Evidence for a Role of Chloroplastic m-Type … for a Role of Chloroplastic m-Type Thioredoxins ......

Evidence for a Role of Chloroplastic m-Type Thioredoxinsin the Biogenesis of Photosystem II in Arabidopsis1[C][W][OPEN]

Peng Wang2, Jun Liu2, Bing Liu, Dongru Feng, Qingen Da, Peng Wang, Shengying Shu, Jianbin Su,Yang Zhang, Jinfa Wang, and Hong-Bin Wang*

State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of LifeSciences, Sun Yat-sen University, 510275 Guangzhou, People’s Republic of China

ORCID ID: 0000-0003-4957-0509 (H.-B.W.).

Chloroplastic m-type thioredoxins (TRX m) are essential redox regulators in the light regulation of photosynthetic metabolism.However, recent genetic studies have revealed novel functions for TRX m in meristem development, chloroplast morphology,cyclic electron flow, and tetrapyrrole synthesis. The focus of this study is on the putative role of TRX m1, TRX m2, and TRX m4in the biogenesis of the photosynthetic apparatus in Arabidopsis (Arabidopsis thaliana). To that end, we investigated the impact ofsingle, double, and triple TRX m deficiency on chloroplast development and the accumulation of thylakoid protein complexes.Intriguingly, only inactivation of three TRX m genes led to pale-green leaves and specifically reduced stability of thephotosystem II (PSII) complex, implying functional redundancy between three TRX m isoforms. In addition, plants silencedfor three TRX m genes displayed elevated levels of reactive oxygen species, which in turn interrupted the transcription ofphotosynthesis-related nuclear genes but not the expression of chloroplast-encoded PSII core proteins. To dissect the functionof TRX m in PSII biogenesis, we showed that TRX m1, TRX m2, and TRX m4 interact physically with minor PSII assemblyintermediates as well as with PSII core subunits D1, D2, and CP47. Furthermore, silencing three TRX m genes disrupted theredox status of intermolecular disulfide bonds in PSII core proteins, most notably resulting in elevated accumulation of oxidizedCP47 oligomers. Taken together, our results suggest an important role for TRX m1, TRX m2, and TRX m4 proteins in thebiogenesis of PSII, and they appear to assist the assembly of CP47 into PSII.

Thioredoxins (TRXs), the ubiquitous small (approxi-mately 12 kD) thiol:disulfide oxidoreductases, are es-sential redox regulatory elements in plant metabolism(Schürmann and Buchanan, 2008; Dietz and Pfannschmidt,2011). All TRXs have a redox-active site that containstwo conserved Cys residues in the peptide motif WC(G/P)C (Holmgren, 1989). In the reduced state, TRXsare able to reduce disulfide bridges in the target pro-teins, thereby modulating their functions and stability(Dietz and Pfannschmidt, 2011).

In contrast with other organisms, plants have a largenumber of TRXs. At least 20 TRX isoforms have beenidentified in Arabidopsis (Arabidopsis thaliana), and theseare targeted to various cellular compartments, including

the chloroplast, mitochondria, and cytosol (Meyer et al.,2005; Lemaire et al., 2007). Depending on their subcel-lular localization, TRXs are reduced by different electrondonor systems. Both cytosolic and mitochondrial TRXs arereduced by compartment-specific NADPH:thioredoxinreductases (NTRs), whereas chloroplastic TRXs arereduced by the ferredoxin:thioredoxin reductase withelectrons provided by photosynthetic electron transport(Schürmann and Jacquot, 2000). In addition, chloroplastshave a modified type of NTR, called NTRC, whichcontains a C-terminal TRX domain and acts as a bifunc-tional enzyme with NTR/TRX activity for the reductionof target proteins (Serrato et al., 2004).

The chloroplastic TRX system is quite complicatedand includes five major groups (2f, 4m, 1x, 2y, and 1z)in Arabidopsis (Collin et al., 2003, 2004; Schürmannand Buchanan, 2008; Arsova et al., 2010). The diversityof TRXs implies the existence of an intricate TRX-modulated redox regulatory network in the chloro-plast. Previously, in vitro studies using purified TRXproteins have shown that the f- and m-type TRXs arerequired for carbon metabolism mainly through theregulation of some Calvin cycle enzymes, while the x- andy-type TRXs seem to serve as hydrogen donors for anti-oxidant enzymes (Collin et al., 2003, 2004; Schürmannand Buchanan, 2008; Chibani et al., 2011). However,the physiological significance and functional specificityof these different TRX isoforms remain to be determined.Recently, the genetic characterization of TRXs in mutantplants has revealed their novel physiological functionsin vivo. The inactivation of TRX f1 leads to decreased

1 This work was supported by the National Natural Science Foun-dation of China (grant nos. 31171173 and 313771237), the GuangdongProvincial Natural Science Foundation of China (grant no.S2012010010533), and the Fundamental Research Funds for the Cen-tral Universities (grant no. 10lgpy34).

2 These authors contributed equally to the article.* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Hong-Bin Wang ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OPEN] Articles can be viewed online without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.113.228353

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light activation of ADP-Glc pyrophosphorylase and al-tered diurnal starch turnover (Thormählen et al., 2013).The trx m3 mutation hampers meristem development,leading to a seedling-lethal phenotype (Benitez-Alfonsoet al., 2009), and TRX y2 functions as an electron donorto Met sulfoxide reductases for protein repair (Laugieret al., 2013). Collectively, these data reveal the potentialfor functional diversity in chloroplastic TRXs.The primary reactions of photosynthesis are mediated

by three pigment-protein complexes, PSII, the cyto-chrome b6f (Cyt b6f) complex, and PSI, which are em-bedded in the thylakoid membranes of chloroplasts andconnected in series by small, mobile electron carriers likeplastoquinone and plastocyanin (Rascher and Nedbal,2006; Eberhard et al., 2008). A characteristic feature ofthese photosynthetic apparatuses is that they all consistof multiple nucleus- and chloroplast-encoded subunitsas well as numerous pigments, such as chlorophylls andxanthophylls. Hence, the biogenesis of the photosyn-thetic complexes depends upon a tight coordinationbetween protein and pigment synthesis as well as thespatially and temporally coordinated assembly of thedifferent subunits and the proper incorporation of vari-ous cofactors (Rochaix, 2011). Notably, mounting evi-dence suggests that TRXs play an essential role in thebiogenesis of the photosynthetic apparatus. Global pro-teomic analyses have revealed that some photosyntheticapparatus subunits, such as D1 and PsbO in PSII, cyto-chrome f and Rieske FeS protein in the Cyt b6f complex,and PsaA, PsaF, and PsaN in PSI, may be TRX partners(Motohashi and Hisabori, 2006; Ströher and Dietz, 2008;Montrichard et al., 2009; Lindahl et al., 2011). TRX z hasbeen shown to redox regulate chloroplastic gene ex-pression and development (Arsova et al., 2010). NTRCparticipates in the posttranslational regulation of mag-nesium protoporphyrin methyltransferase in tetrapyr-role synthesis (Richter et al., 2013). In addition, HIGHCHLOROPHYLL FLUORESCENCE164 (HCF164), a lu-menal TRX-like protein, has been shown to be involvedin the assembly of the Cyt b6f complex (Lennartz et al.,2001; Motohashi and Hisabori, 2006, 2010). In spite ofthis, our knowledge of the regulatory function of TRXsin the biogenesis of the photosynthetic apparatus hasbeen largely limited by the transient nature of interac-tions between TRXs and their target proteins or by theabsence of detectable phenotypes in single TRX mutantsthat are presumably due to functional redundancy withinTRX gene families.In this study, we aimed to further investigate the role

of chloroplastic TRXs in the biogenesis of the photo-synthetic complexes. Among the numerous chloroplas-tic TRXs, the m-type TRX proteins have been suggestedto be involved in leaf development, chloroplast mor-phology, cyclic electron flow, and tetrapyrrole synthesis(Ikegami et al., 2007; Chi et al., 2008; Benitez-Alfonsoet al., 2009; Luo et al., 2012; Courteille et al., 2013). Be-sides these, the TRX m1, TRX m2, and TRX m4 proteinshave been demonstrated to peripherally associate withthe stroma-exposed thylakoid membranes (Peltier et al.,2002; Friso et al., 2004). All of these findings encouraged

us to comprehensively investigate the impact of TRXm1, TRX m2, and TRX m4 deficiency on chloroplastdevelopment and the accumulation of the thylakoidprotein complexes. Based on the pale-green leaf pheno-type and the specifically impaired PSII complex inplants triply silenced for TRX m1, TRX m2, and TRX m4,we have established the redundant role of the TRX m1,TRX m2, and TRX m4 proteins in PSII accumulation.Furthermore, biochemical studies in vivo and in vitroindicate that the three TRX m proteins directly interactwith PSII and modulate the reduction of intermoleculardisulfide bonds in CP47.

