Fine‐tuned regulation of the K+/H+ antiporter KEA3 is ... · et al., 2008). Recently, the localization of these KEAs was conﬁrmed by immunodetection (Armbruster et al., 2014;

Fine-tuned regulation of the K+/H+ antiporter KEA3 isrequired to optimize photosynthesis during induction

Caijuan Wang1, Hiroshi Yamamoto1,2, Fumika Narumiya3,4, Yuri Nakajima Munekage3,5, Giovanni Finazzi6, Ildiko Szabo7 and

Toshiharu Shikanai1,2,*1Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan,2CREST, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0076, Japan,3Graduate School of Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan,4Sakai City Institute of Public Health, Sakai, Osaka 590-0953, Japan,5Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Sandan, Hyogo 669-1337, Japan,6UMR 5168 Laboratoire de Physiologie Cellulaire V�eg�etale (LPCV) CNRS/UJF/INRA/CEA, Institut de Recherches en Technolo-

gies et Sciences pour le Vivant (iRTSV), CEA Grenoble, 38054 Grenoble, France, and7Department of Biology, University of Padova, Padova 35121, Italy

Received 26 May 2016; revised 17 October 2016; accepted 17 October 2016; published online 26 October 2016.

*For corrrrespondence (e-mail [email protected]).

SUMMARY

KEA3 is a thylakoid membrane localized K+/H+ antiporter that regulates photosynthesis by modulating

two components of proton motive force (pmf), the proton gradient (ΔpH) and the electric potential (Δw).We identified a mutant allele of KEA3, disturbed proton gradient regulation (dpgr) based on its reduced

non-photochemical quenching (NPQ) in artificial (CO2-free with low O2) air. This phenotype was enhanced

in the mutant backgrounds of PSI cyclic electron transport (pgr5 and crr2-1). In ambient air, reduced

NPQ was observed during induction of photosynthesis in dpgr, the phenotype that was enhanced after

overnight dark adaptation. In contrast, the knockout allele of kea3-1 exhibited a high-NPQ phenotype

during steady state in ambient air. Consistent with this kea3-1 phenotype in ambient air, the membrane

topology of KEA3 indicated a proton efflux from the thylakoid lumen to the stroma. The dpgr heterozy-

gotes showed a semidominant and dominant phenotype in artificial and ambient air, respectively. In

dpgr, the protein level of KEA3 was unaffected but the downregulation of its activity was probably

disturbed. Our findings suggest that fine regulation of KEA3 activity is necessary for optimizing

photosynthesis.

Keywords: KEA3, Arabidopsis thaliana, ion antiporter, proton motive force, photosynthesis.

INTRODUCTION

In photosynthesis, light reactions produce the first stable

energy intermediates in the forms of NADPH and ATP for

subsequent CO2 assimilation. Linear electron transport

generating NADPH is coupled with ATP synthesis via the

trans-thylakoid proton motive force (pmf), which is com-

posed of a proton gradient component (ΔpH) and an elec-

tric potential component (Δw). In linear electron transport

from water to NADP+, protons are translocated from the

stromal side to the luminal side of the thylakoid membrane

via water splitting in photosystem (PS) II and the Q cycle in

the cytochrome (Cyt) b6f complex, resulting in the forma-

tion of ΔpH. The number of protons translocated across

the thylakoid membrane is fixed per electron movement in

linear electron transport, which does not satisfy the ATP/

NADPH production ratio required by the Calvin–Bensoncycle and photorespiration (Allen, 2002). Other mecha-

nisms to compensate for ATP synthesis are required. In

addition to the function of driving ATP synthesis, ΔpH also

induces an energization-dependent (qE) component of

non-photochemical quenching (NPQ) of chlorophyll fluo-

rescence in PSII (Demmig-Adams et al., 2014). qE is trig-

gered by luminal acidification. Low luminal pH also

downregulates the activity of the Cyt b6f complex, slowing

down the rate of electron transport toward PSI (Stiehl and

Witt, 1969; Munekage et al., 2001). This regulation is espe-

cially important in fluctuating light (Suorsa et al., 2012).

The size of ΔpH must be finely tuned in response to fluctu-

ating environmental conditions, especially light intensity,

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd

540

The Plant Journal (2017) 89, 540–553 doi: 10.1111/tpj.13405

mailto:[email protected]

to maintain efficient photosynthesis. Ion fluxes across the

thylakoid membrane might regulate photosynthesis by

modulating the components of pmf, although the exact

regulatory mechanism for partitioning pmf into Δw and

ΔpH is still elusive. Early studies using isolated chloro-

plasts suggested that illuminated chloroplasts exhibit a

light-induced efflux of H+ and an uptake of K+ (Demmig

and Gimmler, 1983), suggesting that the maintenance of

high stromal K+ and pH is coincident. The movement of K+

is important for allowing optimal functioning of enzymes

catalyzing the photosynthetic carbon reduction cycle (Wu

and Berkowitz, 1992).

Research on the molecular systems that facilitate and

regulate K+ and H+ fluxes through the thylakoid membrane

or the chloroplast envelope has just started. In cyanobacte-

ria, a K+ channel (SynK) was identified in the thylakoid

membrane and was demonstrated to be selective for K+

(Zanetti et al., 2010). The ΔSynK mutant cannot build up

ΔpH as efficiently as wild-type (WT) cells, suggesting the

requirement for a thylakoid ion channel for optimal photo-

synthesis (Checchetto et al., 2012). In Arabidopsis, the two-

pore K+ (TPK) channel TPK3 was also identified in the thy-

lakoid membrane (Zanetti et al., 2010) and its contribution

to the modulation of pmf was confirmed (Carraretto et al.,

2013). Besides K+ channels, K+/H+ antiporters also con-

tribute to K+ transport across photosynthetic membranes

(Wu and Berkowitz, 1992). Proteomic analyses of Arabidop-

sis chloroplast membranes allowed the identification of

three members of the K+ exchange antiporter subfamily

(KEA), which belong to the CPA2 (cation/proton antiporter

2) family, namely KEA1, KEA2 and KEA3 (Ferro et al.,

2010). KEA1 and KEA2 were found in the chloroplast envel-

ope (Ferro et al., 2010; Simm et al., 2013), whereas KEA3

was present in the thylakoid membrane fraction (Zybailov

et al., 2008). Recently, the localization of these KEAs was

confirmed by immunodetection (Armbruster et al., 2014;

Kunz et al., 2014). The KEAs have been proposed to import

K+ into acidic compartments against an electric field, cou-

pled with H+ efflux from the compartments (M€aser et al.,

2001). The shorter version of KEA2 mediated the cation/H+

exchange with preference for K+ = Cs+ > Li+ > Na+ in vitro

and complemented the yeast mutant deficient in the endo-

somal Na+ (K+)/H+ exchanger NHX1p (Aranda-Sicilia et al.,

2012). In recent genetic studies in Arabidopsis, KEA1 and

KEA2 were proposed to function in a H+-driven K+ export

system from the chloroplast stroma to the cytosol,

whereas KEA3 loads K+ into the lumen by using ΔpH

formed across the thylakoid membrane during illumination

(Armbruster et al., 2014; Kunz et al., 2014).

