 KO mutant (Armbruster et al., 2014), the total size of the pmf was not affected in the two KEA3ox lines at any light intensity (Fig. 5A). Unexpectedly, the total size of the pmf was reduced in the DPGRox lines at high light intensities of 331 and 699 μmol photons m−2 s−1 (Fig. 5A). A gH+ parameter was determined by chasing the initial fast relaxation kinetics of the ECS in the dark and is considered to represent the H+ conductivity of ATPase (Fig. 1; Kohzuma et al., 2013). gH+ was remarkably increased in two DPGRox lines, especially at high light intensities (Fig. 5B). However, this phenotype was not apparent in the KEA3ox lines, except for the slight increase observed in KEA3ox line 5 at 101 μmol photons m−2 s−1 (Fig. 5B). A similar tendency was observed in νH+, which represents the initial velocity of H+ flux across the thylakoid membrane (Fig. 5C).

Figure 5.

Figure 5.

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Impact of the overexpression of wild-type and DPGR-type KEA3 on pmf, gH+, and νH+ of the thylakoid membrane. A, The total size of the pmf and the contributions of ΔpH and Δψ to the pmf were determined in the detached leaves from wild-type (WT), kea3-1, KEA3ox (lines 5 and 32), and DPGRox (lines 18 and 36) plants by ECS analysis at the indicated light intensity. After 3 min of illumination at different light intensities, full ECS decay kinetics (ECSt) in the dark were followed to obtain the pmf, ΔpH, and Δψ, as shown in Supplemental Figure S3. All data were standardized against the 515-nm absorbance change induced by a single turnover flash. The error bars represent the SD of the total size of the pmf. Uppercase, italic lowercase, and nonitalic lowercase lettering represent the statistical differences in the total amplitudes of pmf, ΔpH, and Δψ, respectively. B, gH+ represents the H+ conductivity of the thylakoid membranes. Although it mainly depends on ATP synthase activity, the high gH+ observed in DPGRox lines likely reflects enhanced KEA3 activity. C, νH+, calculated as pmf × gH+, represents the total H+ flux through the thylakoid membrane. Data represent means ± sd (n = 6–9 biological replicates). Bars with the same letters are not significantly different between genotypes at each light intensity (Tukey’s honestly significant difference [HSD] mean-separation test, P < 0.05). PFD, photon flux density.

To investigate whether the overexpression of KEA3 altered the partitioning of pmf, we chased the ECS decay kinetics in prolonged darkness (Supplemental Fig. S3). The absolute size of the different components is compared in Figure 5A, whereas the same data are presented as the ratio of ∆pH and ∆ψ to pmf in Supplemental Figure S4. In KEA3ox plants, the absolute size of ∆ψ was significantly increased in line 32, with the highest protein level (×24) at two high light intensities (331 and 699 μmol photon m−2 s−1; Fig. 5A). The ratio of ∆ψ to pmf was also significantly higher in this line than in the wild type at 47 and 331 μmol photons m−2 s−1 (Supplemental Fig. S4). KEA3ox line 5, with a lower protein level (×19), showed a milder effect on the pmf partitioning, although a significant difference in the absolute size of ∆ψ was still observed at 331 μmol photons m−2 s−1 (Fig. 5A). At 47 and 101 μmol photons m−2 s−1, a similar trend was observed in DPGRox plants, although the difference was not statistically significant (Fig. 5A; Supplemental Fig. S4). The total size of the pmf was reduced in the two DPGRox lines at 331 and 699 μmol photons m−2 s−1 (Fig. 5A). Unexpectedly, this was accompanied by a decrease in the absolute size of ∆ψ in line 18 at 331 μmol photons m−2 s−1, which may be due to a compensative regulatory mechanism by other ion channels or transporters in the context of small pmf size under excess light condition (see “Discussion”). This phenotype could not be explained if KEA3-dependent antiport were electronically neutral. Furthermore, the absolute size of ∆ψ was larger in this line than in the wild type at 699 μmol photons m−2 s−1 (Fig. 5A).

