Contribution of Cyclic and Pseudo-cyclic Electron ... - Cell Press

Molecular Plant Review Article

Contribution of Cyclic and Pseudo-cyclic Electron Transport to the Formation of Proton Motive Force in Chloroplasts Toshiharu Shikanai1,2,* and Hiroshi Yamamoto1,2 1

Department of Botany, Graduate School of Science, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 Japan

2

CREST, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0076 Japan

*Correspondence: Toshiharu Shikanai ([email protected]) http://dx.doi.org/10.1016/j.molp.2016.08.004

ABSTRACT Photosynthetic electron transport is coupled to proton translocation across the thylakoid membrane, resulting in the formation of a trans-thylakoid proton gradient (DpH) and membrane potential (Dc). Ion transporters and channels localized to the thylakoid membrane regulate the contribution of each component to the proton motive force (pmf). Although both DpH and Dc contribute to ATP synthesis as pmf, only DpH downregulates photosynthetic electron transport via the acidification of the thylakoid lumen by inducing thermal dissipation of excessive absorbed light energy from photosystem II antennae and slowing down of the electron transport through the cytochrome b6f complex. To optimize the tradeoff between efficient light energy utilization and protection of both photosystems against photodamage, plants have to regulate the pmf amplitude and its components, DpH and Dc. Cyclic electron transport around photosystem I (PSI) is a major regulator of the pmf amplitude by generating pmf independently of the net production of NADPH by linear electron transport. Chloroplast ATP synthase relaxes pmf for ATP synthesis, and its activity should be finely tuned for maintaining the size of the pmf during steady-state photosynthesis. Pseudo-cyclic electron transport mediated by flavodiiron protein (Flv) forms a large electron sink, which is essential for PSI photoprotection in fluctuating light in cyanobacteria. Flv is conserved from cyanobacteria to gymnosperms but not in angiosperms. The Arabidopsis proton gradient regulation 5 (pgr5) mutant is defective in the main pathway of PSI cyclic electron transport. By introducing Physcomitrella patens genes encoding Flvs, the function of PSI cyclic electron transport was substituted by that of Flv-dependent pseudo-cyclic electron transport. In transgenic plants, the size of the pmf was complemented to the wild-type level but the contribution of DpH to the total pmf was lower than that in the wild type. In the pgr5 mutant, the size of the pmf was drastically lowered by the absence of PSI cyclic electron transport. In the mutant, DpH occupied the majority of pmf, suggesting the presence of a mechanism for the homeostasis of luminal pH in the light. To avoid damage to photosynthetic electron transport by periods of excess solar energy, plants employ an intricate regulatory network involving alternative electron transport pathways, ion transporters/channels, and pH-dependent mechanisms for downregulating photosynthetic electron transport. Key words:: alternative electron transport, cyclic electron transport around photosystem I, ion channel, flavodiiron protein, proton motive force, pseudo-cyclic electron transport Shikanai T. and Yamamoto H. (2017). Contribution of Cyclic and Pseudo-cyclic Electron Transport to the Formation of Proton Motive Force in Chloroplasts. Mol. Plant. 10, 20–29.

INTRODUCTION Light reactions of photosynthesis convert solar energy into chemical energy in the form of NADPH and ATP, which are utilized for many cellular reactions, including CO2 assimilation. In oxygenic photosynthesis, NADPH and ATP are mainly synthesized by linear electron transport from water to NADP+ (Figure 1). 20

Molecular Plant 10, 20–29, January 2017 ª The Author 2017.

Coupled to this electron transport, protons (H+) are released by water splitting in photosystem II (PSII) and the quinone (Q) cycle in the cytochrome (Cyt) b6f complex. Movement of two

Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

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pseudoCET NADP+ + H+ NADPH O2 + 4H+ 2H2O FNR Flv SOD

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Figure 1. Schematic Representation of Photosynthetic Electron Transport. Linear electron transport (LET) from water to NADP+ is indicated by the green arrows. Two pathways of cyclic electron transport (CET) around PSI are indicated by the red arrows. The main CET pathway is mediated by the PGR5/PGRL1 complex, which interacts with PSI. On the other hand, the minor pathway depends on chloroplast NDH, which forms the supercomplex with PSI. The blue arrows indicate two pathways of pseudo-cyclic electron transport (pseudoCET). The authentic water–water cycle depends on O2 reduction around PSI. Resulting ROS is scavenged by superoxide dismutase (SOD) and ascorbate peroxidase (APX). Flv is conserved from cyanobacteria to gymnosperms but not in angiosperms. Flv mediates direct reduction of O2 to H2O. The electron donor in vivo is likely NAD(P)H or Fd. CET and pseudoCET are called alternative electron transport and contribute to pmf formation and consequently ATP synthesis without net accumulation of NADPH.