In the present study, we report the results of a screening

of Arabidopsis mutants that regulate the size and compo-

nents of pmf. We isolated and characterized a mutant allele

of KEA3, disturbed proton gradient regulation (dpgr), in

which the regulation of KEA3 activity was modified. On the

basis of the comparison of its mutant phenotype with that

of the knockout allele, kea3-1, we discuss the mechanism

of regulation of the pmf by KEA3 during induction and in

fluctuating light.

RESULTS

The dpgr mutant displays high chlorophyll fluorescence in

CO2-free air with low O2

To clarify alternative electron transport pathways which

regulate the formation of the pmf we screened Arabidopsis

mutants with high chlorophyll fluorescence, indicative of

low NPQ, in CO2-free air containing 5% O2. These condi-

tions were chosen to enhance the activity of alternative

electron flow pathways. Indeed, removal of CO2 from the

air largely prevents linear electron flow involving PSI and

PSII, and the reduced O2 concentration (5%) should block

photorespiration; the Mehler reaction was not completely

inhibited. As the electron transport pathway is easily light

saturated in the absence of these major electron sinks,

measurements were performed under a low actinic light

(AL) intensity (50 lmol photons m�2 sec�1). By screening

45 000 M2 seedlings mutagenized by ethyl methane sul-

fonate, we identified three mutants on the basis of their

high chlorophyll fluorescence phenotype in this artificial

air. Two mutants were allelic to proton gradient regulation

5 (pgr5) (Munekage et al., 2002) and chlororespiratory

reduction 2 (crr2) (Hashimoto et al., 2003) respectively,

confirming that the screening was suitable to identify

mutant with altered alternative electron transport path-

ways. In this study, we focused on the third mutant, dpgr.

This mutant was not allelic to pgr5 or any crr mutants

defective in PSI cyclic electron transport pathways. One

minute after the onset of AL of 50 lmol photons m�2 sec�1

in CO2-free air with 5% O2, the dpgr mutant emitted a high

level of chlorophyll fluorescence (Figure 1).

The level of chlorophyll fluorescence reflects the excita-

tion state of the PSII reaction center. Thus, it can be low-

ered either by an alternative electron sink in the artificial

air employed here or by induction of qE. To test whether

qE induction was affected in dpgr, NPQ was measured in

CO2-free air containing two different concentrations of O2 –ambient (21%) and low O2 level (2%) – as a time-course

after the onset of AL (50 lmol photons m�2 sec�1)

(Figure 2). Although 5% O2 was used in the mutant screen-

ing, we decreased the O2 concentration to 2% so that we

could detect the dpgr phenotype even more clearly in fur-

ther study, except for the chromosome mapping. In CO2-

free air with 21% O2, NPQ was slightly decreased in dpgr

compared to that in the WT for 3 min after the onset of AL,

but recovered to the WT level after 4-min of illumination

(Figure 2b). When the level of O2 was lowered to 2%, NPQ

was decreased to a much lower level in dpgr than that in

the WT (Figure 2a). The reduced NPQ phenotype in dpgr in

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2017), 89, 540–553

K+/H+ antiporter KEA3 and photosynthesis optimization 541

CO2-free air containing 2% or 21% O2 was consistent with

the fact that the mutant was isolated on the basis of its

high chlorophyll fluorescence in CO2-free air containing 5%

O2.

To further characterize the dpgr mutant, we generated

its double mutants with the pgr5 and crr2-1 mutants,

defective in the antimycin A-sensitive and -resistant path-

way of PSI cyclic electron transport, respectively. We used

the original allele of each mutant reported previously

(Munekage et al., 2002; Hashimoto et al., 2003). Consistent

with the fact that the allele of crr2 was isolated by the

same screening as dpgr, the crr2-1 single mutant exhibited

a reduction in NPQ induction in CO2-free air containing 2%

O2 to a level comparable to the dpgr mutant (Figure 2a),

but the phenotype was not observed in the presence of

21% O2 (Figure 2b). NPQ was further reduced in the dpgr

crr2-1 double mutant, compared with that in the single

dpgr or crr2-1 mutants (Figure 2). In the pgr5 single

mutant, the size of NPQ was further reduced even in the

presence of 21% O2 (Figure 2b). In the dpgr pgr5 double

mutant, the size of NPQ was reduced more than in the

pgr5 single mutant, especially at 2% O2 (Figure 2a) and

also after the first 1 min in CO2-free and 21% O2 air (Fig-

ure 2b). These results indicate that the induction of NPQ is

disturbed in the dpgr mutant in artificial air, most likely via

a mechanism independent of PSI cyclic electron transport.

The dpgr mutant was allelic to the kea3 mutant

The dpgr mutant was backcrossed with the corresponding

WT (Col gl1). In the F2 generation, approximately 25% of

plants exhibited the strong high chlorophyll fluorescence

phenotype in CO2-free air containing 5% O2, suggesting a

recessive or semidominant nature of the dpgr mutation. To

map the dpgr locus on the chromosome, the dpgr mutant

was crossed with the WT plant with the Ler background.

The dpgr locus was mapped on chromosome 4. However,

it was difficult to further map the locus on chromosome 4,

mostly because of the semidominant nature of the dpgr

mutation (see below). As the heterogeneous status of chro-

mosomes between two accessions may affect the extent of

NPQ in the F2 population, the dpgr mutant was crossed

with WT Col, in which only chromosome 4 was substituted

by that of Ler. Analysis of the resulting F2 plants finally

mapped the dpgr locus within a 2910-kb region

(1 242 600–4 152 500) including the centromere of chromo-

some 4 (Figure 3a). Due to the sequence diversity in this

region between Col and Ler, further recombination did not

occur. We sequenced the total genome of the dpgr mutant

and compared it with the WT sequence. Numerous nucleo-

tide differences were detected in this region, but most of

Figure 2. The reduced non-photochemical quenching (NPQ) phenotype of

dpgr, dpgr crr2-1 and dpgr pgr5 in artificial air (CO2-free air with 2 or 21%

O2 in a N2 background).

NPQ was measured in the wild type (WT), dpgr, dpgr crr2-1 and dpgr pgr5

leaves in CO2-free air containing 2% O2 (a) and 21% O2 (b) at 50 lmol pho-

tons m�2 sec�1 at each time point after 30-min dark adaptation. Data repre-

sent means � SD (n = 3–9). A Statistically significant difference was

analyzed in WT versus dpgr, crr2-1 versus dpgr crr2-1 and pgr5 versus dpgr

pgr5 (**P < 0.01, *P < 0.05, t-test). [Colour figure can be viewed at wileyonlin-

elibrary.com].

Figure 1. High-chlorophyll-fluorescence phenotype of the dpgr mutant in

CO2-free air containing 5% O2.

M2 seedlings were exposed to actinic light (50 lmol photons m�2 sec�1) for

1 min in artificial air and the chlorophyll fluorescence image was captured

by a CCD camera. The dpgr mutant emitted high chlorophyll fluorescence,

as indicated by the arrow. [Colour figure can be viewed at wileyonlinelibrary.

com].