When the ECS decay kinetics were chased in prolonged darkness, a slower rise of the ECS signal was observed after the initial fast decay (Supplemental Fig. S3). During the fast decay of the ECS, ΔpH-dependent H+ efflux induces depolarization of the thylakoid membrane. The slow inversion phase is interpreted as the relaxation of this depolarization by a compensation effect of counterion fluxes through the thylakoid membrane, such as an efflux of anions (mainly Cl−) and/or an influx of cations (mainly H+ and K+; Bailleul et al., 2010). Eventually, this phase ends once the ECS signal reaches equilibrium. The recovery of the ECS to reach equilibrium was faster in DPGRox plants than in wild-type plants (Supplemental Fig. S3). To test whether this phenomenon is linked with the high-gH+ phenotype, we analyzed the correlation between gH+ and the time needed to recover to half the level of ΔpH (Fig. 6). In all genotypes, including kea3-1, a linear correlation was observed between the two parameters at light intensities of >101 μmol photons m−2 s−1 (Fig. 6, A–C). This tight correlation between the two parameters is likely explained by the involvement of altered KEA3 activity in both phases. DPGR-type KEA3-dependent H+ reuptake may also accelerate the slow inversion phase.

Figure 6.

Figure 6.

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gH+ was linearly correlated with the recovery time to relax half of ΔpH (recovery time1/2 ΔpH) during the slow inversion phase of ECS in the prolonged dark period. Recovery time1/2 ΔpH during the slow inversion phase of ECS in the prolonged dark period, obtained from the data in Supplemental Figure S3, was plotted against the corresponding gH+ under 699 (A), 331 (B), 101 (C), and 47 μmol photons m−2 s−1 (D), obtained from data in Figure 4B. The data from all genotypes were perfectly distributed in the order DPGRox > KEA3ox > wild type (WT) > KO mutant (kea3-1), indicating that KEA3 activity is tightly tied to both the fast decay phase (the gH+ phenotype in Fig. 4B) and the slow inversion phase of ECS (Supplemental Fig. S3) in the dark.

Photosystems Were Sensitive to Fluctuating Light and High Light in DPGRox Plants

∆pH-dependent regulation of electron transport, consisting of qE induction and photosynthetic control, was mildly impaired in KEA3ox and severely impaired in DPGRox plants (Fig. 4, B and E). This discovery motivated us to test the resistance of both photosystems to stressful light conditions, fluctuating light intensity (Fig. 7), and constant high light (Fig. 8).

Figure 7.

Figure 7.

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Precise regulation of pmf components is required for photoprotection of PSI. Effects of fluctuating light on photosynthetic parameters determined by Dual-PAM in detached leaves from wild-type (WT), kea3-1, KEA3ox (lines 5 and 32), and DPGRox (lines 18 and 36) plants. Parameters were Y(I) (A), Y(ND) (B), and Y(NA) of PSI (C), Y(II) (D), and NPQ (E). Detached leaves from plants dark adapted for 30 min (black bar) were exposed to three cycles of fluctuating light. Each cycle consisted of 4 min of low light (47 μmol photons m−2 s−1; white bars) followed by 1 min of high light (1,529 μmol photons m−2 s−1; yellow bars). Data represent means ± sd (n = 6 biological replicates).

Figure 8.

Figure 8.

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Both photosystems were sensitive to constant high light in DPGRox plants. Effects of constant high light (1,000 μmol photons m−2 s−1 for 30 min) on the maximal quantum yield of PSII, Fv/Fm (A) and PSI, Pm (B) were determined in detached leaves from wild-type (WT), kea3-1, KEA3ox (lines 5 and 32), and DPGRox (lines 18 and 36) plants. Prior to the high-light treatment, Fv/Fm and Pm levels were monitored in plant leaves dark adapted for 30 min (0 min). After the high-light treatment, leaves were put on wet tissue paper and adapted to the dark for 30 min before the determinations of Fv/Fm and Pm. Data represent means ± sd (n = 5 biological replicates). Bars with the same letters are not significantly different between genotypes (Tukey’s honestly significant difference [HSD] mean-separation test, P < 0.05).