electrons (e ) originating from water splitting is linked to transfer of six H+ from the stroma to the thylakoid lumen in linear electron transport. To synthesize three molecules of ATP from ADP, 14 H+ are considered to be required (Seelert et al., 2003; Vollmar et al., 2009). The production ratio of ATP/NADP by linear electron transport is approximately 1.29, which cannot satisfy the ratio of 1.5 required by the Calvin-Benson cycle (Allen, 2002). In C3 plants, the operation of photorespiration increases the ratio up to 1.67. To satisfy the ATP/NADPH production ratio, supplemental mechanisms for ATP synthesis (regulation of H+/e stoichiometry) are needed. Alternative electron transport contributes to this compensation of ATP synthesis (Figure 1). In cyclic electron transport around photosystem I (PSI), electrons are transferred from ferredoxin (Fd) to the plastoquinone (PQ) pool, generating a trans-thylakoid H+ gradient (DpH) without net production of NADPH (Shikanai, 2007; Yamori and Shikanai, 2016). In the water–water cycle, electrons from PSI are transferred to O2, generating superoxide (Asada, 2000). The resulting reactive oxygen species (ROS) are scavenged by superoxide dismutase (SOD) and ascorbate peroxidase (APX) (Figure 1). This ROS scavenging system consumes the reducing equivalents generated by PSI, Fd, and NADPH for the regeneration of ascorbate. One idea is that the water–water cycle consumes excessively absorbed light energy and contributes to regulating the H+/e stoichiometry. However, the rate of O2 reduction is low in most steady-state conditions in angiosperms, suggesting that the contribution of the water–water cycle to this regulation is not significant (Badger et al., 2000; Shirao et al., 2013). In addition to DpH, trans-thylakoid membrane potential (Dc) also contributes to ATP synthesis as another component of proton

motive force (pmf). Although both DpH and Dc contribute to ATP synthesis, another function of DpH is the downregulation of photosynthetic electron transport by acidifying the thylakoid lumen (Shikanai, 2014, 2016). The regulation involves two mechanisms. The first is thermal dissipation of excessively absorbed light energy as heat from PSII antennae. This process is monitored as an energization-dependent (qE) component of non-photochemical quenching (NPQ) of chlorophyll fluorescence (Niyogi, 1999). The second is downregulation of Cyt b6f complex activity, which is called photosynthetic regulation (Stiehl and Witt, 1969). This regulation is especially important for protecting PSI by downregulating the rate of electron transport toward PSI under fluctuating light conditions (Suorsa et al., 2012). The Arabidopsis thaliana proton gradient regulation 1 (pgr1) mutant has an amino acid alteration in the Rieske subunit of the Cyt b6f complex, resulting in hypersensitivity of the complex to luminal acidification (Munekage et al., 2001; Jahns et al., 2002). In the pgr1 mutant, PSII was sensitive to high light, suggesting the physiological significance of lowering luminal pH in the light.

CONTRIBUTION OF PSI CYCLIC ELECTRON TRANSPORT TO PMF In PSI cyclic electron transport, electrons are transferred from Fd to the PQ pool, consequently generating DpH via the Q cycle of the Cyt b6f complex without net accumulation of NADPH (Figure 1). In angiosperms including Arabidopsis, two pathways of PSI cyclic electron transport are operating (Munekage et al., 2004). The first cyclic electron transport pathway to be described was discovered by Arnon and coworkers and is sensitive to antimycin A (Tagawa et al., 1963). The Arabidopsis pgr5 mutant is defective in PSI cyclic electron transport Molecular Plant 10, 20–29, January 2017 ª The Author 2017.

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of NdhS was necessary for the electrostatic interaction with Fd (Yamamoto and Shikanai, 2013). In Arabidopsis, chloroplast NDH forms the supercomplex with PSI via two minor lightharvesting complex I proteins, Lhca5 and Lhca6 (Peng et al., 2009). We do not eliminate the possibility that chloroplast NDH accepts electrons from NADPH via Fd coupled to the reverse reaction of Fd-NADP+ oxidoreductase (FNR) in the dark (chlororespiration). Despite the structural similarity to respiratory NADH dehydrogenase, however, the function of chloroplast NDH is more tightly linked to photosynthesis (Shikanai, 2015).

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sensitive to antimycin A (Munekage et al., 2002; Sugimoto et al., 2013). The two discoveries probably involved the same electron transport. Machinery mediating this electron transport (Fd-PQ oxidoreductase [FQR]) has long been unclear. PGR5-Like Photosynthetic Phenotype 1 (PGRL1) protein accepts electrons from Fd and donates them to a PQ analog in vitro (Hertle et al., 2013). PGRL1 interacts with PGR5, which is required to reduce PGRL1 by Fd in vivo (DalCorso et al., 2008), suggesting that the PGR5/PGRL1 complex is an elusive FQR (Hertle et al., 2013), although conflicting ideas still exist.

