Ion antiport accelerates photosynthetic acclimation in fluctuating light environments

Abstract

Many photosynthetic organisms globally, including crops, forests and algae, must grow in environments where the availability of light energy fluctuates dramatically. How photosynthesis maintains high efficiency despite such fluctuations in its energy source remains poorly understood. Here we show that Arabidopsis thaliana K⁺ efflux antiporter (KEA3) is critical for high photosynthetic efficiency under fluctuating light. On a shift from dark to low light, or high to low light, kea3 mutants show prolonged dissipation of absorbed light energy as heat. KEA3 localizes to the thylakoid membrane, and allows proton efflux from the thylakoid lumen by proton/potassium antiport. KEA3's activity accelerates the downregulation of pH-dependent energy dissipation after transitions to low light, leading to faster recovery of high photosystem II quantum efficiency and increased CO₂ assimilation. Our results reveal a mechanism that increases the efficiency of photosynthesis under fluctuating light.

ARTICLE

Received 3 Jun 2014 | Accepted 1 Oct 2014 | Published 13 Nov 2014

DOI: 10.1038/ncomms6439 OPEN

Ion antiport accelerates photosynthetic acclimation in uctuating light environments

Ute Armbruster1, L. Ruby Carrillo2, Kees Venema3, Lazar Pavlovic4, Elisabeth Schmidtmann1, Ari Kornfeld5, Peter Jahns4, Joseph A. Berry5, David M. Kramer2 & Martin C. Jonikas1

Many photosynthetic organisms globally, including crops, forests and algae, must grow in environments where the availability of light energy uctuates dramatically. How photo-synthesis maintains high efciency despite such uctuations in its energy source remains poorly understood. Here we show that Arabidopsis thaliana K efux antiporter (KEA3) is critical for high photosynthetic efciency under uctuating light. On a shift from dark to low light, or high to low light, kea3 mutants show prolonged dissipation of absorbed light energy as heat. KEA3 localizes to the thylakoid membrane, and allows proton efux from the thylakoid lumen by proton/potassium antiport. KEA3s activity accelerates the downregulation of pH-dependent energy dissipation after transitions to low light, leading to faster recovery of high photosystem II quantum efciency and increased CO2 assimilation. Our results reveal a mechanism that increases the efciency of photosynthesis under uctuating light.

1 Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, California 94305, USA. 2 Plant Research Laboratory, Michigan State University, R106 Plant Biology Building, East Lansing, Michigan 48824-1312, USA. 3 Dpto de Bioqumica, Biologa Celular y Molecular de Plantas, Estacin Experimental del Zaidn, Consejo Superior de Investigaciones Cientcas, c/. Profesor Albareda 1, 18008 Granada, Spain. 4 Biochemie der Panzen, Heinrich-Heine-Universitat Dsseldorf, Universitatsstra 1, 40225 Dsseldorf, Germany. 5 Department of Global Ecology, Carnegie Institution for Science, 260 Panama Street, Stanford, California 94305, USA. Correspondence and requests for materials should be addressed to U.A.(email: mailto:[email protected]

Web End [email protected] ) or to M.C.J. (email: mailto:[email protected]

Web End [email protected] ).

NATURE COMMUNICATIONS | 5:5439 | DOI: 10.1038/ncomms6439 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 1

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6439

Efcient conversion of energy is a critical challenge for all organisms. Energy in the environment is available either as chemical potential or light. Uniquely, the energy available as

light can undergo extreme uctuations in amplitude on short time scales. Photon ux can change 100-fold within seconds, for example, due to shade from clouds or leaves moving in the wind1,2. Understanding the mechanisms that underlie efcient photosynthesis in uctuating light can open doors to increased crop yields, and more broadly can reveal fundamental principles of robust energy conversion systems.

The light reactions of photosynthesis produce NADPH and ATP to power carbon xation. Light-harvesting antennae absorb photons and transfer their energy to the photosystems, which pump electrons from water to NADPH. This process is coupled to proton translocation across the thylakoid membrane into the thylakoid lumen, which drives ATP synthesis3,4. Under high light, the high proton concentration in the thylakoid lumen activates energy-dependent quenching (qE), a mechanism that dissipates absorbed light energy in the PSII antenna as heat5. qE is thus part of a negative feedback loop that decreases energy transfer to PSII by up to 75% in response to excess light68.

To maximize yields under continuously changing light intensities, the PSII antenna must rapidly alternate between dissipation of excess absorbed energy under high light, and efcient harvesting of photons for photosynthesis under limiting light9. On transition from high to limiting light, the qE energy dissipation mechanism is downregulated to increase the energy available to PSII. However, this downregulation occurs on a timescale of minutes, producing an extended period where heat dissipation of light energy starves the photosystems of excitation energy. Simulations predict that the slow downregulation of heat dissipation mechanisms can reduce the photosynthetic efciency by B10% in crop canopies10. The design principles that determine the kinetics of qE downregulation remain

unknown11, motivating a search for molecular factors that affect its dynamics.

Here we show that ion antiport activity across the thylakoid membrane accelerates photosynthetic acclimation after a change in light intensity from dark to low light, or from high to low light. In the model plant Arabidopsis thaliana (hereafter Arabidopsis), K efux antiporter 3 (KEA3) is localized in the thylakoid membrane and mediates H/K antiport. After each transition to low light, KEA3 allows proton efux from the lumen, which accelerates the downregulation of pH-dependent energy quenching, and transiently increases photosystem II quantum efciency and CO2 xation. Our ndings demonstrate that a K /H antiport activity mediated by KEA3 increases photosynthetic efciency under uctuating light conditions.

ResultsKEA3 is co-regulated with photosynthesis-related genes. The previously established regulation of qE by thylakoid pH5 led us to hypothesize that proton/cation antiporters in the thylakoid membrane could regulate the dynamics of qE. We therefore searched for proton/cation antiporters in Arabidopsis that were transcriptionally co-regulated with photosynthesis-related genes. The expression of KEA3 was strongly correlated with genes annotated with the gene ontology (GO) term photosynthesis (Supplementary Fig. 1a, Po10 12, MannWhitney U-test).

Conversely, the most signicantly enriched GO term among the 100 genes whose expression was most highly correlated with KEA3 was photosynthesis (Po10 41). KEA3 is highly conserved among photosynthetic eukaryotes (Supplementary

Fig. 1b).