The dynamic responses of PSII and PSI operation were examined under fluctuating light consisting of three cycles (Fig. 7). Each cycle consisted of 4 min of low light (47 μmol photons m−2 s−1) followed by 1 min of high light (1,529 μmol photons m−2 s−1). Y(I) and Y(II) were gradually decreased in two DPGRox lines (Fig. 7, A and D). Contrary to the previous result, PSII was much more tolerant to the fluctuating light intensity in KEA3ox lines, although the reduction of Y(I) was evident in line 32, which accumulated a higher level of KEA3 than line 5 (Fig. 2B). Consistent with reduced NPQ during steady-state photosynthesis (Fig. 4E), NPQ induction was suppressed in both DPGRox lines in all high-light phases as well as the induction of photosynthesis in the first low-light phase, whereas impaired NPQ induction was apparent only during the induction phase in KEA3ox lines (Fig. 7E), suggesting that the activity of wild-type KEA3 could be properly regulated in response to changing light intensity even in KEA3ox lines. In the two DPGRox lines, Y(ND) was dramatically reduced in high-light phases, while Y(NA) was enhanced (Fig. 7, B and C). In contrast to NPQ induction, a similar trend in Y(ND), and to a lesser extent in Y(NA), was observed in KEA3ox plants, especially in line 32. This discrepancy could be explained by the fact that the induction of photosynthetic control requires lower lumenal pH than does qE induction (Kramer et al., 1999).

We also tested how overexpression of KEA3 impacted both photosystems after constant high-light treatment for 30 min (Fig. 8). In this experiment, we also analyzed the pgr5 mutant defective in the main pathway of cyclic electron transport around PSI (Munekage et al., 2002), since we found some similarities among phenotypes between the pgr5 mutant and DPGRox plants in terms of reduced pmf, enhanced gH+ (Wang et al., 2015), and increased Y(NA) (Yamamoto and Shikanai, 2019). The pgr5 mutant is defective in the induction of photosynthetic control and in ensuring sufficient acceptors from PSI (Yamamoto and Shikanai, 2019). We evaluated the maximum efficiency of PSII and PSI photochemistry by measuring the ratio of variable fluorescence to maximum fluorescence (Fv/Fm) and the maximal level of oxidized P700 (P700+) in the dark (Pm) before (time 0) and after, respectively, the high-light treatment (1,000 µmol photons m−2 s−1) for 30 min (Fig. 8). Both Fv/Fm and Pm were markedly reduced to levels comparable to those observed in the pgr5 mutant in two DPGRox lines, but there was no change in the KEA3ox lines after the high-light treatment (Fig. 8, A and B, 30 min). Notably, although the Pm level was reduced in pgr5 before the high-light treatment (Fig. 8B, time 0), the Fv/Fm level was unaffected (Fig. 8A, time 0). On the other hand, both Fv/Fm and Pm levels were slightly decreased in the DPGRox lines before the high-light treatment, and KEAox line 32 also showed a slight decrease in Fv/Fm (Fig. 8, A and B, time 0). In DPGRox lines and KEA3ox line 32, PSII was even sensitive to the standard growth light intensity of 50 µmol photons m−2 s−1.