On the basis of the current model of the PGR5/PGRL1 complex (Labs et al., 2016), the pmf formation of the antimycin A-sensitive pathway solely depends on the Q cycle of the Cyt b6f complex. In contrast, conservation of the membrane subcomplex (P module in respiratory NADH dehydrogenase) suggests that chloroplast NDH pumps H+, coupled with electron transport from Fd to PQ (Baradaran et al., 2013). In the current model, recycling of two e from Fd to PSI is coupled with translocation of four H+ in the PGR5-/PGRL1-dependent pathway via the Q cycle, whereas eight H+ are translocated by the movement of two e , in the NDH-dependent pathway; four H+ depend on the Q cycle and the additional four H+ depend on putative H+-pumping activity of NDH. These theoretical considerations suggest that PSI cyclic electron transport via chloroplast NDH represents a more efficient pathway to generate pmf than PSI cyclic electron transport via PGR5/PGRL1. However, the contribution of the PGR5-/PGRL1-dependent pathway is more significant in C3 plants (Munekage et al., 2004; Okegawa et al., 2008; Wang et al., 2015). Chloroplast NDH probably has H+-pumping activity and may not function efficiently in the presence of large pmf or DpH at high light intensities. This idea is consistent with the fact that the function of chloroplast NDH is significant in low light in Marchantia polymorpha (Ueda et al., 2012) and rice (Oryza sativa) (Yamori et al., 2015). Under high light conditions, NADPH production is more than necessary for CO2 fixation; it is probably not important for plants to keep the same ATP/NADPH production ratio and consequently H+/e stoichiometry between low light and high light conditions. As discussed below, plants relax Dc by opening a K+ channel, TPK3, when the qE component of NPQ is induced for the substitution of Dc with DpH (Carraretto et al., 2013). Under these conditions, light energy is sufficient to keep the size of the pmf even under conditions of partial uncoupling between electron transport and pmf formation via the channel.

Another pathway of PSI cyclic electron transport discovered in angiosperms depends on the chloroplast NADH dehydrogenase-like (NDH) complex (Burrows et al., 1998; Shikanai et al., 1998). The complex was originally discovered by the complete determination of two plastid genomes in tobacco (Nicotiana tabacum) (Shinozaki et al., 1986) and Marchantia polymorpha (Ohyama et al., 1986). Because 11 ndh genes encode homologs of NADH dehydrogenase (complex I) subunits in mitochondria (Matsubayashi et al., 1987), chloroplast NDH has been believed to accept electrons from NADH or NADPH (Shikanai, 2007). However, chloroplast NDH lacks the N module involved in NADH oxidation (Shikanai, 2015; Peltier et al., 2016) and in fact accepts electrons from Fd rather than NADPH (Yamamoto et al., 2011). A positive surface charge

Electrochromic shift (ECS) represents an absorbance change by photosynthetic pigments, which is affected by Dc (Cruz et al., 2001; Bailleul et al., 2010). The difference in ECS signals in light and after a 100-ms dark pulse (ECSt) approximates the total size of the pmf formed in the light. In the pgr5 mutant, the size of the pmf was drastically reduced (Wang et al., 2015; Figure 2). In Arabidopsis, the contribution of NDH-dependent PSI cyclic electron transport to the total PSI cyclic electron transport activity was estimated to be minor (Munekage et al., 2004; Okegawa et al., 2008). However, we detected a statistically significant reduction in the size of pmf in NDH-less mutants (Wang et al., 2015). Because linear electron transport was not affected in these NDH-less mutants, this small reduction in pmf directly reflects the contribution of NDH to pmf formation. In double

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Figure 2. Size and Components of pmf in Arabidopsis Plants Operating Different Alternative Electron Transport. The pgr5 mutant is defective in the main pathway of PSI cyclic electron transport, leading to drastic reduction in the size of the pmf. The contribution of DpH was higher in the pgr5 mutant that that in the wild-type. The pgr5 mutant was transformed by the FlvA and FlvB genes originating from Physcomitrella patens (pgr5+35S; PpFlv no. 13). Although the size of the pmf was complemented to the wild-type level, the contribution of DpH was lower than that in the wild-type, leading to partial complementation of NPQ (Yamamoto et al., 2016). ECS signals were analyzed at different photon flux densities (PFD; mmol photons m 2 s 1). The error bars represent the SD with biological replicates (n = 5). This figure is a different presentation of the same data that was published previously (Yamamoto et al., 2016).

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Regulation of Proton Motive Force mutants defective in both pathways of PSI cyclic electron transport, photosynthesis and plant growth were severely impaired (Munekage et al., 2004). In double mutants, the size of the pmf was more severely reduced than that in single mutants (Wang et al., 2015). Both pathways of PSI cyclic electron transport contribute to pmf formation during steady-state photosynthesis.