542 Caijuan Wang et al.

them were unlikely to be related to the dpgr phenotype

because the nucleotide alterations were associated with

the sequences related to transposable elements or pseudo-

genes. Three exceptions were observed in At4g03090,

At4g03260 and At4g04850. The mutation in At4g03090

does not cause any amino acid alteration and is unlikely to

cause the dpgr phenotype. At4g03260 encodes a putative

nuclear protein, in which the mutation causes an amino

acid alteration from glutamate to lysine. At4g04850

encodes a protein that is annotated as KEA3, a putative K+/

H+ antiporter localized to the thylakoid membrane of

chloroplasts. In the dpgr mutant, a G-to-C substitution

causes an amino acid alteration from glycine (Gly) to argi-

nine (Arg) at the 422nd amino acid position of KEA3 (Fig-

ure 3a). On the basis of the dpgr phenotype specific to

photosynthesis, the mutation in At4g04850/KEA3 most

likely causes the disturbed NPQ regulation. In addition to

the dpgr allele, a T-DNA insertion mutant allele, kea3-1

was also obtained from Arabidopsis Biological Resource

Center and analyzed in this study in order to compare the

effects of the lack of the protein in kea3-1 with those of the

specific point mutation present in dpgr (Figure 3a).

To eliminate the possibility that At4g03260 was related

to the dpgr phenotype, the F2 plants crossed with Col gl1

with the recombination between At4g03260 and

At4g04850/KEA3 were selected by the PCR-based detection

of mutations. Finally, we isolated the dpgr mutant

(dpgr_BC4), in which the mutation in At4g03260 was segre-

gated out. The dpgr_BC4 displayed the same reduced NPQ

phenotype in artificial air as the original dpgr mutant

(dpgr_BC3) (Figure S1 in the Supporting Information).

Thus, the observed dpgr phenotype was caused by the

mutation in At4g04850/KEA3. The dpgr_BC4 mutant (sim-

ply the dpgr mutant from here) was used in the whole

study except for the screening and mapping (Figures 1 and

3).

KEA3 consists of a CPA2 (cation/proton antiporter-2)

domain, which has 13 predicted trans-membrane (TM)

domains, and a C-terminal KTN (K+ transport/nucleotide-

binding) domain. The dpgr mutation caused a single

amino acid alteration in the 11th TM domain (Figure S2a).

An antibody was raised against the recombinant protein

corresponding to the C-terminal KTN domain to character-

ize the gene product (Figure S2a). Thylakoid membranes

isolated from WT, dpgr and kea3-1 plants were subjected

to protein blot analysis, resulting in the detection of two

protein bands (Figure 3b). The upper band (around 75 kDa)

is likely to correspond to the full-length KEA3 after import

into the chloroplast, based on its predicted molecular mass

(80.5 kDa) and a prediction of the transit peptide consisting

of 30 amino acids (PSort and SosuiGv_1.1) (Figure S2a).

This result is in agreement with the localization of KEA3 in

the thylakoid membranes, consistent with previous reports

(Armbruster et al., 2014; Kunz et al., 2014). The lower band

(>50 kDa) is likely to correspond to an isoform produced

by the alternative splicing that results in a truncated KTN

domain, as reported previously (Armbruster et al., 2014).

Both signals were absent in the knockout (KO) allele of

kea3-1 (Figure 3b). Similar levels of proteins were detected

between WT and dpgr plants, indicating that the amino

acid alteration did not affect protein stability in the dpgr

mutant.

Figure 3. Map-based cloning of the dpgr gene.

(a) The dpgr locus was mapped between the molecular markers T5J8 and

F1K3 on chromosome 4. DPGR was determined to At4g05850/KEA3. A sin-

gle nucleotide substitution was found in the 12th exon of At4g04850, caus-

ing an amino acid alteration from Gly422 to Arg. The site of the T-DNA

insertion of the knock-out allele (kea3-1) used in this study was also pro-

vided. Black and gray boxes represent exons and untranslated regions,

respectively.

(b) Protein blot analysis of KEA3 in WT, dpgr and kea3-1. AtKEA3 consists

of a cation/proton exchanger (CPA2) domain and a putative KTN domain.

Antibody was raised against the C-terminal KTN domain (Figure S2a).

Thylakoid membrane proteins from different genotypes were applied to

SDS-PAGE. Immunodetection using the KEA3 antibody showed the

accumulation of KEA3 comparable to the WT level in dpgr, while the

protein was totally absent in kea3-1. The lanes were loaded with intact

thylakoid membranes equivalent to 3 lg chlorophyll (100%), and a series

of dilutions was indicated. A Coomassie stained gel was shown to confirm

the loading (bottom panel: CBB). [Colour figure can be viewed at

wileyonlinelibrary.com]



Topological analysis of KEA3 in the thylakoid membrane

In previous studies, KEA3 was predicted to allow proton

efflux from the thylakoid lumen while allowing the influx

of K+ as a counter ion (Armbruster et al., 2014; Kunz et al.,

2014). However, our dpgr mutant showed a low-NPQ phe-

notype in CO2-free air with low O2 concentrations (Fig-

ure 2), raising the question of whether the actual

orientation of KEA3 differs from that described in previous

reports. The discovery of the dpgr allele motivated us to

determine the topology of KEA3 in the thylakoid mem-

brane (Figure 4).

According to the TM prediction of KEA3 published in

the public database Aramemnon (Schwacke et al., 2003),

KEA3 harbors 12 or 13 TM domains. Because the pres-

ence of the first TM domain depended on the prediction,

the orientation of the C-terminus had to be resolved.

Intact thylakoids isolated from WT plants were subjected

to mild digestion with trypsin (Figure 4a), such that only

the stroma-exposed face was accessible by the protease.

As the KEA3 antibody was raised against the C-terminal

KTN domain, absence of the signal would be expected

after the treatment with trypsin if the C-terminus were

exposed to the stroma side. Otherwise, the KEA3 signal

would be stable during the trypsin assay. After incuba-

tion with trypsin, the upper signal probably correspond-

ing to the full-length KEA3 disappeared within 10 min.

The lower signal that is likely to correspond to the pro-

duct of alternative splicing was more resistant to the pro-

tease but disappeared within 60 min. The faint signal

detected in the position of 25 kDa was unlikely to be

related to KEA3 because the signal was also observed in

the kea3-1 mutant (Figure 3b). In contrast, PsbO, a mar-

ker protein of the thylakoid lumen, was largely resistant

to the protease (Figure 4a). This result indicates that the

C-terminal of KTN is exposed to the stroma side.

KEA3 has five cysteine (Cys) residues (Figure S2a): Two

are located in the predicted transit peptide, one is at the N-

terminus of the mature protein and the other two are in the

middle of two predicted TM domains. Taking advantage of

this feature, a pegylation assay was carried out to deter-

mine the orientation of the N-terminal Cys using methoxy-

polyethylene glycol maleimide (PEG-MAL, 5 kDa) as a

probe (Figure S2b) (Wang et al., 2008; Balsera et al., 2009).

PEG-MAL is a membrane-impermeable chemical modifier

that reacts irreversibly with sulfhydryl groups of Cys, add-

ing 5 kDa to a target protein for each sulfhydryl blocking.