RESULTS

Triple Inactivation of Three M-Type TRX Genes Causesa Pale-Green Leaf Phenotype in Arabidopsis

To examine the physiological functions of the TRXm1,TRX m2, and TRX m4 proteins in vivo, transfer DNA(T-DNA) insertional mutant libraries were screened, andhomozygous trx m1 and trx m4 mutants were identifiedin previous studies (Courteille et al., 2013; Laugier et al.,2013). Although trx m4mutants exhibited elevated cyclicelectron flow capacity, all trx m1 and trx m4 mutantplants showed no obvious growth defects when com-pared with wild-type plants under normal growth con-ditions (Courteille et al., 2013; Laugier et al., 2013),implying that functional redundancy may exist betweenTRX m1, TRX m2, and TRX m4. To test this hypothesisand to avoid the lethality of multiple TRX m deficiency,the tobacco rattle virus (TRV)-based virus-induced genesilencing (VIGS) approach was applied to efficiently si-lence one or more TRX m genes (Liu et al., 2002; Burch-Smith et al., 2006; Arsova et al., 2010; Luo et al., 2012).

To verify the phenotypes in trx m single mutants, thecoding regions of TRX m1, TRX m2, and TRX m4 wereindividually cloned into the pTRV2 vector (pTRV2-TRXm1, pTRV2-TRX m2, and pTRV2-TRX m4) for efficientsilencing of single TRX m genes (Burch-Smith et al.,2006). Three weeks after infection, the time interval usedfor all biological experiments, the mRNA levels of TRXm genes in VIGS plants were analyzed by quantitativereal-time reverse transcription (RT)-PCR. In comparisonwith VIGS-GFP plants (the negative control), the ex-pression of TRX m4 was specifically reduced by morethan 90% in the plants infiltrated with the pTRV2-TRXm4 vector (called VIGS-TRX m4 plants). Unexpectedly,we found that TRX m2mRNA levels in plants infiltratedwith the pTRV2-TRX m1 vector decreased by approxi-mately 74%, and vice versa, whereas the expression ofTRX m4 was not influenced in these plants (Fig. 1B;Supplemental Table S1). This off-target silencing effecton TRX m1 and TRX m2 could be attributed to thesignificantly higher coding sequence homology betweenTRX m1 and TRX m2 (76.1% identity) than between TRXm1 and TRX m4 (57.9% identity; Supplemental Fig. S1A;Jackson et al., 2003; Chibani et al., 2009). Therefore,plants infected with the pTRV2-TRX m1 and pTRV2-TRX m2 vectors exhibited double TRX m1 and TRX

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Redox Regulation of PSII Biogenesis

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m2 silencing and were designated VIGS-TRX m1/m2and VIGS-TRX m2/m1 plants, respectively. Similar tothe single TRX m mutant plants, VIGS-TRX m4, VIGS-TRX m1/m2, and VIGS-TRX m2/m1 plants showedno obvious differences from VIGS-GFP plants (Fig. 1;Supplemental Table S1). Furthermore, we introducedthe coding regions of TRX m1 and TRX m2 into thepTRV2 vector together (pTRV2-TRX m1m2) for simul-taneous inactivation of these two TRX genes. Plantsinfected with the pTRV-TRX m1m2 vector showed morethan 90% silencing efficiency for both TRX m1 and TRXm2 (called VIGS-TRX m1m2 plants) and also exhibitedno visible phenotype (Fig. 1A; Supplemental Table S1).Therefore, we propose that silencing of individual m-typeTRX genes and concurrent silencing of TRX m1 andTRX m2 have no obvious impact on leaf development.

To further investigate the influence of triple TRX mgene silencing on leaf development, we cloned thecoding regions of TRX m4 with TRX m1 or TRX m2into the pTRV2 vector (pTRV2-TRX m1m4 and pTRV2-TRX m2m4 vectors). Plants infected with either pTRV2-TRX m1m4 or pTRV2-TRX m2m4 showed the triple TRXm-silenced phenotype and were designated VIGS-TRXm1m4/m2 or VIGS-TRXm2m4/m1 plants, respectively(Fig. 1B; Supplemental Table S1). Interestingly, theseplants had pale-green leaves with strongly reduced(approximately 45%) chlorophyll contents comparedwith the VIGS-GFP plants; the chlorophyll a/b ratio was1.5 in VIGS-TRX m1m4/m2 and VIGS-TRX m2m4/m1plants versus 2.1 in VIGS-GFP plants (Fig. 1, C and D).Taken together, the phenotypic comparison betweensingle, double, and triple TRX m-silenced plants indicates

Figure 1. Characterization of the Arabidopsis VIGS-TRX m plants. A, Representative photographs of VIGS-GFP, VIGS-TRX m4,VIGS-TRX m1/m2, VIGS-TRX m2/m1, VIGS-TRX m1m2, VIGS-TRX m1m4/m2, and VIGS-TRX m2m4/m1 plants, which wereinfected with the pTRV2-GFP, pTRV2-TRX m4, pTRV2-TRX m1, pTRV2-TRX m2, pTRV2-TRX m1m2, pTRV2-TRX m1m4, andpTRV2-TRX m2m4 vectors, respectively, via agroinfiltration. Photographs of representative leaves from VIGS plants are shownin the insets. All the plants were observed 3 weeks after infiltration. At least three biological replicates were performed, andsimilar results were obtained. Bars = 1 cm. B, The suppression rate of TRX m genes in VIGS-TRX m plants was analyzed byquantitative real-time RT-PCR. The relative transcript levels of TRX m1, TRX m2, and TRX m4 in VIGS-TRX m4, VIGS-TRXm1/m2, VIGS-TRX m2/m1, VIGS-TRX m1m2, VIGS-TRX m1m4/m2, and VIGS-TRX m2m4/m1 plants were normalized to thelevel in VIGS-GFP plants (100%). The data represent means6 SD of three biological replicates. C and D, Total chlorophyll (Chl)content (C) and chlorophyll a/b ratio (D) in VIGS-TRX m and VIGS-GFP plants. FW, Fresh weight. The data represent means 6 SD

of three biological replicates. Statistical significance compared with the VIGS-GFP plants is indicated by asterisks (**P # 0.01,Student’s t test).

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Wang et al.






that the TRX m1, TRX m2, and TRX m4 proteins pos-sess a functional redundancy in leaf greening, and onlythe inactivation of the three m-type TRX genes canresult in pale-green leaves.

