Identification of a Photosystem II Phosphatase Involved inLight Acclimation in Arabidopsis W

Iga Samol,a Alexey Shapiguzov,a,1 Björn Ingelsson,b Geoffrey Fucile,a Michèle Crèvecoeur,a Alexander V. Vener,b

Jean-David Rochaix,a and Michel Goldschmidt-Clermonta,2

a Department of Botany and Plant Biology and Department of Molecular Biology, University of Geneva, 30 quai Ernest Ansermet, 1211Geneva 4, SwitzerlandbDepartment of Clinical and Experimental Medicine, Linköping University, SE-581 85 Linköping, Sweden

Reversible protein phosphorylation plays a major role in the acclimation of the photosynthetic apparatus to changes in light.Two paralogous kinases phosphorylate subsets of thylakoid membrane proteins. STATE TRANSITION7 (STN7)phosphorylates LHCII, the light-harvesting antenna of photosystem II (PSII), to balance the activity of the twophotosystems through state transitions. STN8, which is mainly involved in phosphorylation of PSII core subunits,influences folding of the thylakoid membranes and repair of PSII after photodamage. The rapid reversibility of theseacclimatory responses requires the action of protein phosphatases. In a reverse genetic screen, we identified thechloroplast PP2C phosphatase, PHOTOSYSTEM II CORE PHOSPHATASE (PBCP), which is required for efficientdephosphorylation of PSII proteins. Its targets, identified by immunoblotting and mass spectrometry, largely coincidewith those of the kinase STN8. The recombinant phosphatase is active in vitro on a synthetic substrate or on isolatedthylakoids. Thylakoid folding is affected in the absence of PBCP, while its overexpression alters the kinetics of statetransitions. PBCP and STN8 form an antagonistic kinase and phosphatase pair whose substrate specificity andphysiological functions are distinct from those of STN7 and the counteracting phosphatase PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATED PHOSPHATASE38, but their activities may overlap to some degree.

INTRODUCTION

The primary reactions of plant and algal photosynthesis occur in thethylakoid membranes of chloroplasts. These membranes harborlarge multimeric pigment-protein complexes called photosystemsthat convert light energy into chemical energy with high efficiency,a process that ultimately fuels most of the biosphere. In the linearmode of electron transfer through the photosynthetic chain, pho-tosystem II (PSII) and photosystem I (PSI) are connected in seriesthrough plastoquinone (PQ), the cytochrome b6f complex, andplastocyanin (Eberhard et al., 2008). The activities of PSII and PSImust be balanced for efficient electron transfer, which requires anappropriate redox poise of the intermediate electron carriers. Underlow light, the activity of the two photosystems is tuned througha mechanism called state transitions (Allen, 1992; Wollman, 2001;Aro and Ohad, 2003; Rochaix, 2007; Lemeille and Rochaix, 2010;Minagawa, 2011). A mobile part of the light-harvesting complex II(LHCII) light-harvesting antenna is dynamically allocated to PSII orPSI depending on the redox state of the PQ pool. Binding of re-duced plastoquinol to the Qo site of the b6f complex activates thethylakoid kinase STN7, which phosphorylates components of theLHCII antenna and causes their dissociation from PSII and at least in

part their association with PSI (state 2) (Vener et al., 1997; Zitoet al., 1999; Bellafiore et al., 2005; Bonardi et al., 2005; Minagawa,2011; Tikkanen and Aro, 2012). This process is reversible; whenthe PQ pool is oxidized, dephosphorylation by the recentlyidentified PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATEDPHOSPHATASE38 (PPH1/TAP38) phosphatase (Pribil et al., 2010;Shapiguzov et al., 2010) results in the association of the LHCIIcomponents with PSII (state 1). In the green alga Chlamydomonasreinhardtii, a large fraction of the antenna is implicated in statetransitions, which are controlled by the protein kinase Stt7, theortholog of STN7 (Depège et al., 2003). In the alga, transition tostate 2 induces cyclic electron flow around PSI and favors theproduction of ATP (Finazzi et al., 2002).The thylakoid membranes of plant chloroplasts form a con-

tinuous network of flattened membrane vesicles organized intoregular stacks with many tightly appressed layers, called grana,interconnected by nonappressed stromal lamellae (Dekker andBoekema, 2005). The two photosystems are unevenly distrib-uted in the thylakoid membranes. PSII is confined to grana,while PSI is enriched in stromal lamellae as well as in the mar-gins and ends of grana stacks. State transitions involve changesin thylakoid membrane stacking that become strikingly apparentwhen de-enveloped chloroplasts are examined under the electronmicroscope (Chuartzman et al., 2008). In vivo, phosphorylation ofthylakoid proteins such as the core subunits of PSII by the kinaseSTN8, a paralog of STN7, favors changes in membrane folding(Vainonen et al., 2005; Fristedt et al., 2009a). In vitro, stacking isstrongly promoted by increasing cation concentrations, and thiscan counteract the effect of thylakoid protein phosphorylation(Barber, 1980, 1982; Fristedt et al., 2010).

1 Current address: Institute of Plant Physiology, Russian Academy ofSciences, Botanicheskaya Street, 35, 127276 Moscow, Russia.2 Address correspondence to michel.goldschmidt-clermont@unige.ch.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Michel Goldschmidt-Clermont (michel.goldschmidt-clermont@unige.ch).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.112.095703

The Plant Cell, Vol. 24: 2596–2609, June 2012, www.plantcell.org ã 2012 American Society of Plant Biologists. All rights reserved.

PSII has the remarkable ability to extract electrons from waterusing energy from light. Photooxidation of the reaction centerchlorophyll (P680+) creates one of the strongest oxidants inbiology. This powerful photochemistry can cause collateraldamage to the PSII complex itself, particularly to its D1 subunit(Edelman and Mattoo, 2008). Moreover, molecular oxygen canreact with light-activated chlorophyll molecules or reduced co-factors of the electron transfer chain to produce highly reactiveoxygen species, which can be damaging to the photosyntheticmachinery and to other components of the chloroplast. Thus,photosynthetic organisms, which are subjected to large fluctu-ations in their light environment, must continuously adapt tomaximize photosynthetic activity under low light and to minimizephotooxidative damage under excess light (Eberhard et al.,2008; Li et al., 2009). Photooxidative damage occurs at all lightintensities, but when the rate of damage exceeds the capacityfor repair, photoinhibition is manifested as a decrease in pho-tosynthetic activity and in the proportion of active PSII reactioncenters (Edelman and Mattoo, 2008). Under conditions when theabsorbed light excitation energy exceeds the capacity of thephotosynthetic electron transfer chain or the metabolic de-mands of the cell, a number of different mechanisms interveneto downregulate light harvesting and alleviate photooxidativedamage. Alternative routes of electron flow, which counteractthe excessive reduction of electron carriers, include cyclicelectron flow around PSI, the chlororespiratory pathway in-volving plastid terminal oxidase, as well as the Mehler reactionand the water-water cycle (Eberhard et al., 2008). Non-photochemical quenching (NPQ) is induced in the light-harvestingantenna of PSII (LHCII) to dissipate excess captured light energyas heat (Li et al., 2009). The induction of NPQ limits the reductionof the PQ pool by decreasing the activity of PSII. Thus, in highlight, NPQ takes over a major role instead of state transitions inmaintaining the redox poise of the electron transfer chain(Tikkanen et al., 2011; Tikkanen and Aro, 2012). Whereas phos-phorylation of LHCII by the STN7 kinase is inhibited under highlight, most likely through the thioredoxin pathway (Rintamäki et al.,2000; Martinsuo et al., 2003), phosphorylation of the PSII coresubunits is enhanced (Tikkanen et al., 2008, 2010).