After pegylation, the number of higher mass bands in SDS-

PAGE should correspond to the number of Cys residues

that are modified by PEG-MAL. Intact thylakoid membranes

isolated from WT plants were treated with PEG-MAL, fol-

lowed by SDS-PAGE and immunodetection. No shifted

band was observed in the intact thylakoid samples treated

with PEG-MAL even after incubation for 1 h (Figure S2b). In

the presence of 1% SDS, in which PEG-MAL can react with

all the Cys residues, a ladder of three bands of higher

molecular mass was recognized after incubation for just

5 min (Figure S2b). Most likely, the two N-terminal Cys resi-

dues in the transit peptide were removed due to processing

of the transit peptide and the remaining three Cys residues

were modified. It is noticeable that the mobility of proteins

was affected in the presence of PEG-MAL in the gel and

their molecular weight could not be estimated from the

positions of molecular markers (Figure S2b). The Cys resi-

due at the N-terminus of the mature protein was not acces-

sible by PEG-MAL in the absence of SDS, indicating that it

is oriented towards the luminal side.

Figure 4. Topological analysis of the C-terminal end of KEA3.

(a) The KTN domain of KEA3 is exposed to the stroma side. The thylakoid membrane was isolated from wild-type plants and then incubated with trypsin

(10 lg ml�1) for the indicated period. Samples were separated by 12.5% SDS-PAGE followed by immunoblotting using the antibody against the KTN domain.

An asterisk indicates a non-specific band detected by the KEA3 antibody, which was also observed in the kea3-1 mutant (Figure 3b). PsbO was detected as a

marker for lumen proteins.

(b) The topological model of KEA3 was proposed based on the topological results in Figures 4(a) and S2(b). The N-terminus of KEA3 faces the luminal side,

whereas the C-terminus is exposed to the stroma side. The dpgr mutation (G422R) was in the 11th transmembrane (TM) domain. This model suggests a move-

ment of H+ from the lumen side to the stroma side, with a counter exchange of K+. The asterisks indicate the positions of three Cys residues in the mature pro-

tein. The central hydrophilic regulatory loop between TM9 and TM10 is indicated by a dashed square. [Colour figure can be viewed at wileyonlinelibrary.com]



The knockout allele of kea3-1 displayed the opposite

phenotype in ambient air to the dpgr allele

Although the KO allele of kea3-1 showed the high-NPQ

phenotype in ambient air (Armbruster et al., 2014), the

missense allele of dpgr showed the low-NPQ phenotype in

CO2-free air with low O2 in this study (Figure 2). To

address this discrepancy, we compared the NPQ pheno-

type of kea3-1 with that of dpgr in both artificial and ambi-

ent air (Figure 5). First, we observed that in CO2-free air

containing 2% O2, NPQ was slightly reduced in kea3-1 (Fig-

ure 5a). However, the extent of the reduction was much

less in kea3-1 than in dpgr. A statistically significant differ-

ence was observed at 3 min after the induction of photo-

synthesis in kea3-1 (Figure 5a), although a significant

difference was also observed at 1 min after the induction

in an independent assay (Figure 6b). In any case, the kea3-

1 phenotype in artificial air was weaker than the dpgr phe-

notype, which was most evident at 1 min after the induc-

tion of photosynthesis (Figure 5a). The NPQ phenotype in

dpgr was accompanied by the reduction in PSII yield (Y(II))

(Figure S3), but this was not the case in kea3-1, probably

reflecting the distinct defects in two alleles (see the Discus-

sion).

The dpgr mutant was identified in CO2-free air contain-

ing 5% O2 (Figure 1). To understand the physiological

function of KEA3 and to further compare the phenotypes

of kea3-1 and of dpgr, we investigated the dpgr pheno-

type in ambient air. A clear phenotype of dpgr was

observed, especially after overnight dark adaptation (Fig-

ure 5b). After the long dark adaptation, photosynthesis

was induced at a low light intensity of 50 lmol pho-

tons m�2 sec�1. In WT plants, NPQ was transiently

induced to a maximum level within 1 min, followed by

rapid relaxation in the next minute (Figure 5b). This tran-

sient NPQ induction was absent in the dpgr mutant. This

result is consistent with the low-NPQ phenotype in dpgr

in artificial air (Figure 2). In the kea3-1 allele, transient

NPQ induction was unaffected, indicating that the func-

tion of KEA3 was not required for this process (Fig-

ure 5b). This result suggests that the regulation of KEA3

is disturbed in the dpgr mutant.

At higher light (110 lmol photons m�2 sec�1), the oppo-

site phenotypes between dpgr and kea3-1 alleles were

most clearly observed (Figure 5b). In WT plants NPQ was

rapidly induced to a maximum level within 2 min, followed

by a gradual relaxation, which reflected the full activation

of the Calvin–Benson cycle. As observed at 50 lmol pho-

tons m�2 sec�1, rapid induction of NPQ was impaired in

the dpgr mutant. However, a comparable level of NPQ was

induced at 10 min after the induction of photosynthesis in

both dpgr and WT (Figure 5b). In contrast, a similar level

of NPQ to WT plants was induced within 2 min in the kea3-

1 allele. However, the relaxation of NPQ was slower in

Figure 5. Differences in the non-photochemical quenching (NPQ) pheno-

type between dpgr (missense allele) and kea3-1 (knock-out allele) in artificial

and ambient air.

(a) NPQ was measured in wild type (WT), dpgr and kea3-1 leaves in CO2-

free air containing 2% O2 at 50 lmol photons m�2 sec�1 at each time point

after 30-min dark adaptation. Data represent means � SD (n = 3–4). Aster-isks indicate a statistically significant difference compared with WT

(**P < 0.01, *P < 0.05, t-test).

(b) Time-course of NPQ induction was measured in the detached leaves of

WT, dpgr and kea3-1 plants in ambient air at 50, 110, 700 lmol pho-

tons m�2 sec�1 after overnight dark adaptation. Data represent means � SD

(n = 3–4). [Colour figure can be viewed at wileyonlinelibrary.com].



kea3-1 plants than that in WT plants, resulting in the higher

NPQ phenotype of kea3-1, which was also observed in a

previous study (Armbruster et al., 2014).

A similar trend was also observed at 700 lmol pho-

tons m�2 sec�1, although the maximum NPQ was not

relaxed even in WT plants. Notably, the level of NPQ was

similar at 10 min after the induction of photosynthesis

among three genotypes but the kea3-1 allele showed a

slower relaxation of NPQ in the dark (Figure 5b). When the

same experiment was carried out after dark adaptation for

30-min, the phenotypes of two mutant alleles became less

significant than that observed after overnight dark adapta-

tion, especially in the dpgr mutant (Figure S4).

The dpgr mutant showed a dominant nature in ambient

air

The discrepancy in the NPQ phenotypes between two

mutant alleles in ambient air led us to consider the possi-

bility that the dpgr mutant has a dominant nature. We ree-

valuated the phenotypes of the heterozygous plants of two

mutant alleles in both artificial and ambient air (Figure 6).

As expected, the heterozygotes of kea3-1 displayed the

level and time-course induction of NPQ similar to those in

WT plants in both artificial and ambient air (Figure 6b,d).

In contrast, the dpgr heterozygotes exhibited a reduced

NPQ induction similar to that in the homozygous dpgr

Figure 6. Non-photochemical quenching (NPQ) phenotypes in heterozygotes of two mutant alleles (dpgr and kea3-1) in artificial and ambient air.