KEA3 is localized in the thylakoid membrane. Two splice forms of KEA3 are annotated in Arabidopsis: KEA3.1 and KEA3.2. Both

AtKEA3

cTP KTN

TP Ch

CPA2 domain

KEA3.2 (73.3 kDa) KEA3.1 (58.6 kDa)

DIC

GFP Chlorophyll Merged

75 kDa

KEA3.2-GFP

KEA3

Lhcb1

VDAC

-Tubulin

P.R.

Control

Ch En St Th

KEA3

Toc34

RbcL

Lhcb1

P.R.

Th 1K Gr IM SL

KEA3

PsaD

Lhcb1

Cytf

P.R.

75 kDa

75 kDa

Figure 1 | KEA3 localizes to the thylakoid stromal lamellae. (a) KEA3.2 has an amino-terminal chloroplast-targeting peptide, a cation/proton exchanger (CPA2) domain and a putative KTN domain. (b) Differential interference contrast, GFP and chlorophyll uorescence were imaged in leaf guard cells from Arabidopsis WT plants and kea3-1 plants transformed with KEA3.2-GFP. Scale bar, 10 mm. (ce) KEA3 abundance was measured by western blot in: (c) Total protein (TP) and chloroplast (Ch) protein extracts; (d) Ch subfractionated into envelope (En), stroma (St) and thylakoids (Th); (e) Th separated into the fraction pelleted by 1,000g (1 K), grana (Gr), intermediate membranes (IM) and stromal lamellae (SL). (ce) Ponceau red (P.R.) stains of membranes after protein transfer before immunodetection are shown as loading controls.

2 NATURE COMMUNICATIONS | 5:5439 | DOI: 10.1038/ncomms6439 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6439 ARTICLE

encode a predicted chloroplast transit peptide and a cation/H antiporter (CPA2) domain (Fig. 1a, Supplementary Fig. 2a).

KEA3.2 additionally encodes a carboxyl-terminal (C-terminal) K transport/nucleotide-binding (KTN) domain, which is truncated in

KEA3.1. Size analyses of the endogenous KEA3 protein and tagged versions of both isoforms indicate that KEA3.2 is the major protein isoform in leaves (Supplementary Fig. 2). Recently, C-terminal uorescent protein fusions of KEA3.1 and KEA3.2 were localized to the chloroplast and the Golgi, respectively12,13. In contrast to previous work, we observed KEA3.2-green uorescent protein (GFP) in the chloroplast (Fig. 1b). We conrmed the localization of the endogenous KEA3 to the chloroplast in wild-type (WT) leaves by western blot of total and chloroplast protein extracts (Fig. 1c, Supplementary Fig. 3a). Subfractionation of WT chloroplasts demonstrated that endogenous KEA3 is located in the thylakoid membrane and is enriched in the stromal lamellae (Fig. 1d,e, Supplementary Fig. 3b,c).

KEA3 accelerates photosynthetic acclimation to low light. We analysed the phenotypes of two kea3 mutant alleles, kea3-1 and kea3-2 (Supplementary Fig. 2). These mutants displayed no measurable defects in growth, leaf colouration or protein composition of the photosynthetic apparatus (Supplementary Fig. 4). To characterize potential defects in regulation of photosynthesis, we performed chlorophyll uorescence measurements across various light conditions. We observed a striking perturbation of the kinetics of non-photochemical energy quenching (NPQ) on a shift from dark to low-light conditions. In owering plants, a shift from a dark-acclimated state to light-limiting conditions causes a transient rise in NPQ, which is attributed to qE14. The kea3 mutants displayed a higher maximum NPQ and a delay in its peak and relaxation (Fig. 2a, Supplementary Fig. 5a). This kea3 phenotype was complemented by expressing a KEA3.2-GFP fusion protein that accumulated at levels similar to the native protein (Fig. 2a, Supplementary Fig. 6). Remarkably, plants overexpressing the amino-terminal part of KEA3 containing the chloroplast transit peptide and CPA2 domains (oeKEA3CPA2)

showed accelerated qE relaxation kinetics. When shifted from dark to higher light intensities, kea3 mutants displayed WT-like induction kinetics of NPQ (Supplementary Fig. 5c,e).

The transient nature of the additional NPQ observed on a shift to low light suggested that qE was perturbed in kea3. qE is known to be modulated by the luminal pH and zeaxanthin, and normal qE kinetics require the protein PsbS14. kea3psbs double mutants exhibited the same NPQ kinetics as psbs single mutants (Fig. 2b, Supplementary Fig. 7a), indicating that PsbS is required for the additional NPQ observed in kea3. Double mutants of kea3 with npq1 (lacking zeaxanthin) and npq2 (overaccumulating zeaxanthin) revealed that the NPQ phenotype was modulated by levels of zeaxanthin (Supplementary Fig. 7b,c). Application of high concentrations of nigericin, which inhibit qE15, to leaves, abolished the rapid component of NPQ in both WT and kea3 (Supplementary Fig. 8a). Thus, the additional NPQ observed in kea3 shows the key characteristics of qE.

The transient increase in NPQ was accompanied by lower PSII quantum yield (FII) in kea3 (Po0.004, Students t-test, Fig. 2c), indicating decreased linear electron ow through PSII. This decrease in FII was PsbS dependent (Supplementary Fig. 7e), suggesting that it was caused by an increase in qE. In oeKEA3CPA2,

FII was transiently higher than in WT (Fig. 2c). We conclude that KEA3 accelerates qE relaxation, allowing more absorbed light energy to be used to drive electron ow through PSII.

KEA3 likely mediates K /H antiport. To gain insights into the identity of the cations transported by the CPA2 domain of

KEA3, we used phylogenetics. KEA3 shows more sequence homology to experimentally validated K /H antiporters

AtKEA2 (ref. 16) and EcKefC17 than to Na /H antiporters EcNhaA and TtNapA (Fig. 3a). Comparison of two amino acid residues critical for Na /H binding in Na /H antiporters of the CPA2 family18,19 indicates that presence of an Aspartate

Aspartate motif strictly correlates with Na /H antiport activity, while conversion of the rst residue to Asparagine or

Glutamine correlates with K /H antiport activity16,17,20 (Fig. 3b). Like AtKEA2 and EcKefC, KEA3 has a Glutamine

Aspartate motif. Finally, the presence of the K transport-related KTN domain in KEA3 further supports a K /H antiporter activity21,22.