The D1 subunit of PSII is the primary target of photooxidativedamage, and an active cycle of repair functions to selectivelyreplace the damaged subunit. Because PSII is mainly localizedin the tightly appressed membranes of the thylakoid grana, therepair cycle requires its migration to membrane domains that areexposed to the stroma, allowing the selective degradation ofthe damaged D1 subunit and the synthesis, maturation, andassembly of a new subunit (Baena-González and Aro, 2002;Takahashi and Murata, 2008). This process is facilitated by theprotein kinase STN8 (Tikkanen et al., 2008), which showsspecificity toward proteins of the PSII core (Bonardi et al., 2005;Vainonen et al., 2005). The action of the STN8 kinase favorsunfolding of thylakoid membranes and the ability of the degra-dation and repair machinery to access damaged D1 (Fristedtet al., 2009a).

The kinase STN7 and the phosphatase PPH1/TAP38 forma pair with opposing effects on phosphorylation of the LHCIIantenna, an antagonism that allows the reversible regulation ofstate transitions to maintain the redox poise of the electron

transfer chain. Similarly, it is expected that chloroplast phos-phatase(s) will counterbalance the activity of the STN8 kinase.Here, we identify a protein phosphatase that is required for ef-ficient dephosphorylation of several core subunits of PSII in vivoand show that the purified recombinant protein is active in vitro.Mutants lacking this phosphatase show changes in the foldingof thylakoid membranes. In the mutants, state transitions are notaffected, but they are altered in transgenic lines overexpressingthe phosphatase.

RESULTS

Efficient Dephosphorylation of the PSII Core Requires theProtein Phosphatase PBCP

We designed a genetic screen for chloroplast phosphatases in-volved in dephosphorylation of thylakoid proteins in Arabidopsisthaliana (Shapiguzov et al., 2010). In brief, we first searched ge-nomic databases for putative catalytic or regulatory subunits ofprotein phosphatases and then selected those that were predictedto be targeted to the chloroplast (Suba II; Heazlewood et al., 2007).They were ranked by the strength of these predictions and thedegree of correlated coexpression of their mRNAs with those of theprotein kinases STN7 and STN8 in the Genevestigator microarraydatabase (Zimmermann et al., 2005). Homozygous mutants forthese predicted phosphatases were obtained from the SALK col-lection (Alonso and Ecker, 2006). Seedlings were systematicallyscreened for defective dephosphorylation of thylakoid proteins aftera shift from white to far-red light, using a streamlined immunoblotassay with antiphosphothreonine antibodies (Rintamäki et al.,1997). The PPH1/TAP38 phosphatase previously identified in thisscreen is required for efficient dephosphorylation of LHCII antennaproteins and transition from state 2 to state 1 (Pribil et al., 2010;Shapiguzov et al., 2010).Using the same screen, we identified a different phosphatase

that is required for efficient dephosphorylation of PSII coresubunits. In moderate white light, many thylakoid proteins are-phosphorylated by the kinases STN7 and STN8, includingcomponents of the light-harvesting complex LHCII and of PSII,as well as other thylakoid proteins, such as THYLAKOIDSOLUBLE PROTEIN9 and CALCIUM SENSING (CaS) (Vener,2007; Vainonen et al., 2008; Fristedt et al., 2009b; Reiland et al.,2011). Upon exposure to far-red light, which favors PSI excita-tion, the PQ pool is oxidized, a transition to state 1 is induced,and several thylakoid proteins are dephosphorylated. In the wildtype, strong dephosphorylation of the core subunits of PSII andof the LHCII antenna is apparent within 20 min in immunoblotswith antiphosphothreonine antibodies (from Zymed; Figure 1A).By contrast, phosphorylation of the PSII subunits CP43, D2, andD1 is partly retained even after 40 min in the mutant affectingPBCP (for photosystem II core phosphatase; At2g30170), whilethe dephosphorylation of the LHCII antenna proceeds as effi-ciently as in the wild type. It is interesting to contrast thespecificity of the pbcp-1 phenotype with that of pph1-3, wheredephosphorylation of the PSII subunits proceeds normally whilethe LHCII antenna remains phosphorylated (Figure 1A). Thus,the two phosphatases seem to have complementary roles:

Photosystem II Core Phosphatase 2597

PBCP is primarily required for efficient dephosphorylation ofthe PSII core, while PPH1/TAP38 is required for efficient de-phosphorylation of the LHCII antenna.

Mass Spectrometry Analysis of PBCP-Dependent ThylakoidProtein Dephosphorylation

To measure the phosphorylation of thylakoid proteins specifi-cally and quantitatively, we used two methods based on HPLCcoupled to electrospray ionization mass spectrometry (LC-MS).Thylakoid membranes were isolated from the leaves of mutantand wild-type plants that had been exposed to blue light or far-red light for 30 min. The surface-exposed peptides were thenreleased by extensive proteolytic shaving with trypsin. In thefirst method of analysis, the peptide samples from the wildtype and the mutant were differentially labeled by esterificationof carboxylic groups with hydrogen- or deuterium-containingmethanol, respectively (Vener et al., 2001; Ficarro et al., 2002;

Shapiguzov et al., 2010). A 1:1 mixture of these two preparationswas subjected to immobilized metal ion affinity chromatography(IMAC) to retain the phosphorylated peptide methyl esters.These were then subjected to LC-MS, which allowed simul-taneous measurements of light- and heavy-isotope labeledphosphopeptide pairs and, thus, the determination of the ratio ofphosphorylation of specific peptides in the mutant and the wildtype (Table 1). In the second method of analysis, the surface-exposed peptides released by trypsin were directly analyzed byhigh-resolution LC-MS. Both the phosphorylated and dephos-phorylated forms of peptides from the core subunits of PSII wereidentified and quantified as described previously (Fristedt et al.,2010). The values for phosphorylation of individual peptideswere corrected for the difference in the signal intensities of thephosphorylated and dephosphorylated forms, and the percent-age of phosphorylation of each peptide was calculated (Table 2).Strikingly, after exposure to far-red light, the ratio of phos-

phorylation of the D1 and D2 polypeptides of PSII in the pbcp-1

Figure 1. PSII Dephosphorylation Is Deficient in pbcp Mutants.

(A) Wild-type (WT), pph1-3, and pbcp-1 seedlings were grown in white light for 10 d and then transferred to far-red light (FR) for 20 or 40 min. Totalprotein extracts were analyzed by immunoblotting with antiphosphothreonine antibodies from Zymed and then with antiactin antibodies as a loadingcontrol.(B) Wild-type, pbcp-1, and pbcp-2 seedlings were treated and analyzed as in (A) with antiphosphothreonine antibodies (Cell Signaling). The relativeintensities of the phosphoprotein signals differ with different sources of antiphosphothreonine antibodies (cf. to [A], where the antibody from Zymedwas used).(C) Wild-type, pbcp-1, and rescued (resc; pbcp-1;35S:PBCP:HA) seedlings were treated and analyzed as in (A) with antiphosphothreonine antibodiesfrom Zymed. Anti-PBCP serum was also used to monitor the level of expression of the transgenic protein in the rescued line. Its slower migration is dueto the presence of the triple HA epitope, but the nature of the doublet band that is observed is not known.(D) Wild-type, pbcp-1, and pbcpOE overexpressor seedlings were analyzed as in (C). Minor bands that comigrate with PBCP:HA in the wild-type andpbcp-1 samples (FR) are due to artifactual spillover of the samples from the adjacent lanes.