(a, b) Heterozygous dpgr (dpgr/+) plants showed a semidominant NPQ phenotype in artificial air, whereas heterozygous kea3-1 (kea3-1/+) plants displayed the

wild-type (WT)-like phenotype. NPQ was measured in CO2-free air containing 2% O2 at 50 lmol photons m�2 sec�1 at each time point after 30-min dark adapta-

tion. Data represent means � SD (n = 5–10). Asterisks indicate a statistically significant difference compared with WT (**P < 0.01, *P < 0.05, t-test).

(c, d) Heterozygous dpgr (dpgr/+) plants showed a dpgr-like NPQ phenotype in ambient air, whereas heterozygous kea3-1 (kea3-1/+) plants displayed the WT-like

phenotype. The induction of NPQ was measured in the detached leaves in ambient air at 110 lmol photons m�2 sec�1 after overnight dark adaptation. Data rep-

resent means � SD (n = 3–5). [Colour figure can be viewed at wileyonlinelibrary.com].



allele in ambient air (Figure 6c). Originally, the dpgr

mutant was mapped on chromosome 4 by genotyping the

F2 population (homozygous dpgr), which exhibited the

strong phenotype in CO2-free air containing 5% O2 (Fig-

ures 1 and 2). However, careful reevaluation of the dpgr

phenotype in CO2-free air containing 2% O2 clarified that

the level of NPQ in the dpgr heterozygotes was intermedi-

ate between that in the WT and homozygous dpgr plants,

suggesting the semidominant nature. We conclude that

the dpgr mutation showed a dominant nature in ambient

air, probably due to the disturbed regulation of KEA3 activ-

ity. On the contrary, the KO allele of kea3-1 showed a

recessive nature under both air conditions.

The full-length genomic sequence of the WT KEA3 gene

was introduced into the dpgr mutant. Because of the domi-

nant/semidominant nature of the dpgr mutation, analysis

of the phenotypes of the transgenic lines was complex but

suggestive. Six independent T2 lines were finally analyzed

in total (Figure 7). In artificial air (CO2-free, 2% O2), NPQ

recovered to different intermediate levels between those in

WT and dpgr plants in these lines (Figure 7a). The highest

complementation levels were observed in lines #1 and #16,

whereas complementation was not obvious in line #15. We

also analyzed the induction of NPQ in ambient air, and

classified these lines into three groups based on their NPQ

phenotypes (Figure 7b). Group 1 includes lines #1 and #16,

in which NPQ was enhanced as in the kea3-1 allele. Consis-

tent with this phenotype, the level of KEA3 protein was

severely reduced in lines #1 and #16 (Figure 7c). Co-sup-

pression of the KEA3 genes (both the transgenic WT gene

and the endogenous dpgr gene) in lines #1 and #16 proba-

bly resulted in a phenotype similar to the kea3-1 allele in

both artificial and ambient air. In Group 2, line #15 showed

the dpgr-like phenotype in ambient air (Figure 7b). The

KEA3 protein level was not dramatically changed in line

#15 with respect to WT and dpgr (Figure 7c). To analyze

the expression levels of the transgene, RT-PCR products of

KEA3 mRNA were digested with a restriction enzyme

which could recognize the transcripts originating from the

endogenous dpgr gene (Figure S5). In line #15, transcript

originating from the dpgr gene was dominant. The trans-

gene was not expressed efficiently in line #15, resulting in

the dpgr-like phenotype in both artificial and ambient air.

Plants in Group 3 showed the WT-like phenotype in ambi-

ent air (lines #21, #10 and #11) (Figure 7b). In all of these

lines, transcripts originating from the WT transgene were

detected (Figure S5). Taken together with the fact that

these lines accumulated the WT level or even higher levels

of KEA3, the WT KEA3 protein was likely to be dominant in

stoichiometry in lines #21, #10 and #11 (Figure 7c). The

transgene in these lines recovered the dpgr phenotype in

ambient air but puzzlingly only slightly complemented the

dpgr phenotype in CO2-free air with 2% O2, especially in

line #11.

The size of the pmf was not affected in two alleles

During the induction of photosynthesis, NPQ induction

was differently altered in two mutant alleles (Figure 5b). To

determine whether the size of the pmf is affected, elec-

trochromic shift (ECS) measurements were carried out.

The induction of the pmf, the size of which is represented

by ECSt (ECSt/ECSST), was measured under the same con-

ditions used in the analysis of NPQ induction in ambient

air (at 110 lmol photons m�2 sec�1 after overnight dark

adaptation). In line with the induction curve of NPQ, a

maximum level of pmf was transiently induced within

1 min in the WT plants, followed by relaxation to a steady-

state level (Figure 8). Similar patterns to the WT were

observed in the dpgr and kea3-1 alleles in the time-course

of pmf induction. Thus, the phenotypes of two alleles

observed in NPQ induction were not due to the size of the

pmf but most likely to the different partitioning of pmf

components.

DISCUSSION

In this study we have described a mutant allele of KEA3,

dpgr, which has a semidominant/dominant mutation in the

11th TM domain of this protein. The dpgr mutant displayed

a contrasting phenotype in ambient air with the KO allele

of kea3-1 (Figure 5b), whereas a similar but stronger phe-

notype in CO2-free air with low O2 than kea3-1 (Figure 5a).

Based on the characterization of two mutant alleles, we

discuss the possible molecular basis resulting in the differ-

ent genetic makeup under two different air conditions

between two mutant alleles. Finally, we discuss the physio-

logical function of KEA3 in fine-tuning the induction and

relaxation of NPQ, which is required for optimal photosyn-

thesis.

KEA3 mediates H+ efflux from the thylakoid lumen

In this study, we determined the membrane topology of

KEA3 by finding the orientations of both the N-terminus

and C-terminus (Figures 4 and S2). We propose a model in

which the N-terminal end is facing to the lumen whereas

the C-terminal KTN domain is exposed to the stroma (Fig-

ure 4b). During the preparation of this manuscript, a differ-

ent topology model of KEA3, in which the KTN domain

was localized to the luminal side, was proposed on the

basis of the results of a thermolysin protection assay of a

GFP-tagged KEA3 (Armbruster et al., 2016). Despite the

opposite membrane topology, the same orientation of H+/

K+ movement was proposed in their work. However, their

idea is inconsistent with the activity and membrane topol-

ogy of the closely related KefC (Healy et al., 2014). Further-

more, the KTN domain is expected to sense the NAD(P)H

level and is unlikely to face the luminal side.

In the trypsin assay, the lower signal, probably corre-

sponding to the peptide originating from the alternatively





spliced transcript, was more resistant to the protease than

the upper signal that corresponds to the full-length KEA3.