DISCUSSION

In this study, we demonstrated that overexpression of two versions of KEA3 disturbed the H+ circuit in photosynthesis. Mainly based on the different impacts on the total size and partitioning of the pmf (Fig. 5A), it may be necessary to consider different reasons for the phenotypes observed between the two versions of KEA3 (Fig. 9). DPGR-type KEA3 contains a dominant point mutation in the 11th transmembrane domain and we still do not know how this mutation affects KEA3 activity. However, recently it has been confirmed that the G422R mutation in DPGR-type KEA3 increased K+ transport activity in the E. coli expression system (Tsujii et al., 2019). In KEA3ox plants, the size of the pmf was not affected, but the contribution of ∆ψ to the pmf was increased (Fig. 5A). A slight reduction in pmf may have been compensated by upregulation of electron transport in the absence of strong induction of Y(ND) in these lines (Fig. 4, A and D). This observation is consistent with previous results reported by Armbruster et al. (2016). In DPGRox plants, reduction in the total size of the pmf may also be simply explained by enhanced activity of KEA3 (Fig. 5A). However, H+ conductivity of the thylakoid membrane (gH+) was enhanced in DPGRox lines at high light intensities (Fig. 5B). In general, gH+ is believed to represent H+ conductivity of ATPase (Fig. 1; Kohzuma et al., 2013). However, the level of ATPase was unaffected (Fig. 2B) and the rate of electron transport was not upregulated in DPGRox plants (Fig. 4, A and D), suggesting that upregulation of ATPase activity was unlikely. A more straightforward idea is that the enhanced gH+ also contributed to the H+ leak through DPGR-type KEA3 (Fig. 9). If this H+ leak was coupled to the uptake of equimolar K+, this antiport would be electronically neutral and would not affect ECS decay. We thus speculate that DPGR-type KEA3 may leak more H+ than it takes up K+ (Fig. 9). If this is true, DPGR-type KEA3 is likely a version of KEA3 with an unequal antiport stoichiometry of H+ with K+. Overexpressing DPGR-type KEA3 may exaggerate the disturbed stoichiometry of antiporting, resulting in an uncoupling effect on the pmf in DPGRox plants. This idea also explains the close link between gH+ and the kinetics of the slow inversion phase of ECS kinetics in the dark (Fig. 6). H+ or K+ may be taken up by the DPGR-type KEA3 across the thylakoid membrane to restore the Δψ to its level in darkness. A critical question is whether this uncoupling activity is specific to DPGR-type KEA3. Alternatively, this activity may be intrinsic to KEA3, which is simply enhanced by the dpgr mutation. We cannot eliminate the latter possibility, because gH+ was also closely linked with the recovery time of the slow inversion phase of ECS kinetics in the dark in KEA3ox plants at 101 µmol photons m−2 s−1 (Fig. 6C). Because the protein level and activity of KEA3 were tightly regulated, the phenotypes of KEA3ox plants were evident only at moderate light intensities (101 and 331 µmol photons m−2 s−1), under which KEA3 was probably fully activated in wild-type plants (Figs. 4, 6, and 9).

Figure 9.

Figure 9.

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Schematic summary of the phenotypes in wild-type (WT), KEA3ox, and DPGRox plants under moderate-light and high-light conditions. Under limiting light, active KEA3 optimizes pmf components, whereas KEA3 is proposed to be repressed under excess light for maintaining sufficient lumenal acidification. In KEA3ox plants, overaccumulating KEA3 is still regulated during the transition from moderate to high light, and reduced NPQ and Y(ND) were evident at the moderate light intensity, under which KEA3 is active in wild-type plants. In contrast, the total size of the pmf was reduced at high light intensities in DPGRox plants, resulting in severe reductions in NPQ and Y(ND). Our hypothesis is that the disturbed stoichiometry of H+/K+ antiport caused by a point mutation in the transmembrane domain of DPGR-type KEA3 resulted in unregulated H+ leak from the thylakoid lumen, which may contribute to the high-gH+ phenotype.

When the size of the pmf is reduced, plants likely regulate the partitioning of pmf components so that ∆pH does not decrease below a certain threshold. We also observed this effect in the pgr5 mutant (Yamamoto et al., 2016). Unspecified channels or transporters localized to the thylakoid membrane are likely involved in this compensation. One candidate is TPK3, a thylakoid-localized two-pore K+ channel that mediates K+ efflux from the thylakoid membrane (Carraretto et al., 2013), although TPK3 was recently shown to localize to the vacuole (Höhner et al., 2019). Other candidates include two bestrophin-like anion channels, AtBest1 and AtBest2 (Duan et al., 2016), which were renamed voltage-dependent chloride channels VCCN1 and VCCN2 (Herdean et al., 2016b) and which mediate Cl− influx into the lumen. A final candidate is CLCe, a member of the Cl− channel/transporter CLC family that was also localized to the thylakoid membrane (Marmagne et al., 2007; Lv et al., 2009). Phenotypic characterization supports its role in Cl− homeostasis in chloroplasts for regulating electron transport during dark-to-light and light-to-dark transitions (Herdean et al., 2016a). Reduction in the contribution of Δψ to the pmf in DPGRox lines at 331 μmol photons m−2 s−1 may be explained by this compensatory mechanism (Fig. 5A).