PSII Activity Is Dramatically Reduced in Plants TriplySilenced for TRX m1, TRX m2, and TRX m4

To gain insight into the primary target of the tripleTRX m1, TRX m2, and TRX m4 gene silencing, nonin-vasive chlorophyll fluorometric analyses were per-formed to investigate photosynthetic electron transportin the VIGS plants (Baker, 2008). The ratio of variablefluorescence to maximal fluorescence (Fv/Fm), which isan indicator of the maximum efficiency of PSII photo-chemistry, was drastically decreased in the VIGS-TRXm1m4/m2 and VIGS-TRX m2m4/m1 plants (0.46 6 0.02versus 0.83 6 0.01 in the VIGS-GFP controls; Fig. 2A;Table I). The decreased Fv/Fm below 0.5 indicates thatthe triple TRX m-silenced plants have primary defectsin electron transfer within PSII or a partial loss of PSIIcapacity (Meurer et al., 1996). Consistently, the actualPSII photochemical efficiency (FPSII) and photochemi-cal quenching (qP) could not be measured in VIGS-TRXm1m4/m2 and VIGS-TRX m2m4/m1 plants (Table I).In contrast, nonphotochemical quenching (NPQ) in theVIGS-TRX m1m4/m2 and VIGS-TRX m2m4/m1 plantswas higher than in the VIGS-GFP plants (Table I); NPQ is

viewed as a compensatory mechanism in response tooverexcited chlorophyll to prevent irreversible damageto PSII (Takahashi and Badger, 2011). Furthermore,inactive PSII always affects the light-induced redox kineticsof PSI (Meurer et al., 1996). The redox kinetics of P700showed that P700 can be oxidized in VIGS-TRX m1m4/m2and VIGS-TRX m2m4/m1 plants, but the amplitude ofchanges in A820 induced by far-red light were lowerthan in the VIGS-GFP plants, and the DA/DAmax valuewas greater than 1 (Fig. 2B), implying that PSI in thetriple TRX m-silenced plants is functional but with lowactivity. These spectroscopic analyses suggest that thephotosynthetic changes in the triple TRX m-silenced plantsprimarily reflect a reduction in PSII activity.

Accumulation of the PSII Complex Is Specifically Impairedin Plants Triply Silenced for TRX m1, TRX m2, and TRX m4

The defect in PSII photosynthetic electron transportcould be associated with altered protein levels in thePSII complex. To examine the steady-state levels ofthylakoid proteins in the VIGS plants, we performedimmunoblot analyses using antibodies raised againstdiagnostic subunits from four thylakoidal membranephotosynthetic protein complexes (i.e. PSII, Cyt b6fcomplex, PSI, and ATP synthase). Our results showedthat levels of the chloroplast-encoded PSII core sub-units D1, D2, CP43, and CP47 in thylakoids isolated

Figure 2. Spectroscopic characterization of VIGS-TRX m and VIGS-GFP plants. A, Chlorophyll a fluor-escence induction analyses in VIGS plants. Theminimal level of fluorescence (F0) and the maximallevel of fluorescence (Fm) are indicated for eachgenotype. The lightning arrows indicate the appli-cation of saturating light pulses (SP). The white barindicates exposure to actinic light (AL), and the blankbar indicates exposure to measuring light (ML).B, Redox kinetics of P700 at 820 nm induced by far-redlight (FR; 720 nm) in VIGS plants. DA and DAmax arein vivo absorbance changes of P700 at 820 nm in-duced by actinic light and far-red light illumination,respectively. Three biological replicates were per-formed, and similar results were obtained.





from VIGS-TRX m1m4/m2 and VIGS-TRX m2m4/m1plants were reduced to approximately 58%, 58%, 18%,and 28% of VIGS-GFP control levels, respectively (Fig. 3).The nucleus-encoded 33-kD subunit of the oxygen-evolving complex (PsbO) accumulated to approximately64% of VIGS-GFP levels, and the light-harvesting com-plex II (LHCII) and PSI antenna protein (Lhca1) werereduced to approximately 68% and 65% of control levels,respectively (Fig. 3). In contrast, the diagnostic subunits ofPSI (PsaA), the Cyt b6f complex (cytochrome f), and ATPsynthase (CF1 b) in the VIGS-TRX m1m4/m2 and VIGS-TRX m2m4/m1 plants accumulated to levels similar tothose in the control VIGS-GFP plants (Fig. 3).

To further investigate the putative structural altera-tions of thylakoid membrane protein complexes in thetriple TRX m gene-silenced plants, thylakoid membranesfrom VIGS plants were solubilized with n-dodecyl b-D-maltoside (DM), and the chlorophyll-protein complexes(with equal amounts of chlorophyll) were separated byblue native (BN)-PAGE (Schägger et al., 1994). Sevenprotein complexes were resolved in the first dimensionin the presence of Coomassie blue dye; these representPSII supercomplexes (band I), PSI monomers and PSIIdimers (band II), PSII monomers (band III), CP43-minusPSII (band IV), trimeric (band V) andmonomeric (band VI)LHCII, and unassembled proteins (band VII; Fig. 4A).The BN-PAGE analysis clearly showed that the amountof PSII complexes (bands I, II, III, and IV) per unit ofchlorophyll was significantly lower in thylakoids iso-lated from the VIGS-TRX m1m4/m2 and VIGS-TRXm2m4/m1 plants than that from the VIGS-GFP con-trol plants (Fig. 4A). Analyses of the second-dimensionSDS-urea-PAGE gels after Coomassie blue stainingconfirmed that the relative amounts of the PSII coresubunits D1, D2, CP43, and CP47 were considerablyreduced in the VIGS-TRX m1m4/m2 and VIGS-TRXm2m4/m1 plants, while no significant changes wereobserved in the amounts of Cyt b6f and ATP synthase(Fig. 4, D and E). Collectively, our results indicate thatthe dramatically decreased PSII activity seen in plantssilenced for the three TRX m genes could be attributedto the impaired accumulation of the PSII complex.

To assess whether the triple silencing of TRX mgenes causes ultrastructural changes in chloroplasts,transmission electron micrographs of ultrathin sectionsof leaves from various VIGS plants were analyzed. Inagreement with the phenotypic comparisons betweenVIGS-TRX m and VIGS-GFP plants, chloroplasts from

VIGS-TRX m4, VIGS-TRX m1/m2, VIGS-TRX m2/m1,and VIGS-TRX m1m2 plants were similar in size tothose from VIGS-GFP plants, and they contained well-developed membrane systems composed of granaconnected by stroma lamellae (Fig. 5). In contrast, thethylakoid membranes from VIGS-TRX m1m4/m2 andVIGS-TRX m2m4/m1 chloroplasts were disrupted,such that the grana stacks in VIGS-TRX m1m4/m2 andVIGS-TRX m2m4/m1 chloroplasts appeared to be lessconnected by stroma lamellae than those in VIGS-GFPcontrol chloroplasts (Fig. 5).

Expression of Chloroplast-Encoded PSII Subunits Is NotAltered in Plants Triply Silenced for TRX m1, TRX m2,and TRX m4

The chloroplast transcriptional regulation system inArabidopsis depends upon both the plastid-encodedplastid RNA polymerase (PEP) and the nucleus-encoded plastid RNA polymerase (NEP), which havedifferent target genes (Shiina et al., 2005). Chloroplastgenes, therefore, can be classified into three groupsaccording to their promoter structures: most of thephotosynthetic genes depend on PEP (class I), whereasRNA Polymerase Subunit Alpha (RpoA), RpoB, Acetyl-CoA Carboxylase Carboxyl Transferase Subunit Beta(AccD), and Uncharacterised Protein Family Ycf2 (Ycf2)genes are exclusively transcribed by NEP (class III),and the nonphotosynthetic housekeeping genes aregenerally transcribed by both PEP and NEP (class II;Hajdukiewicz et al., 1997).