We reasoned that if KEA3 is a K /H antiporter, low concentrations of the small molecule electroneutral K /H antiporter nigericin might rescue the kea3 phenotype. Strikingly, inltration of leaves with 0.03 mM nigericin fully rescued the qE phenotype (Fig. 3c, Supplementary Fig. 8a). This result strongly suggests that KEA3 accelerates the qE relaxation by electroneutral K /H antiport.

KEA3 decreases the DpH component of the proton motive force. If KEA3 is a K /H antiporter, it could alter the proton concentration (DpH) and/or electric potential (Dc) components

70 mol photons m2 s1

a

1.5

Col-0

Col-0

1.2

kea3-1

kea3-1

KEA3.2

KEA3.2

0.9

NPQ

oeKEA3CPA2

oeKEA3CPA2

0.6

0.3

0.0

NPQ

b

c

0.6

psbs kea3-1 psbs

0.3

0.0

0

0

50

50

100

100

150

150

200

200

250

250

300

300

0.8

PSII quantum yield ( II)

0.6

** *

*

*** ***

*** ***

*** *

** **

***

0.4

0.2

*

Time (s)

Figure 2 | KEA3 accelerates qE relaxation on transition from dark to low light. (a) NPQ induction in detached leaves of 3-week-old Col-0, kea3-1 and kea3-1 expressing KEA3.2-GFP (KEA3.2) or overexpressing KEA3CPA2-GFP

(oeKEA3CPA2) was measured at 70 mmol photons m 2 s 1 after 30 min dark incubation. (b) The extra NPQ in kea3 mutants requires PsbS. NPQ induction in detached leaves of 2-week-old psbs and kea3-1 psbs mutant plants was measured as in a. (c) The higher transient NPQ in kea3 decreases the PSII quantum yield (FII), and oeKEA3CPA2 show transiently increased FII. FII was calculated from the same experiment as a. Asterisks indicate time points where kea3-1 or oeKEA3CPA2 differ signicantly from WT(*0.01oPo0.04, **0.001oPo0.01, ***Po0.001, Students t-test). (ac) Error bars represent s.e.m. (n 6).

NATURE COMMUNICATIONS | 5:5439 | DOI: 10.1038/ncomms6439 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 3

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6439

CPA2 domain

90 mol photons m2 s1

AtKEA3

AtKEA2

EcKefC

EcNhaA

50

0

46

35

20

23

1

WT kea3

%Identity

41

22 23

24 21 21

0.8

NPQ

ECS t(*10

0.6

TtNapA

0.4

AtKEA3 CPA2 domain

0.2

3.0

3 )

2.0

Cavity

1.0

D274

Q273

TM5

0.0

100 200 300 400Time (s)

0

TM5 of CPA2

**

0.5

TtNhaA PLALKIFLMALAIIDDLGAIIIIALFYEcNapA SRPYSRIILGAAVIDDVLGLIVLAVVN

BcGerN KTRESTTMLGAAVFDDILVVILLAFAMSynNhaS3 SSNEGQIIIGAAVLDDILGIIVLAVVGSynNhaS4 RTPLGSLALTCAAIDDLTAWCLLAIAIAtCHX17 TTDIGKIALSAAAVNDVAAWILLALAV

ScKHA1 KDRAGIVVLAAGIINDIMGWILLALSIAtKEA2 TSRHGRATFSVLLFQDLAVVVLLILIPEcKefB RSESGQLGFSVLLFQDLAVIPALALVPEcKefC VTQMGRSAFAVLLFQDIAAIPLVAMIPAtKEA3 PTRFGSATLGILLLQDIAVVPLLVILP

Na+

K+

3 )

A ECS(*10

0

pH

0.5

70 mol photons m2 s1

90 100 110 120 130

Time (s)

1.2

*

1

WT mock kea3 mock

WT 0.03 M nigericin kea3 0.03 M nigericin

0.8

WT kea3

*

0.8

0.6

NPQ

0.6

0.4

0.4

0.2

0.2

0 0 100 200 300 600

400 500 Time (s)

pH/(pH+)

0 90 160 310 600mol photons m2 s1

Figure 3 | KEA3 regulates the composition of the p.m.f. by mediating potassium/proton antiport. (a) The CPA2 domain of KEA3 is homologous to that of known K /H antiporters. A percent identity matrix is shown for Arabidopsis KEA2 and KEA3, E. coli KefC and NhaA and T. thermophilus NapA. (b) The locations of two key substrate-binding amino acid residues in TM5 of the CPA2 domain19 are shown on a model of KEA3. (c) Low levels of Nigericin complement the kea3 NPQ phenotype. NPQ induction on transition from dark to 70 mmol m 2 s 1 light was measured in WT (Ws) and kea3-2 leaves incubated in water (mock) or 0.03 mM nigericin. (d,e) p.m.f. kinetics in kea3 mutants are largely unaffected. NPQ and ECSt (which reports the magnitude of the p.m.f.) were measured near-simultaneously in WT (Ws) and kea3-2 by Chl uorescence and dark-induced relaxation kinetics, respectively, in single leaves during a transition from dark to 90 mmol m 2 s 1. (f) After 100 s of low light, kea3 mutants show increased DpH and decreased Dc. Full ECS decay kinetics were recorded after 100 s of low light (90 mmol m 2 s 1) to measure DpH and Dc in WT (Col-0) and kea3-1. The average of six independent measurements per genotype was plotted as a moving average with interval 5 (see also Supplementary Fig. 9). (g) The experiment in f was repeated at different light intensities, and the fraction of the p.m.f. contributed by DpH was plotted. Asterisks indicate where WTand kea3 differ signicantly (*Po0.04,

Students t-test). (ce,g) Error bars represent s.e.m. (n 6).

of the proton motive force (p.m.f.). We performed near-simultaneous measurements of NPQ as a readout of DpH, and of the magnitude of the carotenoid electrochromic shift (ECSt)23 as a readout of total p.m.f. (DpH Dc), during the transition from

dark to low light (Fig. 3d,e). The dynamics of the p.m.f. were largely unchanged in kea3, but the ratio of NPQ to ECSt was higher than in WT, suggesting that KEA3 transiently decreases the contribution of DpH to the proton motive force.