In this study, we aimed to clarify why plants may select ∆pH rather than Δψ as the main component of the pmf. Because of the disturbed activity of the DPGR-type KEA3, the size of the pmf was reduced in DPGRox plants at high light intensities (Fig. 5A). In KEA3ox plants, however, ∆pH was partially substituted by Δψ and the size of the pmf was not affected. On the basis of the phenotypes of KEA3ox plants, we conclude that full induction of Y(ND) is dispensable under constant moderate light intensities. Reduced induction of Y(ND) slightly increased yields of both photosystems (Fig. 4, A and D), but a higher contribution of Δψ to the pmf made PSI more sensitive to fluctuating light in KEA3ox plants (Fig. 7). Unexpectedly, the maximum activity of PSII (Fv/Fm) was slightly lower even under the growth light (∼50 μmol photons m−2 s−1) in the DPGRox lines and KEA3ox line 32 (Fig. 8B). We observed a reduced contribution of ∆pH to the pmf in the same lines and at light intensity close to the growth light (47 μmol photons m−2 s−1; Fig. 5A; Supplemental Fig. S4). This finding likely supports the idea that an elevated Δψ component of pmf, rather than lumen acidification, increases PSII charge recombination rates, producing singlet oxygen and subsequent photodamage (Davis et al., 2016). In addition to PSII photodamage, photodamage of PSI also occurred in DPGRox lines under growth-light conditions. Even at low light intensities, a certain level of ∆pH may be necessary to protect PSII (Takahashi et al., 2009) and PSI.

Notably, we found some similarities in phenotypes between DPGRox plants and the Arabidopsis pgr5 mutant, which is defective in the main route of PSI cyclic electron transport (Munekage et al., 2002), in particular with respect to impaired ∆pH-dependent regulation of electron transport, reduced pmf, enhanced gH+ at high light intensities, and, consequently, sensitivity to stressful light conditions (Figs. 4, 5, 7, and 8). However, the level of gH+ is higher in DPGRox plants (∼70 s−1) than in pgr5 (∼45 s−1) under comparable light intensities (Wang et al., 2015). In contrast, Y(ND) and Y(NA) levels are more severely affected in pgr5 than in the DPGRox lines (Yamamoto and Shikanai, 2019), likely reflecting the distinct reasons for the related phenotypes. Regulation of the size and composition of the pmf is probably closely linked via a network consisting of the regulatory machinery for electron transport and ion transport.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) kea3-1 mutant (GABI_170G09 line) has been described previously in Wang et al. (2017). To generate kea3-1 plants overexpressing the KEA3 variants, the wild-type or dpgr genomic sequence encoding KEA3 was amplified using the primers 5′-ATG​GCA​ATT​AGT​ACT​ATG​TTA​GG-3′ and 5′-TTA​ATC​TTG​AGC​TTT​ATC​AGC​TT-3′. The PCR product was cloned into pDONR/Zeo by BP Clonase reaction (Invitrogen). The resulting plasmid was confirmed by sequencing and then transferred to the binary vector pGWB2 by LR Clonase reaction (Invitrogen). Agrobacterium tumefaciens was transformed with the plasmids by electroporation and the bacteria were used to transform kea3-1 mutant plants via the floral dip method (Clough and Bent, 1998). Transformed plants were selected on Murashige and Skoog medium containing 50 μg/mL kanamycin, and their T2 generation was used for analyses. Arabidopsis wild type Columbia gl1 and mutant plants were grown on soil for 4–5 weeks under long-day chamber conditions (50 μmol photons m−2 s−1, 16-h light/8-h dark cycles at 23°C).

SDS-PAGE and Immunoblot Analyses

Thylakoid membranes were purified from leaves of 4- to 5-week-old plants as previously described by Wang et al. (2017). Chlorophyll concentration was determined as described previously in Porra et al. (1989). Membrane proteins were solubilized in the 2×SDS-PAGE sample buffer. Proteins corresponding to 2 μg chlorophyll were separated by 12.5% (w/v) SDS-PAGE (Schägger, 2006) and electrotransferred onto polyvinylidene fluoride membranes. The antibodies were added and the protein-antibody complexes were labeled using an ECL Prime 48 western blotting detection system (GE Healthcare). Chemiluminescence was detected with a lumino-image analyzer LAS4000 (GE Healthcare).