The recently characterized TRX z participates in thePEP-dependent expression of chloroplast genes throughthe redox regulation of two fructokinase-like proteins(Arsova et al., 2010). To test whether the significantreduction in PSII core subunits observed in the tripleTRX m-silenced plants resulted from impaired tran-scription of chloroplast genes, we used quantitativereal-time RT-PCR to analyze mRNA levels of genes thatencode the representative chloroplast proteins tran-scribed by NEP and/or PEP in the VIGS plants (Table II).The PsaA, PsbA, PsbB, PsbC, and PsbD genes that encodephotosystem subunits were selected as representativesof the PEP-dependent genes (class I), RpoA, AccD, andYcf2 were selected as NEP-dependent genes (class III),and Caseinolytic Protease P (ClpP) and NADH Dehydrog-enase ND2 were chosen as class II genes. Quantitative

Table I. Chlorophyll fluorescence parameters in VIGS-TRX m and VIGS-GFP plants

Measurements of chlorophyll fluorescence parameters were done on VIGS plants after 15 min of dark adaptation. Actinic light intensity was110 mmol photons m22 s21. 1 2 qP represents excitation pressure. At least six leaves from VIGS plants were measured. Data are given as means6 SE

of three biological replicates. Statistical significance compared with the VIGS-GFP plants is indicated by asterisks (**P # 0.01, Student’s t test).

Parameter VIGS-GFP VIGS-TRX m4 VIGS-TRX m1/m2 VIGS-TRX m2/m1 VIGS-TRX m1m2 VIGS-TRX m1m4/m2 VIGS-TRX m2m4/m1

Fv/Fm 0.830 6 0.001 0.825 6 0.003 0.824 6 0.004 0.824 6 0.006 0.835 6 0.001 0.463 6 0.021** 0.461 6 0.030**ФPSII 0.532 6 0.014 0.553 6 0.018 0.554 6 0.015 0.589 6 0.011 0.536 6 0.005 0** 0**NPQ 0.721 6 0.071 0.753 6 0.067 0.684 6 0.035 0.601 6 0.037 0.633 6 0.076 1.877 6 0.256** 1.913 6 0.226**1 2 qP 0.281 6 0.118 0.242 6 0.023 0.249 6 0.021 0.209 6 0.015 0.277 6 0.015 1** 1**


Wang et al.



real-time RT-PCR analysis revealed that the expressionof class I genes in VIGS-TRX m plants was no differentfrom that in VIGS-GFP control plants (Table II), sug-gesting that TRX m proteins are not involved in theregulation of PEP-dependent gene expression. In spiteof this, triple silencing of TRX m1, TRX m2, and TRX m4altered the mRNA levels of some chloroplast-encodedgenes. The ClpP and Ycf2 transcript levels were signifi-cantly different in comparison with levels in the VIGS-GFP plants (Table II). In addition, expression of thenucleus-encoded genes PsaE, PsbO, and PSII LightHarvesting Complex Gene2.4 (Lhcb2.4), which encodeproteins that are targeted to the chloroplast, was sub-stantially reduced in VIGS-TRX m1m4/m2 and VIGS-TRX m2m4/m1 plants (Table II). These data indicatethat silencing the three TRX m genes does not result in ageneral inhibitory effect on the transcription of chloro-plast protein genes.

VIGS-TRX m Plants Accumulate More Reactive OxygenSpecies Than VIGS-GFP Plants

TRX m proteins function as electron donors for Cys-containing proteins and regulate the functions of targetproteins by reducing their disulfide bonds. Among the

target proteins of TRX m, 2-Cys peroxiredoxin is animportant antioxidant for hydrogen peroxide (H2O2)detoxification in chloroplast (König et al., 2002; Dietzand Pfannschmidt, 2011; Tovar-Méndez et al., 2011).Previous studies suggest that TRX m proteins are ableto reduce the oxidative state of 2-Cys peroxiredoxin tomaintain the metabolic balance of reactive oxygenspecies (ROS; Collin et al., 2003; Chi et al., 2008).Therefore, we hypothesized that simultaneous inacti-vation of the Arabidopsis TRX m1, TRX m2, and TRXm4 genes could influence the ROS level in vivo. Theresults of histochemical staining demonstrated thatelevated levels of H2O2 and superoxide anion radical(O2

2) were detected in all VIGS-TRX m plants, espe-cially in the triply silenced VIGS-TRX m1m4/m2 andVIGS-TRX m2m4/m1 plants (Fig. 6). ROS accumula-tion at similar levels has been detected in other TRX mgene-silenced plants, such as the Arabidopsis TRX m3knockout mutant (Benitez-Alfonso et al., 2009) and inrice (Oryza sativa) OsTRX m RNA interference plants(Chi et al., 2008). Furthermore, it is widely acceptedthat ROS serve as an essential redox signal for thetranscriptional regulation of photosynthesis-relatednuclear genes (Nott et al., 2006; Ruckle et al., 2007).In line with this, our expression analysis showed thatmRNA levels for the nucleus-encoded chloroplast

Figure 3. Immunoblot analyses of thylakoid proteinsfrom VIGS plants. A, Immunoblot analysis of thyla-koid proteins loaded on the basis of equal total thy-lakoid proteins. Aliquots of 2.5 mg (¼ VIGS-GFP),5 mg (½ VIGS-GFP), 10 mg (VIGS-GFP), and 10 mg(VIGS-TRX m) of total thylakoid proteins were loadedon the gels. Designations of thylakoid membraneprotein complexes and their diagnostic componentsare labeled on the left. Three biological replicateswere performed, and similar results were obtained.B, Semiquantitative analysis of thylakoid proteins.Immunoblots in A from three biological replicateswere analyzed with Phoretix 1D software (PhoretixInternational). The protein contents (per unit of thy-lakoid protein) of the thylakoid membranes fromVIGS plants were normalized to the level of theb-subunit of the ATP synthase (CF1 b). Protein levelsin the VIGS-TRX m plants are shown relative to thelevels in the VIGS-GFP leaves (100%). Data are givenas means 6 SD of three biological replicates.





protein genes PsaE, PsbO, and Lhcb2were down-regulated(Table II). Taken together, our data suggest that triplesilencing of TRX m1, TRX m2, and TRX m4 not onlyresults in a defect in the PSII complex but also affectsROS metabolism.

Confirmation of Impaired PSII Accumulation in TripleTRX m-Silenced Plants

The above results strongly indicated that the tripleinactivation of TRX m1, TRX m2, and TRX m4 leads topale-green leaves and impaired PSII accumulation. Tofurther confirm these observations in TRX m mutantplants, the homozygous trx m1trx m4 double mutantwas isolated by crossing and was found to have asimilar phenotype to wild-type plants (SupplementalFig. S2; Supplemental Tables S1 and S2). In combinationwith the phenotype observed in plants silenced for bothTRX m1 and TRX m2, we propose that the simultaneousinactivation of TRX m1 and either TRX m2 or TRX m4has no conspicuous effect on leaf development.

Furthermore, we introduced the TRV-TRX m2 vectorinto trx m1trx m4 double mutant plants to silence theTRX m2 gene. Three weeks after infection, plants infil-trated with the pTRV2-TRX m2 vector displayed evi-dently reduced expression of TRX m1 and TRX m2genes in comparison with VIGS-GFP (wild-type) plantsand were designated VIGS-TRX m2/m1 (WT) and

VIGS-TRX m2/m1 (trx m1trx m4) plants (Fig. 7B;Supplemental Table S1). The VIGS-TRX m2/m1 (trxm1trx m4) plants also had pale-green leaves and sharplyreduced chlorophyll contents (Fig. 7, A–D). The two-dimensional BN/SDS-PAGE analysis indicated that theaccumulation of PSII complexes (bands I, II, III, and IV)and PSII core proteins was specifically and markedlyreduced in thylakoid membranes isolated from VIGS-TRXm2/m1 (trx m1trx m4) plants (Fig. 7, E–I).