However, the degradation of the shorter form of KEA3

was still faster than that of PsbO (Figure 4a). Because the

alternative splicing only affected the integrity of the KTN

domain and did not alter the CPA2 domain that mediated

the H+/K+ antiport, it is unlikely that the alternative splic-

ing may result in the production of a KEA3 isoform orien-

tated in the opposite way. Therefore, we propose a

topological model of KEA3 with its C-terminal KTN

domain exposed to the stromal side (Figure 4b). Although

we also propose that the N-terminal end is facing to the

lumen, this conclusion depends on the prediction of the

length of the transit peptide consisting of 30 amino acids

and that the third Cys residue is present in the mature

KEA3. In fact, in contrast to PSort, ChloroP predicts a 93-

amino-acid long targeting peptide. We therefore do not

completely exclude the possibility that the third Cys resi-

due was absent in the mature protein, even though the

observation that following detergent treatment three Cys

residues seem to react with PEG-MAL contradicts the

above hypothesis. Finally, it is still possible that KEA3 has

12 TM domains instead of 13 and its N-terminal end is

facing the stroma.

Since knowledge of the gating and regulatory mecha-

nisms of KEA3 activity remains limited, it is necessary to

gain insight from its bacterial homolog, the K+ efflux sys-

tem (Kef) C. KefC is a ligand-gated K+ efflux transporter

(Roosild et al., 2002), which is important for bacterial sur-

vival during exposure to toxic electrophilic compounds

(Ferguson et al., 1995). KefC activity is inhibited by glu-

tathione (GSH) but is activated by glutathione-S-conjugates

(GS-X) formed in the presence of electrophiles. GSH or

GS-X directly bind to the overlapping sites on the KTN

domain of KefC, which is located in the cytoplasmic side of

the bacterial membrane (Roosild et al., 2009). Activation of

KefC results in K+ efflux and concomitant H+ influx via the

conformational changes in Escherichia coli (Healy et al.,

2014), suggesting the direction of H+ flux is towards the

KTN domain side. Our data suggest a similar situation for

KEA3 in the thylakoid membrane.

The KTN domain is conserved in many K+ transporters

including KEA1, KEA2 and KEA3 and is proposed to be

important in the regulation of their activity (Figure S2a)

(Roosild et al., 2002, 2004, 2009). The residues forming the

glutathione-binding pocket are less conserved in KEA3,

indicating that the plant KEA3 might be regulated in a dif-

ferent way (Chanroj et al., 2012). Regardless of the specific

ligand that triggers conformational changes within KTN

domains, the direction of H+ transport appears to be con-

served in KEA3 due to the highly conserved TM architec-

ture (Figure S2a). On the basis of the topological

information on KEA3 and the orientation of H+ movement

in KefC, we conclude that H+ is transported from the lumi-

nal side to the stromal side via KEA3, while K+ is co-trans-

ported into the lumen (Figure 4b). This idea is consistent

with the kea3-1 phenotype reported in this study and also

with the results of previous studies (Armbruster et al.,

2014; Kunz et al., 2014). KEA3 might substitute ΔpH with

Δw, behaving like a photosynthetic uncoupler (e.g. nigeri-

cin). Thus, its activity should be precisely regulated.

Indeed, lack of KEA3 presumably leads to accumulation of

H+ in the thylakoid membrane, leading to higher NPQ

levels (at least in ambient air after overnight dark adapta-

tion; Figure 5b). In contrast, we presume that in dpgr the

Figure 8. Induction of proton motive force (pmf) evaluated by the total elec-

trochromic shift (ECSt) in ambient air after overnight dark adaptation.

ECS analysis was done in the detached leaves of wild-type (WT), dpgr and

kea3-1 plants at 110 lmol photons m�2 sec�1 in ambient air after overnight

dark adaptation. The total ECS (ECSt) was standardized against the 515-nm

absorbance change induced by a single turnover flash (ECSST). Data

represent means � SD (n = 3). [Colour figure can be viewed at wileyonline-

library.com].

Figure 7. Non-photochemical quenching (NPQ) phenotypes in the T2 lines of dpgr transformed with the full-length of genomic wild-type (WT) KEA3.

(a) In artificial air (CO2-free, 2% O2), the reduced NPQ phenotype of dpgr recovered to different levels in the independent T2 lines, except for line #15. NPQ was

measured in CO2-free air containing 2% O2 at 50 lmol photons m�2 sec�1 at each time point after 30-min dark adaptation. Data represent means � SD (n = 3).

Asterisks indicate a statistically significant difference compared with dpgr (*P < 0.05, t-test).

(b) In ambient air, the independent T2 lines displayed different NPQ induction phenotypes. Lines #1, #16 (Group 1) showed kea3-1-like NPQ induction, while line

#15 (Group 2) displayed the dpgr-like phenotype. The time courses of NPQ induction in lines categorized into Group 3 resembled the WT plants. The NPQ induc-

tion was measured in detached leaves in ambient air at 110 lmol photons m�2 sec�1 after overnight dark adaptation. Data represent means � SD (n = 3).

(c) Protein blot analysis of KEA3 in T2 lines. Co-suppression of KEA3 occurred in some T2 lines (#1 and #16). For each line, three individual T3 plants used in the

chlorophyll fluorescence analysis were independently analyzed. The lanes were loaded with intact thylakoid membranes equivalent to 3 or 2 lg chlorophyll for

detecting KEA3 or Cytf, respectively. [Colour figure can be viewed at wileyonlinelibrary.com].



mutation leading to a lower NPQ is a gain-of-function

mutation, although direct proof in favor of this hypothesis

has still to be obtained.

The KTN domain of KefC is expected to bind NADH, pro-

viding another regulatory feature in E. coli (Schlosser

et al., 1993; Roosild et al., 2002). NADH showed significant

synergy with GSH in suppressing the antiporter activity of

KefC, thereby providing a linkage between the NADH/NAD+

ratio and the antiporter activity of KefC (Fujisawa et al.,

2007). A similar mechanism could be expected in KEA3,

and the redox state of the stroma might affect KEA3 activ-

ity via the KTN domain, possibly by monitoring the

NADPH/NADP+ ratio.

Besides the ligand-mediated control mechanism, a short

central hydrophilic regulatory loop present between TM9

and TM10 (Figure S2a) was proposed to regulate the activ-

ity of KefC by interacting with the KTN domain (Miller et al.,

1997; Ness and Booth, 1999). The highly acidic nature of

this regulatory loop suggests that its probable interplay

with the KTN domain might be mediated by salt bridge con-

nections. This physical interaction seems to be involved in

the gating of KefC (Roosild et al., 2010). This regulatory

loop is partially conserved in the plant KEA1, KEA2 and

KEA3 sequences (Figure S2a), suggesting the possibility of

a similar regulatory mechanism in KEA3. The regulatory

loop is located between the 9th and 10th transmembrane

domains, while the amino acid substitution (G422R) in dpgr

is in the adjacent 11th transmembrane domain (Figures 4b

and S2a). As the dpgr mutant showed a semidominant nat-

ure in artificial air rather than the dominant nature observed

in ambient air (Figure 6), we presume that KEA3 was regu-

lated by its KTN domain in dpgr (see further discussion

below). A phenotype similar to the dpgr mutant was

reported in plants accumulating KEA3 lacking the C-term-

inal KTN domain (Armbruster et al., 2014). This truncated

version of KEA3 is likely to be insensitive to the negative

regulation exerted by the KTN domain. Further research is

needed to understand whether the dpgr mutation might

disrupt the interaction between the regulatory loop and the

KTN domain by affecting the structure of the CPA2 domain.