HOW IS THE PMF REGULATION DISTURBED IN THE ARABIDOPSIS PGR5 MUTANT? PSI cyclic electron transport contributes to pmf formation independently of NADPH production, balancing the production ratio of ATP/NADPH (Shikanai, 2007). In Arabidopsis pgr5 mutants, NPQ induction was impaired and the electron transport rate at PSII was saturated at lower levels at lower light intensity than that in the wild-type (Munekage et al., 2002). These phenotypes can be explained by the reduced size of pmf, consequently limiting CO2 fixation at the ATP level. Unexpectedly, the size of H+ conductivity (gH+) of the thylakoid membrane was increased in the pgr5 mutant (Avenson et al., 2005). The gH+ parameter in ECS analysis is considered to mainly represent H+ conductivity of ATP synthase (Kramer et al., 2004). Despite the higher H+ conductivity of ATP synthase, the smaller size of the pmf resulted in a decrease in the actual rate of ATP synthesis (vH+) in the pgr5 mutant. On the basis of the reduction in vH+, Avenson et al. (2005) calculated a 13% contribution of PGR5-/ PGRL1-dependent PSI cyclic electron transport to the total rate of ATP synthesis under steady-state conditions. Upregulation of gH+ was mainly observed at high light intensities in the pgr5 mutant. The high gH+ level induced by high light treatment was lowered to a low light level for 15 min (Wang et al., 2015). This result suggests that the high gH+ observed in the pgr5 mutant at high light intensities is unlikely to be due to photodamage of the thylakoid membrane. Notably, the same response of gH+ to high light was observed in wild-type plants but with a much lower amplitude of response than that in the pgr5 mutant (Wang et al., 2015). The high level of gH+ observed in the pgr5 mutant likely reflects the regulatory process, which also takes place in the wild-type but with a lower amplitude. H+ conductivity of ATP synthase may be controlled by monitoring the ATP level. A possible explanation for the pgr5 phenotype in gH+ is regulation via high concentrations of inorganic phosphate in the stroma (Avenson et al., 2005). However, this idea has not been supported by any biochemical evidence. It has only been clarified that ATP synthase activity is regulated by several light– dark switches of activity, including thiol modification of the g subunit, preventing wasteful consumption of ATP in the dark (Hisabori et al., 2013). In bacterial F-type ATP synthase, the ATP-binding site present in the 3 subunit may function as a sensor of the ATP level (Krah and Takada, 2016). In leaves, the level of gH+ is drastically reduced in CO2-free air, increasing the sensitivity of the qE machinery to the rate of electron transport generating pmf (Kanazawa and Kramer, 2002). Under drought stress, stomata closure limits CO2 uptake into chloroplasts, slowing down the rate of CO2 fixation. Because the rate of linear electron transport is also slowed down, it is physiologically reasonable to increase the sensitivity of NPQ induction to the rate of linear electron transport. For this

Molecular Plant purpose, there are two strategies: (1) activation of alternative electron transport including PSI cyclic electron transport, and (2) downregulation of ATP synthase activity to keep a high level of DpH (Kanazawa and Kramer, 2002). On the regulation of ATP synthase activity in chloroplasts, there is a gap between spectroscopic studies and biochemistry. The biochemical characterization of the pgr5 phenotype in gH+ might bridge this gap. The high gH+ observed in pgr5 suggests that plants may have a compensatory mechanism for a reduced rate of ATP synthesis. This mechanism may also function in wild-type plants during the transition from low light to high light, because a slight increase in gH+ was observed in the wild-type after transition to high light (Wang et al., 2015). In the pgr5 mutant, however, this mechanism failed to compensate for the reduced ATP level to the wild-type level. Furthermore, the increase in gH+ likely causes a further decrease in the pmf level during steady-state photosynthesis. Most seriously, the reduced level of DpH (high lumen pH) disturbed the downregulation of electron transport via qE induction and downregulation of the Cyt b6f complex (Shikanai, 2014). The resulting increase in electron transfer to PSI is accompanied by lower levels of ATP synthesis. This in turn limits the CalvinBenson cycle and consequently leads to a shortage in electron acceptors from PSI, NADP+. Consequently, electrons are trapped in PSI, causing serious PSI photodamage (Munekage et al., 2002). Because of this problem, the pgr5 mutant could not survive in fluctuating light (Tikkanen et al., 2010).

PSEUDO-CYCLIC ELECTRON TRANSPORT MEDIATED BY FLAVODIIRON PROTEINS Flavodiiron protein (Flv) forms a large family of enzymes catalyzing O2 or NO reduction to H2O and N2O, respectively (Allahverdiyeva et al., 2015). A lactamase-like domain containing a non-heme diiron center is involved in substrate reduction, whereas a flavodoxin-like domain, containing one flavin mononucleotide moiety, accepts electrons from donors. Class C Flvs conserved in phototrophs are further equipped with a C-terminal flavin-reductase-like domain, allowing these proteins to accept electrons directly from NAD(P)H. The electron donors in vivo have still not been conclusively determined, although recombinant protein accepts electrons from NAD(P)H (Vicente et al., 2002). Flv2 and Flv4 are conserved in a limited group of cyanobacteria and function in the photoprotection of PSII, although their exact molecular function is unclear (Zhang et al., 2012; Shimakawa et al., 2015). In contrast, the Flv1/Flv3 heterodimer mediates the Mehler-like reaction, mediating a kind of water–water cycle to protect PSI under fluctuating light conditions (Helman et al., 2003; Allahverdiyeva et al., 2013). In Synechocystis sp. PCC 6803, 20% of electrons originating from water splitting were used to reduce O2 via the Flv1/Flv3 heterodimer (Allahverdiyeva et al., 2011). Flv1 and Flv3 are conserved from cyanobacteria to gymnosperms, including green algae and bryophytes, but not in angiosperms (Allahverdiyeva et al., 2015). Notably, higher activity of O2 reduction was observed in gymnosperms (corresponding to 10% of the maximum O2 evolution at PSII) than in angiosperms (corresponding to 1% of the maximum O2 evolution at PSII) Molecular Plant 10, 20–29, January 2017 ª The Author 2017.