We measured both DpH and Dc after 100 s illumination by turning off the light and observing the extended kinetics of the ECS24. These measurements indicated that after 100 s of low light, the DpH is greater in kea3 than in WT (Po0.04, Students t-test,

Fig. 3f,g, Supplementary Fig. 8b), consistent with the observed

increased NPQ in kea3 at this time point (Figs 2a and 3d, Supplementary Fig. 5a). This difference was not present at higher light intensities. These data further support the idea that KEA3 decreases the contribution of DpH and increases the contribution of Dc to p.m.f.

KEA3 accelerates acclimation from high to low light. Transitions from high to low light are common in many growth environments, including crop canopies1. We therefore asked whether KEA3 inuences the dynamics of photosynthesis during transitions from high to low light. After a sudden shift from high to low light, NPQ in WT Arabidopsis plants decayed over B80 s

4 NATURE COMMUNICATIONS | 5:5439 | DOI: 10.1038/ncomms6439 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6439 ARTICLE

to a new, lower level (Fig. 4a,b, Supplementary Fig. 9a). Strikingly, NPQ decayed more slowly in kea3, while high and low light NPQ values were comparable to WT. The half time of the NPQ decay in kea3 was twice as long as that in WT (Col-0: 24.81.4 s, kea3-1: 47.63.0 s, P 4.1 10 5; Ws: 25.91.4 s, kea3-2:

51.82.0 s P 1.8 10 6 ( s.e.m., Students t-test)). These

data suggest that KEA3 plays a key role in accelerating the decay of NPQ during transitions from excess to limiting light conditions.

We observed decreased FII in kea3 (Po0.02, Students t-test) over a similar time interval to the NPQ defect (Fig. 4c, Supplementary Fig. 9b). As during transition from dark to low light (Fig. 2a,c), these data suggest that the slower relaxation of NPQ in kea3 results in a transient decrease in linear electron ow relative to WT.

We measured CO2 assimilation during the same transition. While CO2 assimilation rates of kea3 and WT plants were similar under steady-state high and low light, kea3 exhibited a period of lower CO2 assimilation than WT immediately after a transition from high to low light (Fig. 4d,e, Po0.007, Supplementary Fig. 9c,d, Po0.04). We conclude that following a high- to low-light transition, KEA3 accelerates the downregulation of qE, thus allowing a larger fraction of the absorbed light energy to drive linear electron ow and CO2 xation.

KEA3 improves photosynthetic efciency in uctuating light. To investigate the role of KEA3 under continuously uctuating light, we exposed plants to alternating cycles of 140 s at high light and 100 s at low light. kea3 mutants consistently showed slower NPQ relaxation and FII recovery than WT after each transition (Fig. 4f,g and Supplementary Fig. 10). These phenotypes were present regardless of whether the uctuations were preceded by 10 min of high light or 10 min of low light, suggesting that KEA3s function is not extensively affected by preceding light conditions. NPQ and FII in kea3 reached WT levels after the uctuations stopped, further supporting the role of KEA3 specically in light transitions. We conclude that KEA3 improves photosynthetic efciency under continuously uctuating light.

DiscussionOur data are consistent with the following intriguing model: (i) The dynamics of qE relaxation are dictated by the dynamics of the thylakoid luminal pH. (ii) After a sudden transition to low light, the ATP synthase alone cannot sustain sufcient proton efux to enable rapid downregulation of qE. (iii) KEA3 overcomes this limitation by providing an additional route for proton efux, thereby accelerating qE relaxation and increasing photosynthetic yield. KEA3s electroneutral activity would provide two advantages for proton efux over a simple proton channel: (i) it retains the Dc component of the p.m.f. and (ii) it can rapidly mediate a large ux because its activity does not generate an opposing electric eld.

We note that the combined activities of KEA3 and the recently identied thylakoid potassium channel TPK3 (ref. 25) would dissipate the p.m.f. We thus anticipate that under most conditions, the activities of these two proteins are tightly regulated to minimize waste of energy. However, under some conditions causing harmful excess DpH26, KEA3 and TPK3 together may form a safety valve to allow dissipation of p.m.f.

Intriguingly, the KTN domain of KEA3 could regulate KEA3 activity in response to the redox state of the stroma. In E. coli, NADH binds to the KefC KTN domain and represses KefC activity17. The observation that the kea3 phenotype is specic to transitions to low light suggests that KEA3 might similarly be repressed by NADPH (or alternatively, activated by NADP).

0

3

0

WTkea3

NPQ

0 60 120 180 (s)

(s)

700

70 mol photons m2 s1

3

WT

kea3

Non-photochemical

quenching (NPQ)

2

1

0.6

***** **

PSII quantum yield ( II)

******

0.4

0.2

0 40 0 40 80 120 160 200

(s) after transition from HL to LL

8

140

4180

81120

121160

161200

CO 2assimilation rate

(mol CO 2m2s1)

6

WT kea3

**

0

5

10

15

20

Normalized CO 2

assimilation rate (%)

4

2

**

0 700 70 mol photons m2 s1

***

5 70 mol photons m2 s1

1,200 mol photons m2 s1

600 1,200 1,800

4

NPQ

3

2

1

WT

kea3

0

0.5

II

0.2

0

0.1

0

Time (s)

Figure 4 | KEA3 regulates the dynamics of photosynthesis during transitions from high to low light. (a,b) NPQ relaxation on transition from high to low light is delayed in kea3 (kea3-2) as compared with WT (Ws). (c)

FII is decreased during transition from high to low light. FII was calculated from the same measurement as in a,b. (d) WT (Ws) and kea3 (kea3-2) show similar CO2 assimilation rates during steady-state high (HL, 700 mmol photons m 2 s 1) and low light (LL, 70 mmol photons m 2 s 1). (e) The normalized CO2 assimilation rate (shown as a percentage of the difference between HL and LL) is decreased in kea3 (kea3-2). (f) In uctuating light, NPQ relaxation is delayed after each transition from high to low light in kea3-2. (g) FII is decreased in kea3-2 during each transition from high to low light.

FII was calculated from the same measurement as in (f). (c,e) Asterisks represent signicantly lower values in kea3-2 (**0.001oPo0.01, ***Po0.001,

Students t-test). (bg) Error bars represent s.e.m. (be: n 7; f,g: n 4).