M-Type TRX Proteins Associate with Minor PSIIAssembly Intermediates and Interact with PSII CoreSubunits D1, D2, and CP47

The observed reduction in the abundance of PSIIcomplexes in the triple TRX m-silenced plants implies adirect regulatory function for m-type TRX proteins inthe biogenesis of PSII. To test this hypothesis, we askedwhether the TRXm1, TRXm2, and TRXm4 proteins areassociated with the PSII complex. Because of the highdegrees of similarity between the amino acid sequencesof these three TRX proteins (Supplemental Fig. S1B), wewere unable to prepare antibodies raised specificallyagainst each of the three individual TRX m proteins. Al-ternatively, we combined 23 FLAG-tagged proteins at theC termini of TRX m amino acid sequences for immuno-blot analysis. Moreover, to strengthen the interactionbetween TRX m and their target proteins, the C-terminal

Figure 4. Analyses of thylakoid protein complexes from VIGS plants. A, BN-PAGE analyses of thylakoid chlorophyll-proteincomplexes. Equal amounts of thylakoid membranes (8 mg of chlorophyll) from VIGS-TRX m and VIGS-GFP plants were sol-ubilized with 1% (w/v) DM and separated by BN-PAGE. Assignments of the thylakoid membrane macromolecular proteincomplexes indicated at right were identified according to a previous study (Peng et al., 2006). B to E, Two-dimensional BN/SDS-PAGE fractionation of thylakoid protein complexes. Individual lanes from the BN-PAGE gels in A were subjected to second-dimension SDS-urea-PAGE followed by Coomassie blue staining. Identities of the relevant proteins are indicated by arrows. Threebiological replicates were performed, and similar results were obtained. [See online article for color version of this figure.]


Wang et al.








Cys residue in the TRX active-site motif (WCGPC) thatcatalyzes the dissociation of target proteins from TRXswas substituted with a Ser residue (Motohashi et al.,2001; Balmer et al., 2003). The TRX m1C107S-FLAG, TRXm2C113S-FLAG, and TRX m4C119S-FLAG fusion proteinswere transiently expressed separately in Arabidopsis

mesophyll protoplasts, and TRX m3C100S-FLAG wasused as the reference protein (Supplemental Fig. S3A).Thylakoid membranes were then isolated from protoplastsand solubilized using 1% (w/v) DM for two-dimensionalBN/SDS-PAGE analysis (Fig. 8A). After electrophoresis,immunoblot analyses were performed using antibodies

Figure 5. Electron micrographs of chloroplasts from VIGS plants. Images of intact chloroplasts and closeup views wereobtained from VIGS-TRX m and VIGS-GFP plants. Leaves were collected 3 weeks after infection. Two biological replicates wereperformed, and similar results were obtained. Scale bars are as indicated.

Table II. Changes in transcript abundance of chloroplast-encoded and nucleus-encoded genes in VIGS-TRX m plants and VIGS-GFP plants

The transcript levels of representative genes encoding chloroplast proteins were analyzed by quantitative real-time RT-PCR. For individual genes,relative mRNA levels were normalized with respect to ACTIN2 (At3g18780) and then to VIGS-GFP levels. The log2 (VIGS-TRX m/VIGS-GFP) value isgiven, where 3.32 corresponds to a 10-fold up-regulation and 23.32 corresponds to a 10-fold down-regulation in VIGS-TRX m plants relative toVIGS-GFP plants. UBIQUITIN4 (At5g20620) was also used as a reference gene with similar results (data not shown). N.D., Not detectable. The datarepresent means 6 SD of three biological replicates. Statistical significance compared with the VIGS-GFP plants is indicated by asterisks (**P # 0.01,Student’s t test).

Gene Accession No. VIGS-TRX m4 VIGS-TRX m1/m2 VIGS-TRX m2/m1 VIGS-TRX m1m2 VIGS-TRX m1m4/m2 VIGS-TRX m2m4/m1

Class I genesPsaA Atcg00350 0.215 6 0.127 20.040 6 0.134 20.145 6 0.021 20.085 6 0.021 20.228 6 0.244 20.412 6 0.087PsbA Atcg00020 0.350 6 0.113 0.325 6 0.085 0.235 6 0.226 0.270 6 0.014 20.150 6 0.237 20.065 6 0.239PsbB Atcg00680 0.068 6 0.214 20.202 6 0.427 20.188 6 0.238 20.202 6 0.427 20.105 6 0.200 20.200 6 0.075PsbC Atcg00280 20.308 6 0.351 0.070 6 0.627 20.152 6 0.837 20.310 6 0.113 20.478 6 0.511 20.368 6 0.046PsbD Atcg00270 0.285 6 0.071 0.130 6 0.177 20.153 6 0.032 0.210 6 0.156 20.367 6 0.618 20.183 6 0.154

Class II genesClpP Atcg00670 20.615 6 0.163 20.245 6 0.219 20.485 6 0.120 20.225 6 0.057 21.595 6 0.054** 21.627 6 0.267**NADH Atcg00890 0.230 6 0.007 0.060 6 0.177 20.120 6 0.021 20.190 6 0.120 20.065 6 0.100 20.045 6 0.072Dehydrogenase ND2

Class III genesRpoA Atcg00740 0.203 6 0.247 20.113 6 0.028 0.213 6 0.049 0.183 6 0.148 20.733 6 0.208 20.606 6 0.100AccD Atcg00500 0.140 6 0.035 0.475 6 0.001 N.D. 0.390 6 0.205 0.290 6 0.173 0.160 6 0.064Ycf2 Atcg00860 20.295 6 0.106 20.335 6 0.099 0.145 6 0.042 0.190 6 0.078 1.885 6 0.232** 1.918 6 0.206**

Nucleus-encoded genesPsaE At4g28750 0.115 6 0.163 20.085 6 0.106 0.090 6 0.057 0.185 6 0.014 21.212 6 0.174** 21.248 6 0.215**PsbO At5g66570 0.095 6 0.163 20.050 6 0.064 20.075 6 0.014 0.140 6 0.007 21.050 6 0.228** 20.868 6 0.070**Lhcb2.4 At3g27690 20.128 6 0.061 20.337 6 0.083 20.128 6 0.061 0.480 6 0.045 23.563 6 0.191** 23.310 6 0.394**






raised against FLAG-tagged TRX m proteins and PSIIsubunits (D1, D2, CP43, and CP47). The results showedthat the vast majority of the TRX m1, TRX m2, and TRXm4 proteins, but not TRX m3, could comigrate withminor PSII assembly intermediates such as the PSII re-action center (RC), the CP43 precomplex, and the CP47precomplex, and the immunoblot signals for the TRXm1, TRX m2, and TRX m4 proteins were also detectedin the CP43-minus PSII monomers (band IV; Fig. 8B). Toconfirm the association of TRX m with the PSII com-plex, we examined the interactions of TRX m with PSIIsubunits by means of pull-down assays. For this purpose,the immobilized His-TRX m1C107S, His-TRX m2C113S,His-TRX m4C119S, and His-TRX m3C100S recombinantproteins were used as baits to trap potential targets inthe thylakoid membranes (Supplemental Fig. S3B). Thetrapped TRX m target proteins were eluted and sub-jected to immunoblot analysis. The results showed thatTRX m1, TRX m2, and TRX m4, but not TRX m3, coulddirectly interact with PSII core subunits D1, D2, andCP47, but not CP43. In addition, the four TRX mproteins could interact with the ATP synthase sub-unit (CF1 b), which is a well-known TRX targetprotein (Motohashi and Hisabori, 2006; Ströherand Dietz, 2008; Montrichard et al., 2009; Lindahlet al., 2011), but not with representative subunitsof PSI (PsaA) and the Cyt b6f complex (cytochrome f;Fig. 8C).

The Deficiency of TRX m1, TRX m2, and TRX m4Interrupts the Redox Status of PSII Core Subunits

Based on the interactions between TRX m and PSII,we wondered whether the triple inactivation of TRXm1, TRX m2, and TRX m4 could have an effect on theredox status of the PSII complex in vivo. Amino acidsequence analyses showed that many PSII subunits,including D1, D2, CP43, CP47, and PsbO, have at leasttwo conserved Cys residues that can form the intra-molecular or intermolecular disulfide bridges (Shimadaet al., 2007). Therefore, we investigated the redox states

of intramolecular and intermolecular disulfide bonds inPSII proteins isolated from VIGS plants.