2598 The Plant Cell

mutant versus the wild type was approximately two (Table 1), inagreement with the results of immunoblotting. In the wild type,the percentage of phosphorylation of D1 and D2 was twofoldlower after far-red light treatment compared with blue light(Table 2). By contrast, in the pbcp-1 mutant phosphorylationdecreased only slightly compared with the decrease in the wildtype. We also noted that in blue light D1 and D2 were phos-phorylated to a higher extent in the mutant than in the wild type(Tables 1 and 2).

We could also detect and quantify two phosphorylated formsof PsbH. After far-red treatment, the isoform of PsbH that ismonophosphorylated at Thr-2 was over twofold more abundantin the mutant than in the wild type, reflecting its higher degree ofphosphorylation in the pbcp-1 mutant (Table 2). The form that isdiphosphorylated at Thr-2 and Thr-4 showed a different be-havior. It was present in similar proportions in the mutant andthe wild type after far-red treatment, even though in the mutant itmay have been slightly overphosphorylated in blue light (Table2). The proportion of diphosphorylated PsbH in the wild type andin pbcp-1 diminished to a similar extent in far-red compared withblue, suggesting that PBCP is not required for dephosphorylationof P-Thr at position 4.

After far-red treatment, there was no significant differencebetween the mutant and the wild type in the level of phos-phorylation of the LHCII antenna proteins Lhcb1 and Lhcb2(Table 1), in agreement with the results obtained by immuno-blotting with antiphosphothreonine antibodies (Figure 1A). Un-der blue light, the mutant showed reduced levels of Lhcb1phosphorylation compared with the wild type. One interpretationof this observation is that compensatory changes in the activityof other phosphatases could occur when PBCP is deficient. Thephosphorylation levels of the CaS protein, a presumptive targetof STN8, were also unaffected in the mutant (Vainonen et al.,2008). Phosphopeptides from CP43 were not detected in thismass spectrometry (MS) analysis; conversely, PsbH was notanalyzed by immunoblotting. Taken together, the data fromthe two methods indicate that PBCP is required for efficient

dephosphorylation of the core subunits of PSII (CP43, D1, D2,and PsbH-Thr2) but not of the LHCII proteins, such as Lhcb1and Lhcb2.

PBCP Belongs to the Family of PP2C Phosphatases

We obtained two alleles of PBCP: pbcp-1 carries a SALK T-DNAinsertion in the fourth exon and pbcp-2 contains a WiscDs-Loxinsert in the 59 untranslated region (see Supplemental Figure 1online) (Alonso and Ecker, 2006; Woody et al., 2007). Both ho-mozygous mutants show similar delays in dephosphorylation ofthe core proteins of PSII (Figure 1B). In this panel, an anti-phosphothreonine antibody from a different commercial source(Cell Signaling) was used for comparison with Figure 1A (anti-body from Zymed). Different antibodies have distinct recognitionspecificities, as demonstrated by contrasting the relative signalintensities of the main phosphoproteins detected in Figures 1Aand 1B (Vainonen et al., 2005). In particular, the antibodies fromZymed detect phosphorylation of the PSII subunits more effi-ciently; conversely, those from Cell Signaling recognize LHCIImore strongly.

Table 1. Ratios of Phosphopeptides in pbcp-1 versus Wild-Type Leaves

Protein Name Identified Phosphopeptide At Gene Identifier

pbcp-1/Wild Type Ratio

Blue Far Red

D1 Ac-tAILER AtCg00020 1.4 6 0.1** 2.2 6 0.2**D2 Ac-tIALGK AtCg00270 1.3 6 0.1** 2.0 6 0.2**PsbH AtQTVEDSSR AtCg00710 1.3 6 0.5 2.0 6 0.9PsbH AtQtVEDSSR AtCg00710 1.6 6 0.5 1.5 6 0.5LHCB 1.1 Ac-RKtVAKPK At1g29910 0.7 6 0.1** 0.9 6 0.2LHCB 1.2 At1g29920LHCB 1.3 At1g29930LHCB 1.5 At2g34420LHCB 2.1 Ac-RRtVK At2g05100 0.8 6 0.2 0.8 6 0.1LHCB 2.2 At2g05070LHCB 2.4 At3g27690CaS SGtKFLPSSD At5g23060 1.0 6 0.3 1.1 6 0.3

Some members of the LHCB family share a common tryptic peptide as shown; phosphorylation cannot be ascribed to a specific gene product. “Ac”denotes N-terminal acetylation and “t” represents P-Thr. Results are from four IMAC experiments with differential stable isotope labeling. Asterisksindicate a statistically significant difference at P < 0.01 in the Student’s t test.

Table 2. In Vivo Phosphorylation Stoichiometry (Percentage ofPhosphorylation) for the PSII Core Proteins from Wild-Type and pbcp-1Leaves

Protein

Blue Light Far-Red Light

Wild Type pbcp-1 t Test Wild Type pbcp-1 t Test

P-D1 42 6 4 60 6 6 ** 20 6 3 45 6 4 **P-D2 42 6 7 56 6 9 * 17 6 4 32 6 2 **P-PsbH 35 6 6 40 6 2 20 6 3 42 6 3 **PP-PsbH 23 6 9 37 6 4 ** 12 6 6 19 6 5

A single asterisk and a double asterisk represent statistically significantdifferences between the pbcp-1 mutant and the wild type at P < 0.05 andP < 0.01, respectively, in the Student’s t test.

Photosystem II Core Phosphatase 2599

We used Agrobacterium tumefaciens to transform the ho-mozygous pbcp-1 mutant with a wild-type copy of the PBCPcDNA tagged with a triple-haemagglutinin (HA) epitope andplaced under the control of the cauliflower mosaic virus 35Spromoter. Among the pbcp-1;35S:PBCP:HA transformed lines,one was selected in which PBCP accumulates to levels onlyslightly higher than the wild type (dubbed “rescued”). As shownin Figure 1C, rapid dephosphorylation of the PSII core subunits,induced by a transfer from white to far-red light, was restored toa large extent in the rescued line. Taken together, these datashow that the defect in dephosphorylation of the PSII coreis indeed caused by a mutation in PBCP. A second pbcp-1;35S:PBCP:HA line was also obtained, in which PBCP is stronglyoverexpressed (pbcpOE; Figure 1D). In this line, dephospho-rylation of the core subunits of PSII was more rapid than in thewild type. Unexpectedly, the LHCII antenna was also somewhatless phosphorylated compared with the wild type in white lightor after 20 min of far-red treatment. Since phosphorylation ofLHCII is not elevated in the pbcp mutants, it is not clear whetherdephosphorylation of LHCII in pbcpOE indicates that the antennais a target of PBCP or whether overexpression induces a lossof specificity and a promiscuous activity of the phosphatasetoward LHCII.

The PBCP polypeptide deduced from the cDNA sequence has298 residues, a molecular mass of 32 kD, and is predicted tocarry an N-terminal transit peptide for chloroplast targeting bythe algorithms TargetP (Emanuelsson et al., 2000), Wolfpsort(Horton et al., 2007), and Multiloc (Höglund et al., 2006). ThePBCP protein is not predicted to contain any transmembranedomains. The mature protein belongs to the family of PP2Cprotein phosphatases, which comprises ;80 members in plants(Kerk et al., 2008; Xue et al., 2008) and includes the PPH1/TAP38 phosphatase. PBCP and its orthologs belong to a phy-logenetic clade that extends from monocots and dicots to thelycopod Selaginella moellendorffii, the moss Physcomitrellapatens, and the green algae C. reinhardtii and Volvox carteri (seeSupplemental Figure 2, Supplemental References 1, andSupplemental Data Set 1 online). This phylogenetic analysis thusshows that PBCP has been conserved throughout the evolutionof plants (Viridiplantae). PBCP shares only ;16% sequenceidentity with PPH1/TAP38 and is more closely related to otherPP2C phosphatases, such as At4G33500, which is part ofa neighboring phylogenetic clade. However, insertion mutants ofthe latter did not show any detectable phenotype in a de-phosphorylation assay with far-red light similar to those pre-sented in Figure 1.