NATURE COMMUNICATIONS | 5:5439 | DOI: 10.1038/ncomms6439 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 5

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6439

KEA3 could thus be a key component of a feedback loop, which detects an oxidized stroma and facilitates reduction of oxidized PSI electron acceptors by downregulating qE.

Our work demonstrates that the kinetics of qE decay inuence photosynthetic efciency. Our discovery that plants have a builtin machinery to accelerate these kinetics makes KEA3 a new target for efforts to improve crop productivity. Excitingly, overexpression of the KEA3 CPA2 domain produces a transient increase in the relative CO2 assimilation rate after transition from high to low light (Supplementary Fig. 9). However, the over-expression of the CPA2 domain appears to also result in undesirable photodamage in high light. It is possible that improved regulation of KEA3 would overcome this limitation. Thus, the future investigation of KEA3 should yield both fascinating insights into the core logic of photosynthesis regulation, as well as keys to possible enhancements in crop yields.

Methods

Plant material, propagation and growth conditions. The kea3-1 mutant (Gabi_170G09) in the Col-0 genetic background originates from the GABI-KAT collection27. The kea3-2 mutant (FLAG_493_C01) in the Wassilewskija (Ws) genetic background originates from the FLAGdb/FST T-DNA collection28. The aba1 mutant (Salk_059469) in the Col-0 genetic background originates from the SALK T-DNA collection29. The npq1 and psbs mutants have been described previously7,8.

Plants expressing KEA3.1 and KEA3.2 with a C-terminal HA tag were generated by introducing the KEA3 coding regions (for primers, see Supplementary Table 1) into the Gateway plant expression vector pJCV55 (ref. 30) downstream of a Cauliower mosaic virus 35S promoter. Constructs for expressing KEA3.02 and KEA3CPA2 with a C-terminal eGFP tag were generated in a similar manner, but with vector pB7FWG2. Flowers of kea3-1 mutant plants were transformed with the KEA3 constructs by oral dip31. The plants were then transferred to the greenhouse and seeds were collected after 3 weeks. Individual transgenic plants were selected on the basis of their resistance to kanamycin (pJCV55) or Basta (pB7FWG2) on sterile 1% agar media containing 1 MS nutrients (PhytoTechnology

Laboratories) and 0.05 g l 01 MES, adjusted to pH 5.7 with KOH. Before transfer to soil, Kanamycin- and Basta-resistant plants were additionally screened for red uorescent protein and GFP expression, respectively, using an M165 FC uorescence microscope (Leica) to verify expression of the transgene (the T-DNA deriving from pJCV55 carries an additional red uorescent protein-encoding gene). The presence of the KEA3 transgene in transformants was conrmed by PCR and its expression was conrmed by protein immunoblot analyses.

WT and mutant Arabidopsis plants were grown on soil under controlled conditions in the greenhouse (matted daylight supplemented with high pressure sodium 400 W lamps, B100 mmol photons m 2 s 1 on leaf surfaces, 14/10 h light/dark cycle, constant temperature of 20 C) or in growth chambers (10 h light, 22 C/14 h dark, 23 C, with a light intensity of 100 mmol photons m 2 s 1).

Nucleic acid analysis. Arabidopsis DNA was extracted using RedExtract-N-Amp (Sigma). T-DNA insertion junction sites were recovered by PCR using combinations of insertion- and gene-specic primers (see Supplementary Table 1), and then sequenced. For reverse transcriptase-PCR (RTPCR) analysis and cDNA synthesis, RNA was extracted from leaves using the RNeasy plant mini kit (Qiagen) according to the manufacturers instructions.

The cDNA for cloning purposes was synthesized using Superscript III (Invitrogen). RTPCR was performed with SuperScript III One-Step RTPCR System (Invitrogen) and gene-specic primers (see Supplementary Table 1), both according to the manufacturers instructions.

Fluorescence measurements. Room temperature Chl a uorescence of whole leaves of 23-week-old Arabidopsis plants was measured using an imaging chlorophyll uorometer (Open FluorCam 800-O/1010; Photon Systems Instruments) or the Dual-PAM 100 (Walz). NPQ during dark-to-light transitions was determined using the FluorCam by acclimating plants for 30 min to dark, followed by 510 min at indicated light intensities of red light. To measure Fm and Fm0, white light pulses (4,000 mmol photons m 2 s 1, duration 800 ms) were applied. NPQ induction and relaxation during dark to 50, 220 and 660 mmol photons m 2 s 1 transitions were monitored using the Dual-PAM by applying red actinic light and white light pulses (4,000 mmol photons m 2 s 1, duration 800 ms) at 20 s intervals. NPQ was calculated as (Fm Fm0)/Fm0 and FII as (Fm Fs)/Fm.

NPQ relaxation half times were calculated as follows. A steady-state low light average NPQ value was subtracted from the rst ve data points during the NPQ relaxation period. The binary logarithm of these values was taken, and a linear tting was performed to obtain a slope s. t1/2 was calculated as t1/2 (NPQ) 1/ s.

For nigericin rescue experiments, plants were dark adapted for 30 min. Then, leaves were pressed against sand paper to facilitate nigericin uptake and incubated in indicated concentrations of nigericin for 3 h in the dark before measuring NPQ during dark-to-light transitions at 70 mmol photons m 2 s 1.

ECS measurements. Near-simultaneous ECS and uorescence measurements as well as p.m.f. partitioning analysis were performed on a custom made spectrophotometer/chlorophyll uorometer chamber with non-focusing optics using three different wavelengths (505, 520 and 535 nm) for deconvoluted ECS measurements23,32. Leaves were dark adapted for 30 min before the actinic light of 90 mmol photons m 2 s 1 was turned on. Each dark interval relaxation kinetic measurement was made by turning off the actinic light for 200 ms. p.m.f. parsing traces were obtained by turning off the actinic light at 100 s. The ECS steady-state (Dc) and ECS inverse (DpH) were extracted from these traces as described previously24,33.