To analyze the redox status of intramolecular di-sulfide bridges in PSII subunits, 4-acetoamido-4-maleimidylstilbene-2,2-disulfonate (AMS; Invitrogen)was used to bind the reduced Cys residues (thiolgroup/sulfhydryl group) in reduced PSII proteins. Incontrast, oxidized proteins with disulfide bridges orsulfenic acids cannot bind AMS; thus, the AMS-labeled reduced proteins migrate slower on nonreduc-ing SDS-PAGE gels than do the oxidized proteins(Ikegami et al., 2007; Karamoko et al., 2011; Richteret al., 2013). To comprehensively analyze the ex-changes of intramolecular disulfide bonds in PSIIsubunits, protein extracts from VIGS-GFP plants weretreated with either the oxidant CuCl2, to induce di-sulfide bond formation, or the reducing agent dithio-threitol (DTT), for reduction of Cys residues, prior toAMS treatment. The mobility of AMS-labeled PSIIsubunits on nonreducing SDS-PAGE gels was thenanalyzed by immunoblotting. For D1, D2, CP43, andCP47, only one band was detected in the VIGS-GFPprotein extracts, and the protein mobility was unal-tered following CuCl2 or DTT treatment, whereas themobility of AMS-labeled PsbO proteins showed a re-sponse to different redox conditions in that two oxi-dized PsbO bands were detected in the CuCl2-treatedand nontreated VIGS-GFP protein extracts, and DTTcould reduce partially oxidized PsbO to reduced PsbO(Supplemental Fig. S4). This suggested that only PsbOproteins are capable of forming intramolecular disulfidebridges and is supported by the evidence that LUMENTHIOL OXIDOREDUCTASE1 (LTO1) mediates the for-mation of intramolecular disulfide bonds in PsbO, whichis essential for the assembly of PSII (Karamoko et al.,2011). However, the mobility of AMS-labeled PsbO inVIGS-TRX m1m4/m2 and VIGS-TRX m2m4/m1 plantswas not different from that in VIGS-GFP and VIGS-TRXm1m2 plants (Supplemental Fig. S4). These resultssuggest that the triple inactivation of TRX m genes doesnot influence the reduction of intramolecular disulfidebonds in PSII subunits.

Figure 6. ROS accumulation in leaves of VIGSplants. Fully expanded leaves from VIGS-TRX m andVIGS-GFP plants grown under normal conditionswere harvested for in situ histochemical stainingusing 3,39-diaminobenzidine (DAB) and nitrobluetetrazolium (NBT) to show the accumulated H2O2

(top row) and O22 (bottom row), respectively. Three

biological replicates were performed, and similarresults were obtained.


Wang et al.






Figure 7. Characterization of VIGS-TRX m2 (trx m1trx m4) plants. A, Representative photographs of the wild-type VIGS-GFP(WT), VIGS-GFP (trx m1trx m4), VIGS-TRX m1/m2 (WT), and VIGS-TRX m2/m1 (trx m1trx m4) plants, which were infected withthe pTRV2-GFP and pTRV2-TRX m2 vectors via agroinfiltration in wild-type and trx m1trx m4 double mutant backgrounds,respectively. Representative leaves are shown in the insets. At least three biological replicates were performed, and similarresults were obtained. Bars = 1 cm. B, The suppression rate of TRX m genes in VIGS-TRX m plants was analyzed by quantitativereal-time RT-PCR. The relative mRNA levels of TRX m1, TRX m2, and TRX m4 in the VIGS-TRX m2/m1 (WT), VIGS-GFP (trxm1trx m4), and VIGS-TRX m2/m1 (trx m1trx m4) plants were normalized to the levels in VIGS-GFP (WT) plants (100%). Thedata represent means6 SD of three biological replicates. C and D, Total chlorophyll (Chl) contents (C) and chlorophyll a/b ratios(D) of the VIGS plants in wild-type and trx m1trx m4 double mutant backgrounds. FW, Fresh weight. The data representmeans6 SD of three independent experiments. Statistical significance compared with the VIGS-GFP (WT) plants is indicated byasterisks (**P # 0.01, Student’s t test). E, BN-PAGE analyses of thylakoid chlorophyll-protein complexes. Equal amounts ofthylakoid membranes (8 mg of chlorophyll) from the VIGS plants in wild-type and trx m1trx m4 double mutant backgrounds





Furthermore, we also examined the role of TRX mproteins in reducing the intermolecular disulfide bridgesin PSII core subunits. With the formation of intermo-lecular disulfide bonds, two or more proteins can beaggregated to form homooligomeric/heterooligomericcomplexes (Buchanan and Balmer, 2005; Dietz andPfannschmidt, 2011). To improve the mobility and vi-sualization of intermolecular disulfide bond-mediatedmacromolecular complexes, protein extracts from VIGSplants were separated by nonreducing SDS-PAGEwithout AMS labeling (Kirchsteiger et al., 2009; Arsovaet al., 2010; Richter et al., 2013). On nonreducing SDS-PAGE gels, the monomers and oligomers of PSII coreproteins D1, D2, CP43, and CP47 were observed inVIGS-GFP protein extracts, and the vast majority ofPSII core subunits were present as monomeric proteins(Fig. 9). Moreover, we found that the oligomerizationof D1 and CP47 was redox sensitive. CuCl2 treatmentcould significantly increase the aggregation of D1 andCP47 proteins, while DTT could reduce the oligomers tomonomers (Fig. 9, A and C), suggesting that D1 andCP47 can form redox-regulated intermolecular disulfidebridges. One possible explanation for the observedoligomeric D2 and CP43 proteins could be the physicalinteractions between the four PSII core subunits (Wollmanet al., 1999; Iwata and Barber, 2004; Nelson and Yocum,2006). Interestingly, more oxidized CP47 oligomerswere detected in VIGS-TRX m1m4/m2 and VIGS-TRXm2m4/m1 plants in comparison with VIGS-GFP andVIGS-TRX m1m2 plants. In contrast, the levels of oli-gomers and monomers of D1, D2, and CP43 detectedin VIGS-TRX m1m4/m2 and VIGS-TRX m2m4/m1plants were significantly lower than in VIGS-GFP andVIGS-TRX m1m2 plants (Fig. 9C). These data suggestthat TRX m1, TRX m2, and TRX m4 are required formodulating the redox status of intermolecular disul-fide bridges in PSII core subunits, and the simulta-neous inactivation of the three TRX m genes leads toelevated levels of oxidized CP47 oligomers.

DISCUSSION

PSII is an integral membrane, multisubunit proteincomplex that catalyzes the light-driven water-splittingreaction and reduction of plastoquinone to initiatephotosynthetic electron flow. In addition to structuralstudies of PSII, an emphasis has been placed on theidentification of auxiliary proteins involved in the bio-genesis and maintenance of the PSII complex (Muloet al., 2008; Nixon et al., 2010; Komenda et al., 2012).Understandably, the identification of PSII accessory

factors and the characterization of their functions willgreatly improve our understanding of regulatory mech-anisms by which the PSII complex accumulates and ismaintained. Here, we report previously unknownfunctions for the classical chloroplastic TRX m1, TRXm2, and TRX m4 in the regulation of PSII biogenesis inArabidopsis.