In Vivo Measurements of Chlorophyll Fluorescence and P700 Absorption Changes

Chlorophyll fluorescence parameters were measured using a MINI-PAM portable chlorophyll fluorometer (Walz) as shown in Figure 2 or a Dual-PAM 100 (Walz) as shown in Figures 3, 6, and 7. Plants were dark adapted for 30 min before measurements. Chlorophyll fluorescence parameters were measured as described previously (Shikanai et al., 1999). The maximum quantum yield of PSII and NPQ were calculated as Fv/Fm and (Fm – Fm′)/Fm′, respectively. Y(II) was calculated as (Fm – Fs)/Fm, where Fs is the steady-state fluorescence. The redox change of P700 was assessed by monitoring the absorbance changes of transmission light at 830 and 875 nm. Pm was determined by the application of a saturation pulse (SP) in the presence of far-red light (720 nm). The maximal level of P700+ during actinic light (AL) illumination (Pm′) was determined by an SP application. The steady-state P700+ level, P, was recorded just before applying a SP. The quantum yield of PSI, Y(I), was calculated as (Pm′ − P)/Pm. Y(NA) was calculated as (Pm − Pm′)/Pm. Y(ND) was calculated as P/Pm. Three complementary quantum yields were defined: Y(I) + Y(NA) + Y(ND) = 1 (Klughammer and Schreiber, 1994).

To analyze the effect of fluctuating light on photosynthetic electron transport, three repetitions of fluctuating light cycle composed of 4 min of low light (47 µmol photons m−2 s−1) and 1 min of high light (1,529 µmol photons m−2 s−1) were applied by changing the AL intensity, as described previously (Yamamoto et al., 2016). Detached leaves from plants dark adapted for 30 min were subjected to fluctuating light treatment. To test the effect of constant high light exposure on photoinhibition, detached leaves from plants dark adapted for 30 min were exposed to high light of 1,000 μmol photons m−2 s−1 for 30 min after the measurement of initial Fv/Fm and Pm levels. After the treatment, the leaves were sandwiched between wet tissue papers and incubated in the dark for 30 min, and then Fv/Fm and Pm levels were measured again to evaluate PSII and PSI photoinhibition.

ECS Measurements

The ECS measurements were carried out using a Dual-PAM 100 equipped with a P515/535 module (Walz). Plants were first dark adapted for 30 min and then their detached leaves were analyzed. The ECS signal was obtained after 3 min of illumination at different AL intensities and then AL was turned off for 1 min to record ECSt and to chase the decay curve in the dark. ECSt represents the size of the light-induced pmf and was estimated from the total amplitude of the rapid decay of the ECS signal in the dark, as described previously (Wang et al., 2015). ECSt levels were normalized against a 515-nm absorbance change induced by a single turnover flash, as measured in dark-adapted leaves before recording. This normalization accounted for variations in leaf thickness and chloroplast density between the leaves (Takizawa et al., 2008). gH+ was estimated by fitting the first 300 ms of the decay curve with a first-order exponential decay kinetic as the inverse of the decay time constant, and νH+ was calculated as pmf × gH+, as described earlier (Avenson et al., 2005). The relative partitioning of the pmf into ΔpH and Δψ was analyzed as previously described (Cruz et al., 2005a). The ECS steady state and ECS inverse were extracted from the traces to estimate ΔpH and Δψ.

Statistical Analyses

One-way ANOVA (Tukey’s honestly significant difference [HSD] mean-separation test) was used to analyze two averages of the data in Figures 5 and 8. The linear correlation of gH+ with the recovery time to relax half the level of ΔpH during the slow inversion phase of ECS in the prolonged dark period (Fig. 6) was analyzed by linear regression, not constrained to pass through the origin.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers NP_001190675.1 (KEA3, gene ID 825822) and NP_001318202.1 (PGR5, gene ID 815111).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Dr. Ute Armbruster (Max Planck Institute for Plant Physiology), Dr. Shigeichi Kumazaki (Kyoto University), Nobuyuki Uozumi (Tohoku University), and Giovanni Finazzi (Commissariat à l'Énergie atomique-Grenoble) for their critical discussion. We thank Brody Frink in our group for his help with English editing.

Footnotes

1

This work was supported by grants from the Human Frontier Science Project and the Japan Society for the Promotion of Science (16H06555 and 19H00992).

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