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Molecular Plant (Shirao et al., 2013). This higher O2 uptake activity in gymnosperms may depend on the function of Flv.

COMPLEMENTATION OF PSI CYCLIC ELECTRON TRANSPORT BY FLV Why have angiosperms not conserved Flv in evolution? To address this question, we cloned two genes encoding Flvs, FlvA and FlvB, from a bryophyte, Physcomitrella patens, and introduced these genes into the Arabidopsis pgr5 mutant defective in the main antimycin A-sensitive pathway of PSI cyclic electron transport, and also into wild-type Arabidopsis (Yamamoto et al., 2016). Exogenous Flvs formed the heterotetramer in Arabidopsis chloroplasts. The main question is whether pseudo-cyclic electron transport (Flv-dependent water–water cycle) can complement the function of PGR5-/ PGRL1-dependent PSI cyclic electron transport in balancing the ATP/NADPH production ratio. In the pgr5 mutant, P700, a PSI reaction center chlorophyll dimer, is reduced by electrons in the light, although P700 is mostly oxidized in the wild-type (Munekage et al., 2002). This is because of the limitation of electron acceptors from PSI and the disturbed downregulation of electron transport through the Cyt b6f complex. In the pgr5 mutant accumulating Flv, P700 was oxidized, as in the wild-type (Yamamoto et al., 2016). Consistent with this fact, the level of pmf was also restored to the wild-type level (Figure 2). Direct rate analysis of Flv-dependent O2 reduction in a pgr5 mutant background indicated that 25% of electrons originating from water splitting at PSII were used to reduce O2 via Flv (Yamamoto et al., 2016). The rate observed in the pgr5 background is similar to that observed in cyanobacteria (Allahverdiyeva et al., 2011) and higher than that in gymnosperms (Shirao et al., 2013). The Flv-dependent O2 uptake was accompanied by an increase in O2 evolution at PSII, resulting in a 25% increase in PSII yield at high light intensities compared with that in the wild-type. Although Flv consumes the reducing power accumulating in the stroma, Flv does not compete with the Calvin-Benson cycle. The rate of CO2 fixation was higher in the pgr5 mutant accumulating Flv than that in the nontransgenic pgr5 mutant and even higher than that in the wildtype under high CO2 conditions, which was necessary to avoid photorespiration-dependent O2 uptake in the assay system (Yamamoto et al., 2016). This phenotype may be explained by the reduced restriction of electron transport at the Cyt b6f complex because of the lower contribution of DpH to total pmf (higher lumen pH) than that in the wild-type. Flv-dependent pseudo-cyclic electron transport is accompanied by water splitting at PSII in addition to the Q cycle in the Cyt b6f complex. Pseudo-cyclic electron transport should be more efficient in pmf formation than PGR5-/PGRL1-dependent PSI cyclic electron transport. Because the rate of PGR5-/PGRL1dependent PSI cyclic electron transport was estimated to contribute to 13% of the total rate of ATP synthesis in wild-type plants, Flv-dependent pseudo-cyclic electron transport, operating at a 25% rate of linear electron transport, likely fully complements pmf formation. Despite the full complementation in the size 24


Regulation of Proton Motive Force of the pmf in the pgr5 mutant accumulating Flv, the induction of NPQ was only partially complemented at moderate light intensities (100–800 mmol photons m 2 s 1) but was complemented to the wild-type level at 1200 mmol photons m 2 s 1 (Yamamoto et al., 2016). This discrepancy observed between pmf and NPQ can be explained by the fact that the contribution of Dc was larger in the pgr5 mutant accumulating Flv than that in the wild-type (Figure 2). Despite the full complementation of the total size of the pmf, the lumen pH was higher in the transgenic plants. We do not have any experimental evidence for discussing the molecular mechanism for altering the ratio of DpH and Dc in transgenic lines. A possible explanation is that PGR5-/PGRL1-dependent PSI cyclic electron transport is linked to the regulation of pmf components, ion transporters, or channels (see below). Because Flv-dependent pseudo-cyclic electron transport is an artificial system of pmf formation originating from Physcomitrella patens, it may be independent of the endogenous regulatory system of pmf components. It is necessary to understand this network of pmf regulation.