TRX m1, TRX m2, and TRX m4 Are Required for theAccumulation of PSII in Arabidopsis

To investigate the functions of TRX m1, TRX m2, andTRX m4 in the accumulation of the photosynthetic ap-paratus, we comprehensively characterized the pheno-types of plants silenced for one, two, or three of theseTRX m genes. Interestingly, single deficiencies of eitherTRX m1 or TRX m4, and double inactivation of TRX m1along with either TRX m2 or TRX m4, resulted in nomacroscopic alteration in phenotype, whereas plantssilenced for all three genes had pale-green leaves (Figs. 1and 7; Supplemental Fig. S2; Supplemental Table S1),suggesting the existence of mutual functional compensa-tion between TRX m1, TRX m2, and TRX m4. This couldbe associated with their similar protein structures andanalogous thylakoidmembrane localization (SupplementalFig. S1; Peltier et al., 2002; Friso et al., 2004). In addition,our hypothesis is supported by the fact that the expressionof TRX m1, TRX m2, or TRX m4 in yeast can rescue theoxidant-hypersensitive phenotype of the trx null strain(Issakidis-Bourguet et al., 2001). Functional compensationbetween TRX isoforms has also been observed in tetra-pyrrole biosynthesis (Ikegami et al., 2007; Luo et al., 2012).

Furthermore, triple inactivation of the three TRX mgenes has pleiotropic effects on photosynthetic electrontransport and chloroplast development. Chlorophyllfluorescence measurements showed that Fv/Fm wasdramatically reduced below 0.5 in the triple-silencedplants (Fig. 2; Table I). The decrease in Fv/Fm is due tospecific impairment of the PSII complexes (Figs. 4 and 7).In line with this, no FPSII and elevated NPQ were detected,indicating that the electron flow within PSII is severelyinhibited in the triple-silenced plants (Table I). In ad-dition, determination of absorbance changes of P700 at820 nm demonstrates that PSI in the triple-silenced plantsis functional but has low activity (Fig. 2B). These spec-troscopic properties also have been observed in a num-ber of mutants with defects in PSII, such as low PSIIaccumulation1 (lpa1; Peng et al., 2006), lpa2 (Ma et al.,2007), lpa3 (Cai et al., 2010), photosynthesis affected mu-tant68 (pam68; Armbruster et al., 2010), and hcf243(Zhang et al., 2011). Moreover, immunoblot analysisconfirmed that essential subunits of the PSII complex

Figure 7. (Continued.)3 weeks after infiltration were solubilized with 1% (w/v) DM and separated by BN-PAGE. Assignments of the thylakoidmembrane macromolecular protein complexes indicated at right were identified according to a previous study (Peng et al.,2006). F to I, Two-dimensional BN/SDS-PAGE fractionation of thylakoid protein complexes. Individual lanes from the BN gels inE were subjected to second-dimension SDS-urea-PAGE followed by Coomassie blue staining. Identities of the relevant proteinsare indicated by arrowheads. Three biological repeats were performed, and similar results were obtained.


Wang et al.







are severely reduced in plants silenced for TRX m1,TRX m2, and TRX m4 (Fig. 3). Finally, the specificallyimpaired PSII complex in the triple-silenced plants hada strong effect on the organization of the thylakoidmembranes. The amount of stroma lamellae and thestacking of grana thylakoids in the triple-silenced plantsare obviously impaired in comparison with that inVIGS-GFP plants (Fig. 5). Taken together, our data onspectroscopy measurements, biochemical analyses, andchloroplast ultrastructure reveal a specific defect in PSIIaccumulation in the triple TRX m-silenced plants, thusproviding a straightforward explanation for the observedpale-green leaf phenotype (Figs. 1 and 7).The analyses described above indicate that TRX m1,

TRX m2, and TRX m4 together participate in the ac-cumulation of the PSII complex. However, the mech-anism involved remains to be elucidated. Consideringthe hydrophilic nature of these three TRX m proteins,the possibility that they may be integral constituents ofPSII can be excluded. Thus, these three TRX m proteinsprobably interact transiently with the PSII complexand some PSII subunits, in a manner similar to severalPSII auxiliary factors that have been shown to interactwith specific PSII subunits and assist their assembly, butare absent in the functional complexes (Peng et al., 2006;Ma et al., 2007; Armbruster et al., 2010; Cai et al., 2010).

Indeed, TRX m1, TRX m2, and TRX m4, but not TRXm3, comigrate with minor PSII assembly intermediatesand interact with PSII core subunits D1, D2, and CP47(Fig. 8). All of these data suggest that the three TRX mproteins participate directly in the biogenesis of the PSIIcore complex.

Assembly of the PSII Core Complex Is Redox Dependentand Can Be Modulated by m-Type TRXs

Thiol-disulfide chemistry plays an essential role inthe biogenesis of the photosynthetic apparatus and inthe maintenance of photosynthetic efficiency (Dietzand Pfannschmidt, 2011; Rochaix, 2011; Kieselbach,2013). While the formation of disulfide bonds in theoxygen-evolving complex has been shown to be es-sential for the assembly of the PSII complex (Burnapet al., 1994; Karamoko et al., 2011), relatively fewstudies have examined the mechanisms of disulfidebridge exchanges in PSII core proteins. This is ham-pered by multiple limitations such as the hydrophobicnature of PSII subunits and the lack of PSII subunits inmutant plants. Our biochemical analyses in vivo showthat two PSII core subunits, D1 and CP47, can formredox-sensitive intermolecular, but not intramolecular,

Figure 8. Evidence for the interaction of TRX m1, TRX m2, and TRX m4 proteins with PSII protein complexes. A, Analysis ofthylakoid protein complexes by BN-PAGE. Freshly isolated thylakoid membranes from protoplasts transformed with pUC19-TRX m1C107S-FLAG, pUC19-TRX m2C113S-FLAG, pUC19-TRX m4C119S-FLAG, and pUC19-TRX m3C100S-FLAG plasmids weresolubilized with 1% (w/v) DM at a chlorophyll concentration of 15 mg, and the protein samples were separated by BN-PAGE.Protoplasts transformed with the empty transient expression vector were the negative control (CK). Assignments of the thylakoidmembrane macromolecular protein complexes indicated at right were identified according to a previous study (Peng et al.,2006). B, Two-dimensional BN/SDS-PAGE fractionation and immunoblot analysis of PSII protein complexes. Thylakoidmembrane complexes separated in A were further subjected to second-dimension SDS-urea-PAGE, and the proteins wereimmunodetected with specific antibodies against FLAG-tagged TRX m proteins, D1, D2, CP43, and CP47. The asterisks indicatenonspecific signals on the immunoblots. The arrows indicate the FLAG-tagged TRX m proteins. Two biological repeats wereconducted, and similar results were obtained. C, Pull-down assay. His-TRX m1C107S, His-TRX m2C113S, His-TRX m3C100S, andHis-TRX m4C119S recombinant proteins were immobilized on cyanogen bromide-activated Sepharose 4B resin and incubatedwith total thylakoidal membrane proteins solubilized with 1% (w/v) DM; empty cyanogen bromide-activated Sepharose 4Bresin with no coupled His-TRX m protein was used as the negative control. After incubating and washing, proteins interactingwith the His-TRX m mutant proteins were eluted with 10 mM DTT and detected by immunoblot analysis. Identities of thylakoidmembrane protein complexes and their subunits are indicated at right. Three biological repeats were performed, and similarresults were obtained. [See online article for color version of this figure.]





disulfide bridges (Fig. 9; Supplemental Fig. S4), im-plying that the assembly of the PSII core complex alsorequires proper redox status of the PSII core subunitsand the involvement of redox regulators. This hy-pothesis is supported by several lines of evidence. Theassembly of D1 proteins into the PSII RC and PSII corecomplex depends on proper redox conditions. Thethiol reactants DTT, N-ethylmaleimide, and iodoso-benzoic acid could reduce the stability of early PSIIassembly intermediates, inhibit C-terminal processingof the D1 precursor, and prevent the reassembly ofCP43 into PSII (Zhang et al., 2000). Moreover, SNOWYCOTYLEDON2 and LOW QUANTUM YIELD OFPHOTOSYSTEM II1 (LQY1), with protein disulfideisomerase activity, interact with PSII core subunitsCP43 and CP47 and are suggested to function in thebiogenesis of thylakoid membranes in cotyledons andin PSII disassembly and/or reassembly during the PSIIrepair cycle, respectively (Shimada et al., 2007; Lu et al.,2011; Tanz et al., 2012). Intriguingly, the triple silencingof TRX m1, TRX m2, and TRX m4 disrupted the redoxstate of PSII subunits and led to elevated levels ofoxidized CP47 oligomers (Fig. 9C), indicating theimportant role of TRX m proteins in modulating theredox status of PSII core subunits.