A polyclonal antiserum raised against PBCP recognizesa polypeptide of ;32 kD in the wild type (Figures 1C and 1D;see Supplemental Figure 1C online). This is approximately thepredicted molecular mass of the mature PBCP protein afterremoval of the putative transit peptide. The protein is not de-tectable in pbcp-1 and pbcp-2 (see Supplemental Figure 1Conline), indicating that both are strong or null alleles andconfirming that the 32-kD band recognized by the antiserum isindeed the PBCP protein. A slightly retarded migration ofPBCP, which can be ascribed to the presence of the triple-HAtag, is observed in the rescued mutant and in the pbcpOE line(Figures 1C and 1D).

PBCP Is a Chloroplast Protein

The deduced amino acid sequence of PBCP carries a predictedN-terminal transit peptide for chloroplast targeting. Indeed,D. Leister and coworkers (Schliebner et al., 2008) observed thatthe fluorescent protein dsRed fused to the transit peptide of PBCP(At2g30170) clearly localizes to the chloroplasts in transfectedArabidopsis protoplasts. This protein was also identified in puri-fied chloroplast fractions by MS (Zybailov et al., 2008). Toconfirm the localization of PBCP, we prepared chloroplasts fromrosette leaves and further fractionated them by centrifugationinto a stromal supernatant and a membrane pellet. These frac-tions were analyzed by immunoblotting using antiserum againstPBCP, and the quality of the fractionation was assessed withantisera against the thylakoid protein D1, the stromal enzymephosphoribulokinase (PRK), and the cytoskeletal protein actin(Figure 2). The PBCP protein was largely found in the chloroplaststromal fraction, and its distribution closely paralleled the stro-mal marker PRK. A minor amount of PBCP was also observed inthe membrane fraction.

PBCP Dephosphorylates Subunits of PSII in Vitro

As described above, PBCP belongs to the family of PP2Cphosphatases, and thylakoid protein dephosphorylation is af-fected in the pbcp mutants in planta. To confirm that PBCPindeed has phosphatase activity and to gain insight regardingwhether its action on the core subunits of PSII is direct, wepurified recombinant PBCP (rPBCP) and assayed its activity invitro. The mature form of PBCP, without the predicted transitpeptide but with a C-terminal poly-His tag, was expressed inEscherichia coli. The enzyme was purified under denaturingconditions by Ni-affinity chromatography, allowed to refold bydialysis into nondenaturing conditions, and then further purifiedby size exclusion chromatography (Figure 3A). A His-taggedbacterial maltose binding protein (MBP) was expressed andpurified in parallel as a negative control. The phosphatase

Figure 2. PBCP Is a Chloroplast Protein.

Chloroplasts from wild-type plants were isolated and further fractionatedinto a membrane pellet and a stromal supernatant. Equal amounts ofprotein from each fraction (10 µg) were analyzed by immunoblotting withantibodies against PBCP, the stromal protein PRK, the thylakoid mem-brane protein D1 of PSII, and the cytoskeletal protein actin.

2600 The Plant Cell

activity of rPBCP was first demonstrated with the chromogenicsubstrate, para-nitrophenyl phosphate (pNPP) (Figure 3B). Con-sistent with some similar phosphatases of the PP2C class, theactivity of rPBCP was much higher with Mn2+ than with Mg2+ asa cofactor in the assay (Pullen et al., 2004; Wehenkel et al.,2007), and the activity was strongly inhibited by the addition ofEDTA as a chelator. Surprisingly, the activity was almost un-affected by the addition of 10 mM NaF, a general inhibitor of

thylakoid protein phosphatases (Bennett, 1980; Hammer et al.,1995; Vener et al., 1999). Addition of the reducing agent DTTstimulated the activity, raising the possibility that the phospha-tase could be regulated by the redox state of its Cys residues.To test the activity of rPBCP on its natural substrates, thylakoidmembranes were purified from the pbcp-1 mutant and exposedto white light for 15 min in the presence of ATP to maximizethylakoid protein phosphorylation. The membranes were thenincubated in the dark in the presence of purified rPBCP, usingas controls the buffer or the purified MBP protein. De-phosphorylation was monitored by immunoblotting with anti-phosphothreonine antibodies. After 5 min of incubation, thedephosphorylation activity of rPBCP in the presence of Mn2+

was apparent as a decrease in the phosphorylation of CP43, D1,and D2 (Figure 3C, lane 5 versus lane 1). Phosphorylation de-creased by 40 to 50% for D1 and CP43, and ;70% for D2. Theactivity was strongly inhibited by the addition of EDTA (lane 7). Itwas much lower in the presence of Mg2+ and was not signifi-cantly inhibited by NaF (see Supplemental Figure 3 online). Theaddition of DTT did not have a detectable effect on the activity(Figure 3C, lane 6), but the sensitivity of the assay may be in-sufficient to detect the effect observed with pNPP as a sub-strate. Some dephosphorylation of the LHCII proteins wasapparent in this in vitro assay, similar to what was observed invivo for the pbcpOE overexpressor.

Role of PBCP in Thylakoid Membrane Organization

In the stn8 mutant and the stn7 stn8 double mutant, which aredeficient in thylakoid protein phosphorylation, the membranestend to form extended grana stacks (Fristedt et al., 2009a),suggesting that thylakoid protein phosphorylation affectsmembrane folding and grana size. We examined the ultra-structure of the thylakoid membranes in leaf chloroplasts of thepbcp mutants, the pbcpOE overexpressor, and the wild typeby transmission electron microscopy. We compared 11-d-oldseedlings treated with blue light for 3 h to favor thylakoid proteinphosphorylation and further treated for 1.5 h with far-red light topromote dephosphorylation (Figure 4). To avoid any differencesdue to cell type–specific differentiation of the plastids, we al-ways selected chloroplasts in the subepidermal cell layer on theadaxial side, the presumptive palisade mesophyll cells of thesedeveloping leaves. There was a striking difference between thewild type and the pbcp mutants, which showed many regionswhere only a few membranes were appressed and, thus,a concomitant reduction in the number of layers in the granastacks (Figures 4A to 4D). Thylakoid membrane folding in thepbcpOE was similar to the wild type (Figures 4E and 4F). Torepresent these differences quantitatively, we measured thetotal length of thylakoid membranes that were found in singlestromal lamellae or in grana stacks with different number oflayers. The resulting histogram (Figure 4G) shows that in the wildtype the broad distribution extended to stacks with 10 or morelayers, while in the mutants the distribution was clearly dis-placed toward fewer layers and showed only very few granastacks with eight layers or more. Thus, the activities of the PBCPphosphatase and of the STN8 kinase seem to affect the foldingof the thylakoid membrane in opposing ways. The distribution in

Figure 3. Recombinant PBCP Has Phosphatase Activity in Vitro.

(A) SDS-PAGE of purified rPBCP loaded under reducing and denaturingconditions. The sizes of the markers in lane M are indicated on the left(kD).(B) In vitro spectrophotometric assay of PBCP activity. The de-phosphorylation of pNPP was measured by the increase in absorption at405 nm as a function of time in 1-mL reaction volumes containing 5 µgrecombinant PBCP, 300 mM NaCl, 50 mM HEPES, pH 8.0, 10 mMpNPP, and the additives indicated. Error bars represent the SD of tripli-cate measurements.(C) Thylakoid membranes (containing 0.16 µg chlorophyll) isolated fromthe pbcp-1mutant, exposed to white light for 15 min, and then incubatedfor 5 min in the dark in the presence of purified recombinant PBCP (5 µg),or with protein buffer or recombinant MBP as controls, in a final reactionvolume of 100 mL containing 5 mM MnCl2. The effects of DTT (10 mM)and EDTA (5 mM) were tested as indicated. The samples were analyzedby immunoblotting with antiphosphothreonine antibodies from Zymed.