MECHANISM FOR PH HOMEOSTASIS IN THE THYLAKOID LUMEN An unexpected discovery was the extremely high contributions of DpH to total pmf in the pgr5 mutant (Figure 2). In the pgr5 mutant, the total pmf is approximately half of that in the wild-type at more than 312 mmol photons m 2 s 1 (Yamamoto et al., 2016). At these light intensities, the contribution of DpH to the total pmf was approximately 90% in the pgr5 mutant, whereas it was from 70% to 80% depending on light intensity in the wild-type (Figure 2). Consequently, the contribution of Dc in the pgr5 mutant was extremely low (less than 10%). These results suggest the presence of an unknown compensatory mechanism for luminal acidification in the light in the absence of a sufficient size of pmf. Even with this mechanism, the absolute size of DpH in the pgr5 mutant was from 60% to 70% of that formed in the wild-type, resulting in insufficient downregulation of electron transport observed in the pgr5 mutant. In addition to the downregulation of electron transport, low lumen pH contributes to alleviating the photodamage of PSII (Takahashi et al., 2009). In light, the steady-state PSII level is determined by the balance between photodamage of PSII and the repair of damaged PSII. A current idea is that PSII photoinhibition is one of mechanisms for protecting PSI from photoinhibition rather than the inevitable disruption by excessive light energy (Tikkanen et al., 2014; Ja¨rvi et al., 2015). This is a tradeoff between the maximum activity of photosynthesis and photoprotection. PGR5-/PGRL1-dependent PSI cyclic electron transport regulates the rate of PSII photoinhibition by inducing a qE component of NPQ. However, the pgr5 mutant is more sensitive to high light than the Arabidopsis nonphoptochemical quenching 4 (npq4) mutant defective in the pH sensor protein, PsbS, for qE induction. In the presence of inhibitors of the PSII repair cycle (inhibitors of protein synthesis), PSII was still more sensitive to high light in the pgr5 mutant than that in the wildtype, suggesting that PSII photodamage was accelerated more in the pgr5 mutant, probably because of the higher lumen pH in the light (Takahashi et al., 2009). In contrast to mitochondria, in

Regulation of Proton Motive Force which the pmf consists of mainly Dc, the contribution of DpH is higher in chloroplasts. Consequently, the thylakoid lumen is acidified in the light. A low lumen pH induces restriction of electron transport. In addition, the machinery of electron transport has been adapted to this pH range for operating in the light. Alternative electron transport pathways also had to be optimized to facilitate luminal acidification in the light with the aid of regulators of pmf components (see below).

COMPETITION BETWEEN THE PGR5-/ PGRL1-DEPENDENT PATHWAY AND FLV In the pgr5 mutant, Flv formed a large electron sink, resulting in complementation of the size of pmf (Yamamoto et al., 2016). However, we did not detect Flv-dependent O2 uptake in the wild-type background during steady-state photosynthesis. Flv cannot compete with PGR5-/PGRL1-dependent electron transport during steady-state photosynthesis. This may be one of the reasons that angiosperms have not conserved Flv during evolution. Furthermore, the introduction of Flv did not interfere with linear electron transport even at low light intensities (Yamamoto et al., 2016). Affinity of Flv to an electron donor, NADPH (or Fd), may be much lower than that of Calvin-Benson cycle enzymes (or FNR) and the machinery of PSI cyclic electron transport, and consequently Flv can function only when the stroma is more reduced by electrons. Because we introduced the exogenous system transferred from Physcomitrella patens, electron transfer to two alternative pathways may simply reflect the different affinity of each pathway to electron donors rather than the regulation in Arabidopsis. To analyze the crosstalk between alternative electron transport pathways, it is necessary to analyze the impact of Flv knockout in organisms containing both the PGR5-/PGRL1-dependent pathway and Flv, such as Chlamydomonas reinhardtii and Physcomitrella patens. The Chlamydomonas pgrl1 mutant increased the level of Flv and light-dependent O2 uptake, suggesting a functional interaction between PGR5-/ PGRL1-dependent PSI cyclic electron transport and Flv (Dang et al., 2014).

FLV CAN ACT AS A SAFETY VALVE FOR ELECTRONS During steady-state photosynthesis, Flv cannot compete with PGR5-/PGRL1-dependent PSI cyclic electron transport. However, Flv could function as an efficient electron sink during the induction of photosynthesis after overnight dark adaptation (Yamamoto et al., 2016). Flv may compete with the PGR5-/ PGRL1-dependent pathway when the stroma is greatly reduced by electrons. We highlighted PSI photodamage under fluctuating light conditions, in which the stroma is transiently greatly reduced immediately after switching from low light to high light. The Arabidopsis pgr5 mutant is extremely sensitive to the condition and cannot survive it (Tikkanen et al., 2010; Suorsa et al., 2012). In rice, chloroplast NDH is also involved in alleviating oxidative stress under fluctuating light conditions, suggesting the general physiological significance of the regulation of electron transport through the Cyt b6f complex via PSI cyclic electron transport (Yamori et al., 2016). State transitions regulate the excitation balance of two photosystems by optimizing the distribution of mobile light-harvesting complex II (LHCII) proteins (Rochaix,