CO2 assimilation measurements. Plants were grown in 50 ml Falcon tubes with a pierced bottom lled with Sunshine Mix 4 potting mix, placed in a dish containing 13 cm of water, under 100 mmol m 2 s 1 uorescent light. For measurement, the

Falcon tubes were inserted using a foam gasket into a LI-COR 6400-17 Whole Plant Arabidoposis Chamber. The chamber was placed under a Walz MAXI IMAGING-PAM with blue actinic LEDs, which ran the light programs and was used to simultaneously record chlorophyll uorescence data. CO2 assimilation was measured with a reference CO2 concentration of 400 mmol mol 1 and a500 mmol s 1 ow rate, averaging and recording data over 2 s intervals. The steady-state assimilation rates under high and low light were obtained for each plant by averaging the assimilation rate over the intervals ( 40 to 0 s) and ( 560

to 600 s), respectively, and normalizing the assimilation rate by the rosette area

measured from IMAGING-PAM images with ImageJ. The transient CO2 assimilation was calculated as follows. For each plant, the data was normalized so that steady-state high light assimilation rate was 1, and the steady-state low light assimilation rate was 0. The average normalized assimilation rate in each time interval was then calculated for each plant.

Leaf pigment analysis. Pigments were analysed by reverse-phase high-performance liquid chromatography34. For pigment extraction, leaf discs were frozen in liquid nitrogen and disrupted with beads in microcentrifuge tubes in the presence of acetone. After a short centrifugation, pigment extracts were ltered through a membrane lter (pore size 0.2 mm) and either used directly for high-performance liquid chromatography analysis or stored for up to 2 days at 20 C.

Computational analysis. Arabidopsis KEA3.1 and KEA3.2 (At4g04850.1 and.2) DNA and protein sequences were retrieved from TAIR (The Arabidopsis Information Resource; http://www.arabidopsis.org

Web End =www.arabidopsis.org ). Subcellular localization signals were analysed by SUBA3 (http://suba.plantenergy.uwa.edu.au/

Web End =http://suba.plantenergy.uwa.edu.au/), which combines predictions from up to 22 prediction programs35, and by ChloroP (http://www.cbs.dtu.dk/services/ChloroP

Web End =http:// http://www.cbs.dtu.dk/services/ChloroP

Web End =www.cbs.dtu.dk/services/ChloroP )36.

For sequence comparisons, homologues of the Arabidopsis KEA3 protein (AtKEA3, Uniprot: Q9M0Z3) were identied by BLAST. Percent identity matrices were calculated for the CPA2 (Pfam00999) domains of E. coli NhaA (EcNhaA, P13738), T. thermophilus NapA (TtNapA, Q72IM4), E. coli KefC (EcKefC, P03819), AtKEA3, Arabidopsis KEA2 (AtKEA2, Q65272) and homologous AtKEA3 sequences from Oryza sativa (O.s., Q2QM48), Physcomitrella patens (P.p., A9SSR0) and Chlamydomonas reinhardtii (C.r., A8ISE8) by using ClustalOmega (http://www.ebi.ac.uk/Tools/msa/clustalo/

Web End =http://www.ebi.ac.uk/Tools/msa/clustalo/). For the homologous KEA3 sequences, an identity matrix was also calculated for the KTN domain (Pfam02254). To generate the KEA3 three-dimensional model, the TtNapA protein was used as a template by the Phyre2 server (http://www.sbg.bio.ic.ac.uk/~phyre2/

Web End =http://www.sbg.bio.ic.ac.uk/Bphyre2/ )37. Sequence comparison of TM5 of the CPA2 domain of AtKEA3, EcNhaA, EcKefB (P45522) EcKefC, TtNapA, Bacillus cereus GerN (BcGerN, Q9KI10), Synechocystis NhaS3 (Q55190) and NhaS4 (Q5N3F5), Arabidopsis AtCHX17 (Q9SUQ7), AtKEA2 and Saccharomyces cerevisiae KHA1 (P40309) were performed using ClustalOmega.

KEA3 co-expression analysis was performed by determining Pearson correlation coefcients of all Arabidopsis genes with KEA3 using the GeneCAT database (http://genecat.mpg.de

Web End =http://genecat.mpg.de). All genes in the database whose identiers contained At were used in this analysis. The 100 genes with the highest correlation values were analysed for the enrichment of specic GO terms by using AmiGO (amigo.geneontology.org/cgi-bin/amigo/term_enrichment,38).

Protein isolation, PAGE and immunoblot analyses. Total protein was isolated from leaves using the protein extraction buffer and corresponding protocol from Agrisera. Thylakoid membranes were isolated from leaves at 4 C by shredding leaf tissue in 0.1 M Tricine/KOH pH 7.9, 400 mM Sorbitol, protease inhibitor cocktail (Sigma) using a Waring blender and ltering the homogenate through two layers of miracloth. The ow-through was centrifuged at 1,000g for 5 min. The resulting pellet was resuspended and incubated in 20 mM Hepes/KOH pH 7.6, 10 mM EDTA for 30 min to break remaining intact chloroplasts. Thylakoid membranes were then extracted by a centrifugation at 10,000g for 10 min. For SDS-

6 NATURE COMMUNICATIONS | 5:5439 | DOI: 10.1038/ncomms6439 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6439 ARTICLE

polyacrylamide gel electrophoresis, proteins were fractionated on Tris-glycine gels (Bio-Rad). For Blue Native polyacrylamide gel electrophoresis, thylakoid membranes were solubilized with 0.7% b-n-dodecyl-D-maltoside (w/v) and separated by

Blue Native gels (Invitrogen)39. For protein blot analysis, proteins were transferred to a nitrocellulose membrane, blocked with 5% (w/v) nonfat dry milk and hybridized with the antibodies indicated in the gure legends and text. Antibodies (except for a-KEA3, a-GFP and a-HA) were purchased from Agrisera. All antibodies were diluted in 50 mM Tris, 150 mM NaCl, 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dry milk before use (a-Tubulin (AS10680) 1:3,000, VDAC (AS07212)

1:10,000, Lhcb1 (AS01004) 1:10,000, RbcL (AS03037) 1:20,000, TOC34 (AS07238) 1:2,000, PsaD (AS09461) 1:5,000, Cytf (AS08306) 1:10,000, CP47 (AS04038) 1:10,000 and PsbS (AS03032) 1:5,000. a-GFP (Invitrogen, A-6455) was diluted 1:2,000 and a-HA (Sigma, H6908) 1:2,500. Uncropped scans of the western blots are shown in Supplementary Fig. 3. AtpB and PsbS signals were quantied by densitometric analysis of western bands using NIH ImageJ software and associated plug-ins (http://imagej.nih.gov/ij/

Web End =http://imagej.nih.gov/ij/).