Photosystem II Core Phosphatase 2601

Figure 4. Thylakoid Folding Is Altered in pbcp Mutants.

Electron micrographs of thin sections from leaves of 11-d-old seedlings exposed to blue light for 3 h and then shifted to far-red light for 1.5 h to inducedephosphorylation of the PSII core subunits. (B), (D), and (F) show larger magnifications of (A), (C), and (E), respectively. WT, the wild type. Bars = 500nm.(A) and (B) The wild type.(C) and (D) pbcp-2.(E) and (F) pbcpOE.

2602 The Plant Cell

the pbcpOE was similar to the wild type, as might be expectedafter prolonged far-red treatment when thylakoid protein phos-phorylation in the wild type and the overexpressor reachescomparable low levels (Figure 1).

State Transitions in the pbcp Mutants andpbcpOE Overexpressor

In the different pbcp-1 transgenic lines transformed with the35S:PBCP:HA construct, PBCP was expressed at differentlevels (Figures 1C and 1D). It was thus possible to comparedifferent lines forming an allelic series with increasing amountsof PBCP, from the pbcp mutants devoid of PBCP, through thewild type or the rescued line with slightly elevated levels ofPBCP, to the pbcpOE overexpressor with strongly increasedaccumulation of the phosphatase. We observed a consistenttrend in the levels of PSII phosphorylation and the kinetics ofdephosphorylation. In the pbcp-1 mutant exposed to white orblue light, phosphorylation of the thylakoid proteins wassomewhat elevated compared with the wild type (Figure 1, Ta-bles 1 and 2), and dephosphorylation upon transfer to far-redlight was strongly retarded. The rescued line showed normalphosphorylation patterns in white light and restored de-phosphorylation upon transfer to far-red light (Figure 1C). InpbcpOE, we observed slightly reduced phosphorylation in whitelight and rapid dephosphorylation upon transfer to far-red light(Figure 1D). This is consistent with the presumed antagonisticeffects of the STN8 kinase and the PBCP phosphatase in whiteor blue light, where the kinase is active, and the role of PBCP indephosphorylation in far-red light, where the kinase is thought tobe downregulated. However, these observations are also com-patible with the possibility that the activity of PBCP is regulated.

State transitions, which involve phosphorylation of the LHCIIantennae and their dynamic allocation to PSII or PSI, are regu-lated by the STN7 kinase and the PPH1/TAP38 phosphatase.Blue or white light induces a transition to state 2 through theactivation of the STN7 kinase and the association of phos-phorylated LHCII with PSI. This is reversed in far-red light, whichinduces dephosphorylation of the LHCII antennae under theaction of the PPH1/TAP38 phosphatase and allocation of LHCIIto PSII. While the STN8 kinase has substrate specificity distinctfrom STN7, the activities of the two kinases may partly overlap.This is suggested by the residual phosphorylation of LHCII andPSII subunits, which is observed in the single mutants of thekinases, and is more extensively abolished in the double mutantstn7 stn8 (Bonardi et al., 2005; Tikkanen et al., 2008). It wastherefore of interest to investigate whether the PBCP phos-phatase mutants were affected in state transitions, which weremeasured by monitoring chlorophyll fluorescence in two sets ofexperiments, either at room temperature or at 77K.

At room temperature, chlorophyll fluorescence emanates al-most exclusively from PSII and its light-harvesting antennae.Under white light, which favors state 2, part of the LHCII antennais dissociated from PSII so the maximal fluorescence Fm2 in-duced by a saturating flash is lower than the maximal fluores-cence Fm1 in far-red light, which induces state 1 and theassociation of LHCII with PSII (Figure 5A). The state transitionparameter, qT = (Fm12 Fm2)3 100/Fm1 was approximately +3in the wild-type seedlings but decreased to a negative value of22 in the stn7 mutant and was also strongly affected in thepph1-3 mutant, both of which were used as controls that areknown to affect LHCII phosphorylation and state transitions(Figure 5B). qT was comparable to the wild type in the stn8mutant and in the rescued line, as well as in the pbcp-1 andpbcp-2 mutants. Thus, state transitions are not significantly af-fected in the absence of PBCP. Interestingly, qT was stronglyreduced in the pbcpOE overexpressor line, an indication thatstate transitions may be affected by the increased activity ofPBCP. This was also apparent when the kinetics of state tran-sitions were monitored by following the fluorescence parameterFs, which is influenced by the redox state of the PQ pool. Whenthe far-red light is turned off, Fs increases abruptly, reflecting theoverreduction of the PQ pool, and then Fs gradually decreasesas transition to state 2 allows reoxidation of the pool (Figure 5C).Compared with the wild type, this decrease was blocked in thestn7 mutant, was strongly retarded in the pph1-3 mutant, andwas not affected in the stn8, pbcp-1, and pbcp-2 lines. The ki-netics of the Fs decrease in the transition from state 1 to state 2were strikingly slower in the pbcpOE overexpressor, suggestingthat state transitions are affected by the increased activity of thephosphatase. A small effect was also apparent in the rescuedline, which has slightly elevated levels of PBCP (Figure 1C).State transitions can also be monitored by measuring the

fluorescence emission spectra of chlorophyll at 77K. Emission at685 nm can be ascribed to PSII and emission at 732 nm to PSI.As the LHCII antenna is reallocated by state transitions, changesin the cross section of the two photosystems are reflected bychanges in the relative size of the respective fluorescenceemission peaks (see Supplemental Figure 4 online). Whenseedlings were transferred from far-red light to blue light, theamplitude of the transition to state 2 was reduced in the pbcpOEline compared with the wild type, but not as strongly as in thestn7 mutant. Conversely, when the seedlings were returned tofar-red light, the transition to state 1 was more pronounced inthe pbcpOE line than in the wild type. Consistent with the qT andFs measurements at room temperature, this indicates thatoverexpression of PBCP affects state transition. These resultscan be correlated with the observation that the LHCII proteinsare less phosphorylated in pbcpOE compared with the wild type(Figure 1D).

Figure 4. (continued).

(G) The length of thylakoid membranes in stromal lamella and in grana stacks with different numbers of layers were measured in electron micrographs ofthe wild type (n = 30), the pbcp-1mutant (n = 22), the pbcp-2mutant (n = 12), and the pbcpOE overexpressor (n = 9). The histograms show the length ofmembranes in domains with the indicated numbers of layers (one layer for stromal lamellae and two or more layers in grana stacks) as a fraction of thetotal length of membranes measured.