Molecular Plant 2007). In Arabidopsis, state transitions are also important in alleviating oxidative stress under fluctuating light conditions, by keeping the association of mobile LHCII proteins with PSI (state 2) in low light. In the Arabidopsis stn7 mutant defective in a kinase essential for state transitions, LHCII proteins are attached to PSII, resulting in a severe reduction of PSI by electrons in the subsequent shift to high light (Grieco et al., 2012). Under prolonged high light conditions, STN7 is inactivated through the redox signal in the stroma, although redox-sensitive thiol residues are in the lumen side (Lemeille et al., 2009). Mobile LHCII is dephosphorylated by PPH1/TAP38 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010) and is reassociated with PSII (state 1). Under excessive light conditions, it would be safer for chloroplasts in state 1 because excessively absorbed light energy can be dissipated safely via the qE mechanism (Grieco et al., 2012). In the pgr5 mutant, dephosphorylation of LHCII was impaired, suggesting the crosstalk between redox regulation and DpH-dependent regulation (Mekala et al., 2015). Information on PSI photodamage has been limited (Sonoike et al., 1995), but land plants have evolved multiple mechanisms to protect PSI from photodamage under fluctuating light conditions (Tikkanen et al., 2012). Furthermore, once PSI is photodamaged, its FeS clusters can function as energy quenchers (Tiwari et al., 2016). PSI is more resistant to high light stress than PSII because it is protected from oxidative stress by several mechanisms, including PSII photoinhibition. In cyanobacteria, an Flv1/Flv3 heterodimer forms a large electron sink and functions to protect PSI against photodamage under fluctuating light (Allahverdiyeva et al., 2013). Introduction of Flv into the Arabidopsis pgr5 mutant drastically alleviated PSI photodamage under fluctuating light conditions (Yamamoto et al., 2016). Although Flv could not function in the wild-type under constant light, we observed the contribution of Flv to alleviation of light stress in fluctuating light even in a wild-type background. To assess the impact of fluctuating light on wildtype plants, we increased the light intensity of high light to 1942 mmol photons m 2 s 1, whereas the intensity of low light was 45 mmol photons m 2 s 1. The low light was interrupted by 1 min of high light exposure every 5 min, and the cycle was repeated three times. Although the experimental conditions were artificial, this range of fluctuation in light intensity occurs in nature. Wild-type plants accumulating Flv showed more resistance of both photosystems to fluctuating light than nontransgenic wild-type plants (Yamamoto et al., 2016). A drastic phenotype of transgenic plants was observed in the redox state of P700 in the high light phase. In the wild-type, P700 was transiently reduced by electrons immediately after the shift to high light. In wild-type plants accumulating Flv, P700 was oxidized even more in the high light phase than that in the low light phase because of the large electron sink formed by Flv. In Arabidopsis, Physcomitrella Flv cannot compete with PGR5-/ PGRL1-dependent PSI cyclic electron transport during steadystate photosynthesis, but it could form a large electron sink under fluctuating light conditions. To protect PSI from photodamage, angiosperms have evolved multiple mechanisms: PGR5-/ PGRL1-dependent PSI cyclic electron transport, state transitions, and PSII photoinhibition. However, angiosperms did not conserve the strategy depending on Flv, which can form an Molecular Plant 10, 20–29, January 2017 ª The Author 2017.

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Molecular Plant efficient safety valve for excessive electrons. We are still unsure of the reason for this evolution. Flv may compete with CO2 fixation at low light by consuming reducing power. However, Physcomitrella FlvA and FlvB behaved perfectly in Arabidopsis, even enhancing the rate of CO2 fixation probably due to the reduced restriction of electron transport at the Cyt b6f complex due to high lumen pH.