For immunolocalization of KEA3, an antibody against the specic peptide sequence NQLGRKAADFLDERLDPGE (present in both KEA3.1 and KEA3.2 isoforms) was generated in rabbits by Yenzym and a dilution of 1:100 in 50 mM Tris, 150 mM NaCl, 0.01% (v/v) and 5% (w/v) nonfat dry milk was used for hybridization.

Determination of KEA3 localization. Leaves of Col-0 or kea3-1 plants expressing KEA3.2-GFP were analysed using a Leica SP5 AOBS Point Scanning Confocal Microscope. GFP and chlorophyll autouorescence (Chl) were excited at 488 nm and signals were collected at 660736 nm (Chl) and 495515 nm (GFP).

Arabidopsis chloroplasts were isolated by a two-step Percoll gradient centrifugation40. The homogenized leaf tissue was applied to a two-step Percoll gradient and intact chloroplasts were collected from the interphase. For chloroplast fractionation, chloroplasts were hypotonically lysed in 10 mM Tris/HCl, pH 8.0 and 1 mM EDTA, pH 8.0, at a chlorophyll concentration of 2 mg ml 1, and loaded onto a three-step Sucrose gradient41. After centrifugation (30,000g; 4 C, 1 h), the upper phase (containing the stroma), the upper interphase (with the envelope membranes) and the pellet (thylakoids) were collected and the proteins were precipitated42.

Thylakoid fractionation was performed according to Kyle et al.43 with minor modications. Thylakoid membranes at 0.4 mg chlorophyll ml 1 in 15 mM Tricin/

KOH pH7.8, 0.1 M Sorbitol, 15 mM NaCl, 5 mM MgCl2 were treated with 0.15% digitonin (w/v) for 1 min and then diluted 10-fold with the same buffer. Samples were then centrifuged at 1,000; 10,000; 40,000; and 150,000g to yield the different fractions.

References

1. Pearcy, R. W. Sunecks and photosynthesis in plant canopies. Annu. Rev. Plant Physiol. Plant Mol. Biol. 41, 421453 (1990).

2. Reinhard, K., Smith, W. K. & Carter, G. A. Clouds and cloud immersion alter photosynthetic light quality in a temperate mountain cloud forest. Botany 88, 462470 (2010).

3. Kramer, D. M., Cruz, J. A. & Kanazawa, A. Balancing the central roles of the thylakoid proton gradient. Trends Plant. Sci. 8, 2732 (2003).

4. Allen, J. Photosynthesis of ATP-electrons, proton pumps, rotors, and poise. Cell 110, 273276 (2002).

5. Mller, P., Li, X. P. & Niyogi, K. K. Non-photochemical quenching. A response to excess light energy. Plant Physiol. 125, 15581566 (2001).

6. Demmig-Adams, B. et al. Using chlorophyll uorescence to assess the fraction of absorbed light allocated to thermal dissipation of excess excitation. Physiol. Plant. 98, 253264 (1996).

7. Li, X. P. et al. A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature 403, 391395 (2000).

8. Niyogi, K. K., Grossman, A. R. & Bjrkman, O. Arabidopsis mutants dene a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. Plant Cell 10, 11211134 (1998).

9. Demmig-Adams, B., Cohu, C. M., Muller, O. & Adams, 3rd W. W. Modulation of photosynthetic energy conversion efciency in nature: from seconds to seasons. Photosynth. Res. 113, 7588 (2012).

10. Zhu, X. G., Ort, D. R., Whitmarsh, J. & Long, S. P. The slow reversibility of photosystem II thermal energy dissipation on transfer from high to low light may cause large losses in carbon gain by crop canopies: a theoretical analysis.J. Exp. Bot. 55, 11671175 (2004).11. Zaks, J., Amarnath, K., Kramer, D. M., Niyogi, K. K. & Fleming, G. R. A kinetic model of rapidly reversible nonphotochemical quenching. Proc. Natl Acad. Sci. USA 109, 1575715762 (2012).

12. Kunz, H. H. et al. Plastidial transporters KEA1, -2, and -3 are essential for chloroplast osmoregulation, integrity, and pH regulation in Arabidopsis. Proc. Natl Acad. Sci. USA 111, 74807485 (2014).

13. Zheng, S., Pan, T., Fan, L. & Qiu, Q. S. A novel AtKEA gene family, homolog of bacterial K /H antiporters, plays potential roles in K homeostasis and osmotic adjustment in Arabidopsis. PLoS ONE 8, e81463 (2013).

14. Kalituho, L., Beran, K. C. & Jahns, P. The transiently generated nonphotochemical quenching of excitation energy in Arabidopsis leaves is modulated by zeaxanthin. Plant Physiol. 143, 18611870 (2007).

15. Brooks, M. D., Sylak-Glassman, E. J., Fleming, G. R. & Niyogi, K. K. A thioredoxin-like/beta-propeller protein maintains the efciency of light harvesting in Arabidopsis. Proc. Natl Acad. Sci. USA 110, E2733E2740 (2013).

16. Aranda-Sicilia, M. N. et al. Arabidopsis KEA2, a homolog of bacterial KefC, encodes a K( )/H( ) antiporter with a chloroplast transit peptide. Biochim.

Biophys. Acta 1818, 23622371 (2012).17. Fujisawa, M., Ito, M. & Krulwich, T. A. Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity. Proc. Natl Acad. Sci. USA 104, 1328913294 (2007).

18. Arkin, I. T. et al. Mechanism of Na /H antiporting. Science 317, 799803 (2007).

19. Lee, C. et al. A two-domain elevator mechanism for sodium/proton antiport. Nature 501, 573577 (2013).

20. Maresova, L. & Sychrova, H. Arabidopsis thaliana CHX17 gene complements the kha1 deletion phenotypes in Saccharomyces cerevisiae. Yeast 23, 11671171 (2006).

21. Chanroj, S. et al. Conserved and diversied gene families of monovalent cation/ H antiporters from algae to owering plants. Front. Plant Sci. 3, 25 (2012).