Photosystem II Core Phosphatase 2603

DISCUSSION

While PPH1/TAP38 was shown to be necessary for de-phosphorylation of LHCII subunits (LHCB 1 and LHCB 2) (Pribilet al., 2010; Shapiguzov et al., 2010), here, we demonstrate thatPBCP is required for efficient dephosphorylation of the coresubunits CP43, D1, D2, and PsbH of PSII. The substrate spe-cificities of the two phosphatases are similar to those of the twokinases, STN7 and STN8 (Bellafiore et al., 2005; Bonardi et al.,2005; Vainonen et al., 2005; Fristedt and Vener, 2011; Reilandet al., 2011). The PBCP phosphatase and the STN8 kinase thusseem to form an antagonistic pair, whose reciprocal activities onphosphorylation of PSII core subunits could allow a balancedacclimatory response. This is similar to the pair formed by theSTN7 kinase and the PPH1/TAP38 phosphatase, which haveopposing effects on phosphorylation of the LHCII antenna toregulate state transitions and maintain a balanced redox state ofthe electron transfer chain.Although the two phosphatases appear to have distinct spe-

cificities, overexpression of PBCP perturbed state transitionsand the phosphorylation of LHCII, which are the major rolesascribed to PPH1/TAP38. The converse effects were not obvi-ous in the pbcp mutants, which did not show elevated levels ofLHCII phosphorylation in white or blue light (Figure 1, Table 1) orchanges in the kinetics of state transitions (Figure 5; seeSupplemental Figure 4 online). Thus caution should be used ininterpreting the phenotype of the pbcpOE line, which is a dom-inant gain-of-function mutant, since overexpression of PBCPmight cause a loss of its normal specificity. Nevertheless, it isinteresting to consider three more possible interpretations,which are not entirely mutually exclusive, of these observations.One is that PBCP may have some overlapping activity towardLHCII, which only becomes apparent upon overexpression, andthat its specificity may not be entirely restricted to the PSII core.A similar situation seems to prevail for the two kinases, whosesubstrate specificity appears to overlap since the residualphosphorylation that is apparent in the single kinase mutants,stn7 or stn8, is abolished in the double mutant stn7 stn8(Tikkanen et al., 2008; Fristedt et al., 2009a). An alternativeinterpretation is that PBCP may have functional interactions withother members of a regulatory network, for example, PPH1 orSTN7, and influence their activity directly or indirectly. A differentinterpretation could be that phosphorylation of the core subunitsinfluences the mobility of proteins within the thylakoid mem-brane, which in turn may influence the rate of state transitions.Because of steric hindrance, PSI and ATP synthase are ex-cluded from the PSII-enriched appressed membranes of granastacks. This segregation is thought to minimize spillover of en-ergy between the photosystems (Dekker and Boekema, 2005).Changes in protein phosphorylation may affect the function ofthe photosynthetic electron transfer chain by changing the dis-tribution and interactions of the photosynthetic complexes andin particular may influence state transitions by limiting thetransfer of LHCII between PSI and PSII. Phosphorylation by the

Figure 5. State Transitions in the pbcp Mutants and pbcpOE Over-expressor.

(A) The kinetics of state transitions were analyzed by continuouslymonitoring chlorophyll fluorescence with a FluorCam (PhotoSystemsInstruments). After a preincubation of 30 min in darkness, actinic light(represented below as a white bar) was turned on for 17 min to inducestate 2 before maximal fluorescence (Fm2) was measured with a satu-rating flash (arrow). Far-red light was then turned on for 17 min (repre-sented below as a red bar) to induce state 1, and maximal fluorescence(Fm1) was again measured with a saturating flash (arrow) before the far-red light was turned off to return to state 2. The curves (normalized to Fo)are the average of 26 seedlings of each genotype from two experiments.Line breaks indicate that the Fm peaks are out of scale. WT, the wildtype.(B) The state transition parameter qT was calculated from the experi-ments in (A) (qT= (Fm12 Fm2)3 100/Fm1). The bars represent the meanand SD of 26 seedlings of each genotype from two experiments. Theasterisks indicate P < 0.001 in Student’s t test.

(C) Chlorophyll fluorescence was normalized for the value of Fs imme-diately after turning the far-red light off to allow comparisons of the ki-netics of the transition to state 2.

2604 The Plant Cell

STN7 and STN8 kinases was shown to influence the mobility ofthylakoid proteins within the membrane (Goral et al., 2010).

Phosphorylation of PsbH reveals another aspect of the antago-nism between STN8 and PBCP. There are two phosphorylationsites at the N terminus of PsbH, at Thr-2 and Thr-4. We find thatin the pbcpmutant, it is Thr-2 that retains phosphorylation in far-redlight, whereas Thr-4 is dephosphorylated as in the wild type.However, MS analysis of phosphorylation in the stn8 mutant hasshown that the STN8 kinase is responsible for phosphorylationat Thr-4 and that this requires prior phosphorylation at Thr-2(Vainonen et al., 2005; Fristedt and Vener, 2011). Thus, in the caseof PsbH, antagonism of PBCP and STN8 might involve the de-phosphorylation of Thr-2 by PBCP, which in turn would preventphosphorylation by STN8 at Thr-4. Little is known on the physio-logical role of phosphorylation at the individual Thr residues inPsbH or in D2 and CP43. In C. reinhardtii, a mutation of PsbH thatprevents phosphorylation of Thr-2 by replacing it with Ala had noapparent phenotypic consequence (O’Connor et al., 1998). Muta-tion of the D2 subunit changing Thr-2 to Ala allowed normal pho-tosynthetic activity, but mutations to Asp or Glu, which mimicphosphorylation, strongly affected PSII accumulation (Fleischmannand Rochaix, 1999).

What then may be the function of STN8 and PBCP in light ac-climation? In the stn8 mutant, phosphorylation of PSII is reducedand the size of the thylakoid membrane grana increases. Con-versely, we observed in the pbcp mutants that phosphorylation ofPSII is retained upon exposure to far-red light and that the pro-portion of large grana stacks decreases. Furthermore, STN8 mayfacilitate the repair cycle of PSII by promoting unfolding of themembranes and allowing easier access of membrane proteasessuch as FtsH and cotranslational integration of D1 (Tikkanen et al.,2008; Fristedt et al., 2009a). However, our attempts to determinewhether photoinhibition and D1 turnover are affected in the pbcpmutants did not give consistent results.

The rPBCP purified from E. coli showed phosphatase activity invitro using the artificial substrate pNPP. Since rPBCP promoteddephosphorylation of the core subunits of PSII in isolated thyla-koids, it seems likely that these are its direct substrates. However,we cannot entirely exclude the possibility that PBCP is only in-directly required for dephosphorylation of PSII. As with some otherPP2C phosphatases, PBCP is Mn2+ dependent and has negligibleactivity with Mg2+ as the metal cofactor (Pullen et al., 2004;Wehenkel et al., 2007). The activity of the phosphatase is stronglyinhibited by the addition of EDTA. Surprisingly NaF, which isa general inhibitor of thylakoid phosphatases (Bennett, 1980; Veneret al., 1999), has only a relatively minor effect on PBCP activity invitro, both when assayed with pNPP or with thylakoid membranes.This observation is puzzling because in pumpkin (Cucurbita max-ima) leaves NaF could prevent dephosphorylation of the PSII coresubunits and retard degradation of D1 (Rintamäki et al., 1996). It willthus be interesting to determine whether in Arabidopsis the PBCPphosphatase is directly involved in the repair cycle of D1 or whetheranother phosphatase is involved.

The PP2C family of protein phosphatases comprises ;80members in Arabidopsis or rice (Oryza sativa; Xue et al., 2008). Thenumber is much larger than in animals, and this expansion in plantssuggests that PP2C phosphatases have acquired distinct substratespecificities or novel functions (Kerk et al., 2008). Indeed, our data

indicate that for the dephosphorylation of thylakoid membraneproteins, PPH1/TAP38 and PBCP have different substrate prefer-ences. The presence of different phosphatases is one possibleexplanation for the various kinetic classes that were discerned inassays of endogenous phosphatase activity with isolated thylakoidmembranes from pea (Pisum sativum) chloroplasts (Silverstein et al.,1993). The strong preference of PBCP for Mn2+ and its insensitivityto NaF distinguish it from both the stromal and the membranephosphatases active on LHCII phosphopeptides, which were pre-viously purified from pea chloroplasts (Hammer et al., 1995, 1997). Itis expected that further phosphatases involved in regulating thyla-koid membrane structure and function remain to be identified.