REGULATION OF THE PMF COMPOSITION In chloroplasts, pmf consists of both DpH and Dc, which contribute equally to ATP synthesis (Cruz et al., 2001). In contrast, only a DpH component induces the downregulation of electron transport via luminal acidification. The regulation of pmf has to satisfy conflicting physiological demands: (1) meeting ATP requirements for CO2 fixation and (2) downregulating electron transport to avoid photodamage. Plants optimize this tradeoff by regulating the two contributory components of pmf. The simplest example can be seen in the light intensity dependence of NPQ induction. The size of the pmf is saturated at relatively low light intensity between 134 and 312 mmol photons m 2 s 1 in wild-type Arabidopsis plants cultured in a growth chamber (Yamamoto et al., 2016; Figure 2). At these light intensities, the contribution of Dc to the pmf was comparable with that of DpH. Such pmf compositions in limiting light conditions favors high photosynthetic efficiency because the downregulatory mechanisms, qE and photosynthetic control, remain off. Upon increase in light intensity, the contribution of DpH to the pmf becomes larger to induce pH-dependent downregulation of electron transport. To shift from Dc to DpH, some counter cations (mainly K+ and Mg2+) should be exported across the thylakoid membrane (Kramer et al., 2003). Efflux of cations (mainly K+ and Mg2+) from the lumen along the electrical gradient has been observed (Hind et al., 1974). Such counter ion fluxes are thought to allow the pmf to be predominately stored as DpH. Influx of anions (Cl ) to the lumen may contribute similarly. The physiological significance of the regulation of pmf components has been proposed (Kramer et al., 2003), but it was only recently that we could imagine regulation on a molecular basis (Finazzi et al., 2015; Carraretto et al., 2016). TPK3 is a two-pore K+ channel, which effluxes K+ from the thylakoid lumen to the stroma (Carraretto et al., 2013). In tpk3 knockdown plants, the contribution of Dc to the total pmf was larger than that in the wild-type, resulting in the reduced size in NPQ induction. This work demonstrates the physiological significance of the regulation of pmf components in photosynthesis. The Arabidopsis genome encodes three members of K+ efflux antiporters (KEAs) localized to chloroplasts (Kunz et al., 2014). Whereas KEA1 and KEA2 localize to the chloroplast envelope, KEA3 is a thylakoid membrane protein. KEA3 effluxes H+ with the counter influx of K+, exchanging Dc with DpH. In Arabidopsis kea3 mutants, DpH contributed more to the total pmf than that in the wild-type, resulting in the high NPQ phenotype (Kunz et al., 2014; Armbruster et al., 2014). In fluctuating light conditions, it is necessary to rapidly induce the 26


Regulation of Proton Motive Force qE component of NPQ to minimize photoinhibition. At the same time, it is also necessary to rapidly relax qE, once light intensity is lowered, so as not to reduce the efficiency of light energy utilization under light-limiting conditions. The KEA3 function is likely coupled with the qE machinery for efficient photosynthesis under fluctuating light conditions. Chloride (Cl ) is also one of the major ions in the thylakoid membrane and can be a counter ion to regulate the components of the pmf. CLCe is a putative Cl channel localized to the thylakoid membrane, and its knockout mildly affected the regulation of pmf (Herdean et al., 2016a). However, CLCe was also proposed to be involved in the homeostasis of nitrate levels in chloroplasts (Monachello et al., 2009). Another candidate for the Cl channel in the thylakoid membrane is a bestrophin-like protein (Atbest). The knockout of the gene encoding Atbest resulted in disturbance of the pmf components, leading to a reduced rate of NPQ induction (Duan et al., 2016; Herdean et al., 2016b). These data suggest that Cl influx to the lumen plays a role in dissipating the Dc component of the pmf as a proton counter ion.

CONCLUDING REMARKS In two organelles producing chemical energy in a plant cell, chloroplasts and mitochondria, the fundamental mechanism for ATP synthesis is highly conserved (Mitchell, 1977). In mitochondria, pmf is formed across the inner membrane and, consequently, H+ is effluxed into the intermembrane space, which is connected to cytosol. In contrast, H+ is translocated across the thylakoid membrane and H+ is concentrated into the restricted lumen space in chloroplasts. Also, because of the low permeability of ions of the mitochondrial inner membrane, the contribution of Dc is predominant in mitochondria. In contrast, both Dc and DpH are utilized for the pmf in chloroplasts. This difference is unlikely to reflect only the structural difference between the two organelles. In chloroplasts, DpH downregulates photosynthetic electron transport. It is essential for plants to develop this pH-dependent regulatory system of photosynthesis to survive natural light conditions. We have realized that each regulatory machinery cross talks to form the network to optimize photosynthesis. The essence of this network is the regulation of the size and components of the pmf. To substitute Dc with DpH, plants partially uncouple electron transport to pmf formation by transferring ions (at least K+ and Cl ) across the thylakoid membrane. This strategy is possible for plants because solar energy is infinite and does not limit photosynthesis when regulation of electron transport is needed. This is in contrast with the fact that the energy source of electron transport is respiratory substrates in mitochondria. We can find the most fundamental strategy for the survival of phototrophs using solar energy in the regulation of the pmf.

FUNDING T.S. was supported by grants from the Japan Science and Technology Agency (CREST program), Human Frontier Science Program, the Japan Society for the Promotion of Science (25251032), and MEXT KAKENHI (16H06555).

AUTHOR CONTRIBUTIONS Both authors jointly wrote the manuscript.

Regulation of Proton Motive Force ACKNOWLEDGMENTS The authors are grateful to Drs. Ildiko´ Szabo´, Giovanni Finazzi, Chris Chang, and David Kramer for their valuable discussions. No conflict of interest declared. Received: March 24, 2016 Revised: July 28, 2016 Accepted: August 8, 2016 Published: August 26, 2016

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