22. Roosild, T. P., Miller, S., Booth, I. R. & Choe, S. A mechanism of regulating transmembrane potassium ux through a ligand-mediated conformational switch. Cell 109, 781791 (2002).

23. Sacksteder, C. A. & Kramer, D. M. Dark-interval relaxation kinetics (DIRK) of absorbance changes as a quantitative probe of steady-state electron transfer. Photosynth. Res. 66, 145158 (2000).

24. Cruz, J. A., Sacksteder, C. A., Kanazawa, A. & Kramer, D. M. Contributionof electric eld (Dj) to steady-state transthylakoid proton motive force (pmf)

in vitro and in vivo. control of pmf parsing into Dj and DpH by ionic strength. Biochemistry 40, 12261237 (2001).25. Carraretto, L. et al. A thylakoid-located two-pore K channel controls photosynthetic light utilization in plants. Science 342, 114118 (2013).

26. Kramer, D., Sacksteder, C. & Cruz, J. How acidic is the lumen? Photosynth. Res. 60, 151163 (1999).

27. Rosso, M. G. et al. An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for anking sequence tag-based reverse genetics. Plant Mol. Biol. 53, 247259 (2003).

28. Samson, F. et al. FLAGdb/FST: a database of mapped anking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants. Nucleic Acids Res. 30, 9497 (2002).

29. Alonso, J. M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653657 (2003).

30. Karimi, M., Inze, D. & Depicker, A. GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7, 193195 (2002).

31. Clough, S. J. & Bent, A. F. Floral dip: a simplied method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735743 (1998).

32. Sacksteder, C. A., Kanazawa, A., Jacoby, M. E. & Kramer, D. M. The proton to electron stoichiometry of steady-state photosynthesis in living plants: a proton-pumping Q cycle is continuously engaged. Proc. Natl Acad. Sci. USA 97, 1428314288 (2000).

33. Takizawa, K., Cruz, J. A., Kanazawa, A. & Kramer, D. M. The thylakoid proton motive force in vivo. Quantitative, non-invasive probes, energetics, and regulatory consequences of light-induced pmf. Biochim. Biophys. Acta 1767, 12331244 (2007).

34. Farber, A., Young, A. J., Ruban, A. V., Horton, P. & Jahns, P. Dynamics of xanthophyll-cycle activity in different antenna subcomplexes in the photosynthetic membranes of higher plants. Plant Physiol. 115, 16091618 (1997).

35. Tanz, S. K. et al. SUBA3: a database for integrating experimentation and prediction to dene the SUBcellular location of proteins in Arabidopsis. Nucleic Acids Res. 41, D1185D1191 (2013).

36. Emanuelsson, O., Nielsen, H. & von Heijne, G. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci. 8, 978984 (1999).

37. Kelley, L. A. & Sternberg, M. J. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, 363371 (2009).

38. Boyle, E. I. et al. GO::TermFinderopen source software for accessing Gene Ontology information and nding signicantly enriched gene ontology terms associated with a list of genes. Bioinformatics 20, 37103715 (2004).

39. Peng, L., Shimizu, H. & Shikanai, T. The chloroplast NAD(P)H dehydrogenase complex interacts with photosystem I in Arabidopsis. J. Biol. Chem. 283, 3487334879 (2008).

40. Kunst, L. Preparation of physiologically active chloroplasts from Arabidopsis. Methods Mol. Biol. 82, 4348 (1998).

41. Li, H. M., Moore, T. & Keegstra, K. Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts. Plant Cell 3, 709717 (1991).

NATURE COMMUNICATIONS | 5:5439 | DOI: 10.1038/ncomms6439 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 7

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6439

42. Wessel, D. & Flgge, U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141143 (1984).

43. Kyle, D. J., Staehelin, L. A. & Arntzen, C. J. Lateral mobility of the light-harvesting complex in chloroplast membranes controls excitation energy distribution in higher plants. Arch. Biochem. Biophys. 222, 527541 (1983).

Acknowledgements

We thank Luke Mackinder for uorescence microscopy assistance; Jose Dinneny and Lina Duan for Arabidopsis growth and genotyping assistance; Krishna Niyogi for providing psbs mutant seeds, plant growth space and Imaging-PAM; and David Ehrhardt, Virginia Walbot, Trinna Cuellar, Xiaobo Li, Luke Mackinder, Leif Pallesen, Ru Zhang, Robert Jinkerson, Wolf Frommer, Jonathan Weissman and Peter Walter for feedback on this manuscript. This project was funded by the Carnegie Institution for Science, by ERDF-conanced grants from the Ministry of Economy and Competitiveness (BIO2012-33655) and Junta de Andalucia (CVI-7558) to K.V., the Natural Sciences and Engineering Research Council of Canada (NSERC) PGS-D3 scholarship to L.P. and Deutsche Forschungsgemeinschaft grants (JA 665/10-1 and GRK 1525 to P.J.; AR 808/1-1 to U.A.).

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Web End =http://www.nature.com/ http://www.nature.com/naturecommunications

Web End =naturecommunications

Competing nancial interests: The Carnegie Institution for Science has submitted a patent form on behalf of U.A. and M.J. on aspects of the ndings.

Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/

Web End =http://npg.nature.com/ http://npg.nature.com/reprintsandpermissions/

Web End =reprintsandpermissions/

How to cite this article: Armbruster, U. et al. Ion antiport accelerates photosynthetic acclimation in uctuating light environments. Nat. Commun. 5:5439 doi: 10.1038/ ncomms6439 (2014).

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Web End =http://creativecommons.org/licenses/by/4.0/

Author contributions

U.A. and M.C.J. conceived the study; U.A., P.J., K.V., D.M.K. and M.C.J. designed the experiments; U.A. performed KEA3 localization, generation and analysis of double mutants; U.A. and E.S were responsible for the generation of KEA3-GFP expressing plants; U.A. performed the analysis; U.A. and L.P. performed Chl a uorescence

measurements; L.R.C. performed simultaneous Chl a uorescence and ECS measurements, parsing experiments; U.A., M.C.J., A.K. and J.A.B. performed simultaneous Chl a uorescence and gas exchange measurements; L.P. performed the pigment analysis; U.A. and K.V. performed the phylogenetic analyses; U.A. and M.C.J. wrote the paper.

8 NATURE COMMUNICATIONS | 5:5439 | DOI: 10.1038/ncomms6439 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.