METHODS

Plant Material

Arabidopsis thaliana mutant lines in Columbia-0 background wereobtained from the European Arabidopsis Stock Center (pbcp-1 isSALK_127920.31.10.N, and pbcp-2 is WiscDsLox_359F02). They weregenotyped as described in Supplemental Figure 1 online. The stn7(Bellafiore et al., 2005), stn8 (Vainonen et al., 2005; Fristedt et al., 2009a),and pph1-3 mutants (Shapiguzov et al., 2010) were described.

To rescue the pbcp-1 mutant with the wild-type cDNA, the completecoding sequence of PBCP (At2g30170), excluding the stop codon,was amplified by PCR from a cDNA clone (pda00938) obtained from theRiken Institute (Japan). The forward primer (59-ATCTCGAGCGAGA-GAGCAAGCCATTGCAG-39) and reverse primer (59-GTCGACTGAGC-TAACAACTTTGGCAACG-39) included XhoI and SalI sites, respectively.The PCR product was inserted in the XhoI and SalI sites of pCF399 underthe control of the cauliflower mosaic virus 35S promoter and the NOSterminator with a triple HA epitope tag at the C terminus (Lorrain et al.,2008). Plants of Arabidopsis were transformed by floral dipping usingAgrobacterium tumefaciens GV3101 strain. T1, T2, and T3 progenyseedlings were selected onMurashige and Skoogmedium supplementedwith 1.5% Suc and 10 µg mL21 Basta (Pentanal).

Dephosphorylation in Vivo

For the genetic screen, seedlings were grown for 10 d on Murashige andSkoogagar in a growth chamber (Percival CU36L5) in longdays (16 h light and8 h dark) at 22°C under fluorescent white light (50 to 60 µE m22 s21). De-phosphorylation was monitored as described (Shapiguzov et al., 2010) byplacing seedlings under far-red light-emitting diodes (LEDs; L735; Epitex) forthedesignated time. Seedlingswere snap-frozen in liquidnitrogenandgroundtwice for 10 s with glass beads in a triturator (Silamat S5; Ivoclar Vivadent).Protein extracts were prepared by incubating in lysis buffer containing100 mM Tris-HCl, pH 7.7, 2% SDS, 50 mM NaF, and 23 Protease InhibitorMixture (Sigma-Aldrich) for 30 min at 37°C. The samples were separated byTris-Gly SDS-PAGE in 12% acrylamide gels supplemented with 6 M urea,which allowed of better separation of the D1 and D2 phosphoproteins. Im-munoblotting was performed using antiphosphothreonine antibodies (Zymedor Cell Signaling) or antiactin as a control (Sigma-Aldrich).

Analyses of in Vivo Protein Phosphorylation by MS

Thylakoids for MS analyses were isolated from 15-d-old seedlings grownon Murashige and Skoog agar plates. The plants were dark-adapted for1 h and then placed under far-red or blue LEDs for 30 min to induce state1 or state 2, respectively. Thylakoid isolation was performed in the dark(under green light) in the cold room as described (Fristedt et al., 2009a).For trypsin treatment thylakoids were resuspended in 25 mM NH4HCO3

Photosystem II Core Phosphatase 2605

and 10 mM NaF at a concentration of 2 mg mL21 of chlorophyll andincubated with sequencing-grade modified trypsin (Promega) for 3 h atroom temperature. Peptide extracts were cleared by ultracentrifugation,aliquoted, and dried in a SpeedVac.

For relative quantitative MS studies, the collected peptides fromtrypsin-treated wild-type and pbcp-1 mutant thylakoids were subjectedto esterification by either d0-methyl methanol or d3-methyl methanol(Sigma-Aldrich) prior to enrichment of phosphopeptides by IMAC. Bothesterification of peptides and the subsequent phosphopeptide enrich-ment by IMAC were done as described before (Shapiguzov et al., 2010).The esterified samples were mixed 1:1, subjected to IMAC, and analyzedby nano-liquid chromatography electrospray ionization MS, which al-lowed simultaneous measurements of intensities for light and heavyisotope–labeled phosphopeptide pairs and quantitative comparison ofthe phosphorylation differences in the mutant and wild-type proteins. Thereverse labeling of peptides from the wild type and pbcp-1was performedas an internal control. For analysis of the peptides, an online nano-flowHPLC system (EASY-nLC; Bruker Daltonics) was used and MS analysiswith the HCTultra PTM discovery system (Bruker Daltonics) was per-formed using the same settings as before (Shapiguzov et al., 2010). Theautomated, online, tandem MS analyses were made using alternatingcollision-induced dissociation/electron transfer dissociation fragmenta-tion of peptide ions.

Label-free characterization of the phosphorylation status for the PSIIcore proteins was done by LC-MS analyses of the peptides obtained fromthe proteolytic treatment of wild-type and pbcp-1 thylakoids withoutfurther enrichment, as described (Fristedt et al., 2010). LC-MS separationand detection of the phosphorylated peptides and their nonphosphor-ylated counterparts was used for determination of the phosphorylationstoichiometry for PSII proteins. The level of phosphorylation for the PSIIcore proteins was calculated from the ratios of phosphorylated to non-phosphorylated peptide intensities in each LC-MS chromatogram usingnormalization for the difference in the signal intensities of the phos-phorylated and dephosphorylated peptide ions from the D1, D2, andPbsH proteins as determined (Fristedt et al., 2010).

Purification of Recombinant PBCP

The cDNA sequence of the predicted mature PBCP protein was clonedinto pET28a (Invitrogen) to include a C-terminal 6x-His tag and expressedin Escherichia coli strain BL21. Following cell lysis and centrifugation at7600g, the pellet containing recombinant PBCP was solubilized in 6 Mguanidine with phosphate buffer at pH 8.0. Cell debris was pelleted at23,000g, and the denatured proteins were loaded onto Ni-nitrilotriaceticacid (Ni-NTA) resin (Qiagen). The Ni-NTA was washed with 4 M ureacontaining 35 mM imidazole, and rPBCP was eluted with 2 M ureacontaining 300 mM imidazole and 200 mM NaCl. EDTA (10 mM) and20 mM 2-mercaptoethanol were added to the eluate. Denatured PBCPwas refolded by dialyzing the eluate with 300 mM NaCl and 50 mMHEPES, pH 8.0. Following dialysis, the rPBCP eluate was centrifuged at23,000g and further purified by size exclusion chromatography ona preparative grade Superdex-200 column (GE Healthcare) with a mobilephase of 300 mM NaCl and 50 mM HEPES, pH 8.0. Recombinant PBCPeluted at the expected globular particle size of ;30 kD and was con-centrated to stocks of 1 mg mL21. As a source of antigen to raise a rabbitanti-PBCP serum, the recombinant protein was purified by Ni-NTAchromatography as recommended by the manufacturer (Qiagen) followedby preparative SDS-PAGE.

Activity Assays of Recombinant PBCP

Recombinant PBCP phosphatase activity was assayed in vitro bymonitoring the dephosphorylation of pNPP. The reaction rate was

calculated by the increase in absorbance at 405 nm as a function of timeacross the linear portion of the curve. The reaction mixture contained300 mM NaCl, 50 mM HEPES, pH 8.0, 5 mM MnCl2 or MgCl2, 10 mMpNPP, and 5 µg mL21 PBCP (;170 nM). Stocks of pNPP, NaF, DTT, andEDTA were prepared in aqueous solution containing 300 mM NaCl, 50mM HEPES, pH 8.0, and 5 mM MnCl2 or MgCl2.