Complex genetic nature of the dpgr allele under different

air conditions

The dpgr mutant showed a dominant nature in ambient air

and a semidominant nature in CO2-free air with low O2 (Fig-

ure 6). If the regulation of KEA3 were completely distorted,

we would also expect the dominant phenotype in artificial

air. In principle, activity of KEA3 may be downregulated to

some extent in the dpgr mutant in artificial air via the high

NADPH/NADP+ ratio. As the stroma is highly reduced due

to lack of the Calvin–Benson cycle and photorespiration in

artificial air, the NADPH level should be high, resulting in

the inhibition of KEA3 activity in the WT. We prefer a model

in which the dpgr defect unusually elevates the antiporter

activity with a mechanism independent of the regulation

via the KTN domain and the KTN domain still can downreg-

ulate KEA3 activity to some extent in the dpgr mutant.

It is more challenging to explain the phenotypes of the

complementation lines in Group 3 of Figure 7(b) (lines #21,

#10 and #11). These lines showed the WT-like phenotype

in ambient air (Figure 7b). However, the dpgr-type NPQ

phenotype was only partly complemented in these lines in

artificial air (Figure 7a). These lines accumulated high

levels of WT KEA3 originating from the transgene (Fig-

ure 7c), as well as low levels of the mutant protein origi-

nating from the endogenous dpgr allele (Figure S5).

Therefore, the ratio of the mutant KEA3 (DPGR) protein

should be very small compared with the WT KEA3 protein

in these lines. This idea probably explains the complemen-

tation of the dpgr mutant by over-accumulation of the WT

protein in ambient air, by diluting the mutant KEA3. How-

ever, another explanation is needed for the partial comple-

mentation of the NPQ level in artificial air in these lines.

Even in the presence of a high level of NADPH in artificial

air, KEA3 activity may not be fully inhibited in WT plants.

As a result, over-accumulation of WT KEA3 may cause a

leaky H+/K+ exchange. In the context of an extremely low

rate of ΔpH formation in artificial air due to the absence of

large electron sinks, even the slow leak of H+ may affect

the steady-state size of ΔpH in these lines.

Physiological function of KEA3 during the induction of

photosynthesis

The experimentally determined KEA3 topology was consis-

tent with that predicted from the KO mutant phenotype

(Armbruster et al., 2014; Kunz et al., 2014). In ambient air,

the kea3-1 phenotype is easily explained by the increased

accumulation of H+ in the thylakoid lumen during steady-

state photosynthesis, suggesting that KEA3 is required to

relax qE by exchanging ΔpH with Δw, as already proposed in

previous works (Armbruster et al., 2014; Kunz et al., 2014).

However, our results suggest that KEA3 activity is finely

regulated during the induction of photosynthesis. Indeed,

the dpgr mutant showed the low-NPQ phenotype during

the induction of photosynthesis in ambient air (Figure 5b).

As kea3-1 could induce the WT level of transient NPQ dur-

ing the induction of photosynthesis, KEA3 function is not

required in this process. This difference in the dpgr pheno-

type suggests that KEA3 is inactivated during the induction

of photosynthesis but the inactivation is disturbed in the

dpgr mutant. Most likely, the high level of NADPH accumu-

lating during the induction of photosynthesis inhibits KEA3

activity via the KTN domain. The physiological significance

of the regulation of KEA3 activity is most evidently demon-

strated during the induction of photosynthesis by the AL of

110 lmol photons m�2 sec�1 (Figure 5b). The induction

and relaxation of NPQ in WT plants were between those of

kea3-1 and dpgr plants. KEA3 activity was inhibited during



the first minute of induction but then activated to relax

NPQ to the steady-state level in WT plants. The dpgr phe-

notype was stronger after overnight dark adaptation in

ambient air than that after short dark adaptation for 30 min

(Figures 5b and S4). After short dark adaptation, the transi-

tion from NPQ induction to relaxation occurred during the

60–80 sec after the induction of photosynthesis. After over-

night dark adaptation, however, the maximum NPQ level

was sustained for longer than 80–100 sec after the induc-

tion of photosynthesis. We consider that this phenotypic

difference is due to the slower activation of the Calvin–Benson cycle enzymes after long dark adaptation (Spreitzer

and Salvucci, 2002). According to our hypothesis, after the

overnight dark adaptation, KEA3 activity had to be down-

regulated via the KTN domain, the regulation that was dis-

turbed in the dpgr mutant (Figure 5b). However, the

phenotype was less clear after 30-min dark adaptation:

Higher levels of transient NPQ were induced in the dpgr

mutant after 30-min dark adaptation than after overnight

dark adaptation. KEA3 activity may be strictly inhibited dur-

ing the short dark adaptation via another unknown mecha-

nism, but this inhibitory state may be relaxed during

overnight dark adaptation. This regulatory process may be

necessary in the daytime to induce NPQ transiently during

the shift from very low light to high light.

It is not simple to explain the phenotype in CO2-free air

with low O2, especially in kea3-1. In dpgr, the low-NPQ phe-

notype is at least partly explained by the disturbed regula-

tion of KEA3. In the absence of large electron sinks,

induction of NPQ depends mainly on PSI cyclic electron

transport, a process that is essentially independent of the

function of KEA3 (Figure 2). In addition to the reduced NPQ,

the yield of PSII (Y(II)) was also reduced in the dpgr mutant

(Figure S3). Optimization of the pmf components may be

necessary for the operation of non-cyclic alternative electron

transport, although the molecular mechanism is totally

unclear. It is even more puzzling that NPQ induction and Y(II)

were mildly affected in the kea3-1 mutant, although some of

the differences were not statistically significant (Figures 2

and S3). As the kea3-1mutant theoretically stores more H+ in

the thylakoid lumen, the slightly low NPQ cannot be

explained by the lack of KEA3 activity. Chloroplast develop-

ment was severely impaired in the kea1 kea2 kea3 triple

mutant (Kunz et al., 2014). Chloroplast development may be

slightly affected even in the kea3 single mutant, resulting in

a phenotype with a low electron transport rate in artificial air.

It is also possible that the kea3-1 defect may cause the mis-

regulation of other channels or transporters in artificial air.

EXPERIMENTAL PROCEDURES

Plant materials and growth conditions

The dpgr mutant was isolated from Arabidopsis thaliana M2 seeds(ecotype Columbia gl1) mutagenized by ethyl methane sulfonate.

The kea3-1 (GABI_170G09 line) was obtained from the ArabidopsisBiological Resource Center. The gl1 mutation was introduced intokea3-1 by backcrossing with Col gl1 three times to unify thegenetic background with other genotypes. Plants were grown onsoil for 4–5 weeks under long-day chamber conditions(50 lmol photons m�2 sec�1, 16-h light/8-h dark cycles at 23°C).An exception was in the ECS measurement (Figure 8), in whichplants were grown for 8–9 weeks under short-day conditions (8-hlight/16-h dark cycles) to obtain larger leaves.

Mutant screening

Mutant screening was performed using an imaging system ofchlorophyll fluorescence (Shikanai et al., 1999) in CO2-free aircontaining 5% O2 in a N2 background, supplied by a pre-mixedgas cylinder. Seedlings were dark-adapted for 15 min beforephotosynthesis was induced. Measurement was performed in a10-cm3 box. Prior to measurement, the pre-mixed gas was sup-plied to the box with a speed of 1500 cm3 min�1 for 5 min.The chlorophyll fluorescence image was captured by a CCDcamera after 1 min of illumination by AL (50 lmol pho-tons m�2 sec�1).