Plants for thylakoid extraction were cultivated for 3 weeks on soil ingrowth chambers at 24°C under white light (100 µE) in short days (8 h light,16 h dark). Thylakoids were prepared as described (Fristedt et al., 2009a)without addition of NaF, washed in a buffer containing 25 mM Tricine-NaOH, pH 7.8, 100 mM sorbitol, 10 mM KCl, and either 5 mM MgCl2 or5 mM MnCl2. Thylakoids (containing 0.17 mg mL21 chlorophyll) weresupplemented with ATP to a concentration of 0.4 mM and incubated inwhite light (100 mE m22$s21) for 15 min. Recombinant PBCP, MBP, orbuffer was added to the thylakoids and, where indicated, supplementedwith 10 mM DTT or 50 mM NaF and incubated in the dark for the des-ignated times before the reaction was stopped by the addition of the 43gel-loading lysis buffer (10% SDS, 40% Suc, 1 mM EDTA, 50 mM Tris-HCl, pH 6.8, and 20% b-mercaptoethanol). Thylakoid protein phos-phorylation was analyzed by immunoblotting with antiphosphothreonineantibodies and quantified by comparison to a dilution series of an un-treated thylakoid sample.

Isolation of Intact Chloroplasts and Fractionation

Intact chloroplasts were isolated from mature plants grown on soil in longdays (12 h light, 12 h dark). The procedure was performed on ice. Theleaves were ground in 20 mM HEPES-KOH, pH 8.0, 5 mM EDTA, and330 mM sorbitol with a Polytron mixer (Kinematica). The homogenate wasfiltered through two layers of Miracloth (Calbiochem) and centrifuged at1000g at 4°C for 5 min. The chloroplast pellet was resuspended in 0.5 mLof the same buffer, and intact chloroplasts were isolated on a Percollgradient as described (Aronsson and Jarvis, 2002). Washed intactchloroplasts were lysed by hypo-osmotic shock in 10 mM Tricine-NaOH,pH 7.8, 5 mM MgCl2, and 10 mM NaF. The insoluble fraction containingthylakoid membranes was pelleted by centrifugation (21,000g for 10 min).The soluble fraction enriched in chloroplast stromal proteins wascleared by an additional centrifugation step (21,000g for 30 min).The samples were treated with 10% trichloroacetic acid to precipitateprotein.

Transmission Electron Microscopy

Seedlings were grown onMurashige and Skoog agar plates (16 h light, 8 hdark, 50 to 60 µEm22 s21) for 11 d. Four hours after the onset of light, theywere exposed for 3 h to blue LEDs and then for 1.5 h to far-red LEDs (L470and L735; Epitex). Cuttings from the first pair of leaves, taken in themiddle of the lamina, were immediately immersed in 2.5% glutaraldehydein 100mMcacodylate sodium buffer, pH 7.0, containing 0.01%Tween 20.The leaf pieces were fixed overnight at 4°C under agitation. They werethen rinsed sequentially (30 min/step) in cacodylate sodium buffer con-taining (1) 0.1% Tween, (2) 0.05% Tween, and (3) three times in caco-dylate alone. The samples were postfixed in 1.5% OsO4 for 2 h, washedthree times in cacodylate buffer, then once in milliQ water, and furtherfixed in 1%aqueous uranyl acetate. Sample preparationwas performed at4°C. After washing in distilled water, the tissue was dehydrated ina graded ethanol series and embedded in Epon 812. Ultrathin crosssections (85 nm thick) were cut and stained with 2.5% uranyl acetate andthen Reynolds lead citrate. The sections were viewed in a FEI TecnaiG2 Sphera transmission electron microscope at 120 kV. Care wastaken to always select plastids located in adaxial subepidermal cells(presumptive palisade parenchyma cells). The lengths of membranesfrom electron micrographs were measured using ImageJ software (http://

2606 The Plant Cell

rsbweb.nih.gov/ij/). Data presented in Figure 4G are from 30 images of thewild type and 22 of pbcp-1 (from five independent experiments), 12 ofpbcp-2 (two experiments), and nine of pbcpOE (one experiment).

Chlorophyll Fluorescence Analysis

Room temperature chlorophyll fluorescence was monitored with a Fluo-rCam 800MF kinetic imaging fluorimeter (PhotoSystems Instruments)essentially as described (Willig et al., 2011). Seedlings grown on Mura-shige and Skoog agar for 15 dwere dark-adapted for 30min. White actiniclight (8%) was then switched on for 17 min to induce state 2. Far-red light(100%) was then turned on against the background of continuing whitelight for 17 min to stimulate transition from state 2 to state 1. Finally, far-red light was turned off to initiate the reverse transition. Measuring flashescontinued throughout the protocol to record Fs. Saturating light flasheswere applied at the end of the dark, at the end of the 17-min white, and atthe end of the 17-min white plus far-red light periods tomeasure Fm, Fm2,and Fm1, respectively. The fluorescence signal was averaged for 26seedlings. Chlorophyll fluorescence at 77K was measured on seedlingsessentially as described (Shapiguzov et al., 2010). Seedlings were frozenin liquid nitrogen, ground, and resuspended in ice-cold buffer containing50 mM HEPES-KOH, pH 7.5, 100 mM sorbitol, 10 mMMgCl2, and 10 mMNaF. The samples were filtered and frozen in liquid nitrogen, and chlo-rophyll fluorescence at 77K was recorded on a Jasco FP-750 spectro-fluorometer. Excitation was at 480 nm (slit width 5 nm) and emission wasrecorded in the 600- to 800-nm range (slit width of 5 nm). Curves werenormalized for the PSII peak at 685 nm.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under accession numbersAT2G30170.1 and AY063046.1.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Two Mutant Alleles of PBCP.

Supplemental Figure 2. Phylogenetic Analysis of PBCP Homologs.

Supplemental Figure 3. PBCP Activity in Vitro in the Presence ofMg2+ or NaF.

Supplemental Figure 4. Analysis of State Transitions by ChlorophyllFluorescence at 77K.

Supplemental Data Set 1. Text File of Alignment Used for Phyloge-netic Analysis in Supplemental Figure 2.

Supplemental References 1. Supplemental References for SupplementalFigure 2.

ACKNOWLEDGMENTS

We thank Sam Zeeman for advice on devising the genetic screen, ElisabetGas for help with chloroplast isolation, Natalia Cabanas Fiscowich for con-tributing to the chlorophyll fluorescence measurements, Christian Fankhauserfor advice and a gift of pCF399, and Nicolas Roggli for preparing the figures.This work was supported by SystemsX.ch (RTD Plant Growth in a ChangingEnvironment), grants from the Swiss National Foundation (3100AO-117712and 31003A_133089/1), the FP7 Marie-Curie Initial Training Network (ITN)COSI (ITN 2008 GA 215-174), an EMBO postdoctoral fellowship (to G.F.), anda grant from the Swedish Research Council (2008-5490).

AUTHOR CONTRIBUTIONS

M.G.-C., J.-D.R., A.V.V., I.S., A.S, G.F., B.I., and M.C. designed theresearch. I.S., A.S., G.F., B.I., and M.C. performed research. M.G.-C.,J.-D.R., A.V.V., I.S., A.S., G.F., B.I., and M.C. analyzed data. M.G.-C. andJ.-D.R. wrote the article.

Received January 10, 2012; revised May 11, 2012; accepted May 25,2012; published June 15, 2012.

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Photosystem II Core Phosphatase 2609

DOI 10.1105/tpc.112.095703; originally published online June 15, 2012; 2012;24;2596-2609Plant Cell

Vener, Jean-David Rochaix and Michel Goldschmidt-ClermontIga Samol, Alexey Shapiguzov, Björn Ingelsson, Geoffrey Fucile, Michèle Crèvecoeur, Alexander V.

ArabidopsisIdentification of a Photosystem II Phosphatase Involved in Light Acclimation in

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