Analysis of chlorophyll fluorescence

Chlorophyll fluorescence parameters were measured using aMINI-PAM portable chlorophyll fluorometer (Walz, http://www.walz.com/) for measurement in artificial air or a Dual-PAM 100 (Walz)for the induction of NPQ in ambient air. For the measurement inartificial air, CO2-free air with 21% or 2% O2 in a N2 backgroundwas supplied by pre-mixed gas cylinders. Measurement was per-formed in a 10-cm3 box. Prior to measurement, the pre-mixed gaswas supplied to the box with a speed of 1500 cm3 min�1 for5 min. Chlorophyll fluorescence parameters were measured asdescribed previously (Shikanai et al., 1999). NPQ and the quantumyield of PSII (Y(II)) were calculated as (Fm � Fm

0)/Fm0 and(Fm

0 � Fs)/Fm0, respectively.

Thylakoid protein isolation, PAGE and immunoblot

analyses

Leaves from 4 to 5-week-old plants were homogenized on iceusing a blender in 0.33 M sorbitol, 20 mM Tricine/KOH (pH 8.4),5 mM EGTA, 2.5 mM EDTA and 10 mM NaHCO3 (Medium I). Thehomogenate was filtered through two layers of Miracloth and wascentrifuged at 4800 g at 4°C for 5 min to obtain intact chloroplasts.The pellet was resuspended in 20 mM HEPES/KOH (pH 7.6), 5 mM

MgCl2 and 2.5 mM EDTA (Medium II) to rupture intact chloroplas-ts. Thylakoid membranes were isolated by centrifugation at10 000 g at 4°C for 2 min.

Isolated thylakoid proteins were separated by 7.5% (for thepegylation assay) or 12.5% (for the others) SDS-PAGE, then trans-ferred to a polyvinylidene difluoride (PVDF) membrane. Antibod-ies indicated in each figure and the text were used to detect thethylakoid proteins. To detect KEA3, a rabbit polyclonal antibodywas raised against the C-terminal KTN domain, as indicated inFigure S2.

Determination of the topology of KEA3

In the tryptic proteolysis experiment, thylakoid membraneswere resuspended in Medium II at a chlorophyll concentrationof 1 mg ml�1. Trypsin (Sigma-Aldrich, http://www.sigmaaldrich.com/) was added to a final concentration of 10 lg ml�1. Sampleswere taken before, and 10, 30, 60 and 120 min after the addi-tion of trypsin. Reactions were stopped by adding an equal



http://www.walz.com/

http://www.walz.com/

http://www.sigmaaldrich.com/

http://www.sigmaaldrich.com/

volume of Medium II containing 49 cOmplete protease inhibitorcocktail (Roche, http://www.roche.com/). Sample preparationwas finished by adding 29 Laemmli loading buffer. SDS-PAGE(12.5%) was employed to separate the proteins followed byimmunoblotting.

To determine the orientation of the N-terminal cysteine in themature form of KEA3 (indicated in Figures 4b and S2), a pegyla-tion assay was carried out as previously described, with somemodifications (Wang et al., 2008; Balsera et al., 2009). Prior to thepegylation assay, the thylakoids isolated as described above wereincubated with or without 1% SDS for 20 min at room tempera-ture (~25�C). The prepared samples were then incubated with2 mM PEG-MAL (Sigma-Aldrich) in Medium II on ice (in theabsence of SDS) or at room temperature (in the presence of SDS)for 0, 5, 10, 30 and 60 min. Reactions were stopped by addingLaemmli buffer in the presence of 100 mM DTT. SDS-PAGE (7.5%)was employed to separate the proteins followed by immunoblot-ting using the KEA3 antibody.

Complementation of the dpgr mutant

For complementation of the dpgr mutant, the WT genomicsequence including KEA3 was amplified using the primers 50-CATTTGCCATTTGGATACACATGCATG-30 and 50-CTTTTAGTTTG-GAATATTGGCTGCAC-30. The PCR product was cloned intopDONR/Zeo by BP Clonase reaction (Invitrogen, http://www.invitrogen.com/). The resulting plasmid was confirmed by sequenc-ing and then transferred to the binary vector pGWB-NB1(Nakagawa et al., 2007) by LR Clonase reaction (Invitrogen).Agrobacterium tumefaciens was transformed with the resultantplasmids by electroporation, and the bacteria were used totransform dpgr mutant plants via the floral dip method. Trans-formed plants were selected on Murashige–Skoog medium con-taining 7.5 lg ml�1 BASTA, and the T2 generation of transgeniclines was used for analyses.

ECS measurements

The ECS measurements were carried out using a Walz Dual-PAM 100 equipped with a P515/535 module (Walz). Measure-ments were carried out in ambient air. Before measurements,8–9-week-old plants grown under a short-day photoperiod weredark-adapted overnight followed by 10-min illumination with110 lmol photons m�2 sec�1 actinic red light. A 1-sec dark-pulse was applied at the different time points indicated in Fig-ure 8 to record ECSt, which represents the size of the light-induced pmf and was estimated from the total amplitude ofthe rapid decay of the ECS signal during the dark pulse, asdescribed previously (Wang et al., 2014). ECSt levels were nor-malized against a 515-nm absorbance change induced by a sin-gle turnover flash (ECSST), as measured in dark-adapted leavesbefore recording. This normalization allows us to take intoaccount possible changes in leaf thickness and chloroplast den-sity between leaves (Takizawa et al., 2008).

ACKNOWLEDGEMENTS

The authors thank Sayaka Horiguchi and Momoko Okajima for theircontribution to primary mapping of the dpgr mutation. The authorsalso thank Tsuyoshi Nakagawa (Shimane University, Japan) andthe Arabidopsis Biological Resource Center for providing pGWB-NB1 and kea3-1, respectively. We also thank Takehide Kato for hisgift of the Arabidopsis Col-0 line, in which only chromosome 4 wassubstituted by that of Ler. This work was supported by grants fromthe Japan Science and Technology Agency (CREST program), theHuman Frontier Science Program, and the Japan Society for the

Promotion of Science (25251032 and 16H06555). None of theauthors have any conflicts of interest to declare.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online ver-sion of this article.Figure S1. The dpgr phenotype in artificial air conditions (CO2-freeair with 2% or 21% O2 in a N2 background) was linked to the muta-tion in At4g04850/KEA3.Figure S2. Membrane topology analysis of the N-terminal end ofKEA3.Figure S3. Yield of photosystem II (Y(II)) in dpgr and kea3-1 in arti-ficial air.Figure S4. Comparison of the induction of non-photochemicalquenching between dpgr and kea3-1 plants in ambient air after 30-min dark adaptation.Figure S5. RT-PCR analysis of KEA3 mRNA in the T2 lines of dpgrtransformed with the full-length genomic wild type KEA3.

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Fine‐tuned regulation of the K+/H+ antiporter KEA3 is ... · et al., 2008). Recently, the localization of these KEAs was conﬁrmed by immunodetection (Armbruster et al., 2014;