Biogenesis of the PSII complex is a highly orderedprocess in which a series of assembly steps occur se-quentially and are well coordinated in different com-partments of the thylakoid membranes (Komendaet al., 2012; Nickelsen and Rengstl, 2013). Thus, it isinteresting to consider which step(s) of PSII assemblymight involve TRX m1, TRX m2, and TRX m4. In theearly stages of PSII assembly, PSII core subunitsalways combine with distinct low-molecular-massproteins to form minor PSII assembly intermediatesubcomplexes, such as the PSII RC, the CP47 precom-plex, and the CP43 precomplex. In two-dimensionalBN/SDS-PAGE, TRX m1, TRX m2, and TRX m4 canassociate with these PSII assembly intermediates (Fig. 8B).Similar combinations with small PSII complexes havebeen observed for some PSII accessory factors thatare involved in the early steps of PSII biogenesis;HCF136 and YCF48 localize to the thylakoid lumenand participate in the stabilization of newly synthe-sized D1 precursor and mediate the formation of thePSII RC (Plücken et al., 2002; Komenda et al., 2008;Rengstl et al., 2011). The intrinsic thylakoid proteinsPAM68 and SII0933 play an essential role in the con-version of the PSII RC into larger PSII complexes(Armbruster et al., 2010; Rengstl et al., 2011). Furthermore,

Figure 9. Visualization of the redoxstatus of intermolecular disulfide bondsin PSII subunits from VIGS plants.Proteins from VIGS-TRX m1m2, VIGS-TRX m1m4/m2, VIGS-TRX m2m4/m1,and VIGS-GFP leaves were extractedunder nonreducing conditions and weretreated with either 1 mM CuCl2 or 10 mM

DTT. The treated and untreated sampleswere separated on 12% nonreducing(2DTT) or reducing (+DTT) SDS-PAGEgels (15 mg of total leaf proteins perlane) and probed with antibodies raisedagainst D1 (A), D2 (B), CP47 (C), and CP43(D). RbcL (the large subunit of Rubisco)was used as a loading control. The aster-isks indicate a nonspecific signal. Oxi-dized oligomers and reduced monomersof PSII subunits are labeled. Three bio-logical replicates were performed, andsimilar results were obtained.


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the chloroplast thylakoid membrane system is partic-ularly complex in plants, because it is organized intononappressed stroma lamellae and appressed granastacks. The various PSII complexes are characterized indistinct thylakoid membrane domains. The function-ally dimeric PSII, including PSII supercomplexes andPSII core dimers, dominate in the thylakoid grana,

whereas the PSII assembly intermediate subcomplexes,such as PSII monomers, the CP47-containing PSII RC,and the PSII RC, prevail in the stroma lamellae (Dan-ielsson et al., 2006). Thus, it is reasonable that the triplesilencing of the three TRX m genes resulted in theconspicuous impaired stroma thylakoid membranesand grana (Fig. 5). Taken together, these results suggest

Table III. Summary of conserved Cys residues in described PSII assembly factors in Arabidopsis

The PSII assembly factors containing conserved Cys residues are indicated in boldface.

PSII Assembly and

PSII Repair ProcessaAssembly

Factor

Accession

No.Sizeb Localizationc No. of Conserved

Cys ResiduesdSite of Conserved

Cys ResidueseReferences

kDa

PSII RC assembly Alb3f At2g28800 44 Intrinsic TM 1 154 Bellafiore et al. (2002);

Ossenbuhl et al.

(2004); Gohre et al.

(2006)

C-Terminal

Processing

Protease for

D1 Proteinof PSII (CtpA)

At4g17740 46 Extrinsic L 5 224, 299, 410, 412, 420 Anbudurai et al.

(1994)

HCF136 At5g23120 38 Extrinsic L 0 – Plucken et al. (2002)

HCF243 At3g15095 65 Intrinsic TM 19 19, 61, 78, 90, 94, 98,

116,126, 198, 210,

261, 379, 427, 433,

478, 455, 512, 546,

562

Zhang et al. (2011)

LPA1 At1g02910 40 Intrinsic TM 3 28, 29, 42 Peng et al. (2006)

PSII RC47 assembly PAM68 At4g19100 20 Intrinsic TM 1 13 Armbruster et al.

(2010)

Incorporation of CP43 LPA2 At5g51545 15 Intrinsic TM 0 – Ma et al. (2007)

LPA3 At1g73060 34 Extrinsic S 3 64, 140, 242 Cai et al. (2010)

LPA19g At1g03600 12 Extrinsic L 0 – Chen et al. (2006);

Wei et al. (2010)

Assembly of the

oxygen-evolving

Mn4CaO5 cluster

CYCLOPHILIN38

(CYP38)

At3g01480 39 Extrinsic L 1 174 Fu et al. (2007);

Sirpio et al. (2008);

Vasudevan et al.

(2012)

LTO1 At4g35760 35 Intrinsic TM 10 1, 64, 71, 150, 153, 185,248, 251, 271, 286

Karamoko et al. (2011)

PSII dimmer and

PSII supercomplex

assembly

Degradation of

Periplasmic

Proteins1 (Deg1)

At3g27925 42 Extrinsic L 2 40, 365 Kapri-Pardes et al.

(2007); Sun et al.

(2010)

FK-506 BINDING

PROTEIN20-2

(FKBP20-2)

At3g60370 24 Extrinsic L 5 1, 11, 26, 96, 210 Lima et al. (2006)

Psb29/Thylakoid

Formation1

(Thf1)g

At2g20890 27 Extrinsic S 2 159, 217 Wang et al. (2004);

Keren et al. (2005)

PSII repair LQY1 At1g75690 12 Intrinsic TM 8 44, 47, 55, 58, 78, 81,

89, 92

Lu et al. (2011)

PSBP-LIKE

PROTEIN 1

(PPL1)

At3g55330 18 Extrinsic L 0 – Ishihara et al. (2007)

THYLAKOID LUMEN

PROTEIN18.3

(TLP18.3)/Psb32f

At1g54780 23 Intrinsic TM 0 – Sirpio et al. (2007)

aPSII assembly factors are classified according to their proposed functions in the biogenesis and repair of the PSII complex. bPredictedmolecular mass of mature proteins. cL, Lumen; S, stroma; TM, thylakoid membrane. dThe number of conserved Cys residues in the matureproteins. eThe site of conserved Cys residues in amino acid sequences of mature proteins. fAlb3 is involved in the integration of LHCII intoPSII supercomplexes; THYLAKOID LUMEN PROTEIN18.3/Psb32 functions in the assembly of PSII supercomplex. gLPA19 and Psb29/ThylakoidFormation1 play roles in PSII repair.





that TRX m1, TRX m2, and TRX m4 may play an es-sential role in the early steps of PSII biogenesis forproper assembly of the PSII core complex.

Previous studies of PSII mutants, mainly performedin Chlamydomonas reinhardtii, indicated that D2 andCP47 serve a central function in the stable assembly ofthe PSII complex (Erickson et al., 1986; Vermaas et al.,1986; Lang and Haselkorn, 1989). However, the regu-latory mechanism underlying the integration of CP47into the PSII complex is an open question. At present,only two auxiliary proteins, PAM68 in Arabidopsisand Psb28 in cyanobacterium (Synechocystis PCC 6803),have been identified as being involved in the assemblyof CP47 into PSII (Dobakova et al., 2009; Armbrusteret al., 2010). Considering that TRX m1, TRX m2, andTRX m4 interact with PSII assembly intermediatesand CP47 proteins, and that their deficiency leads todrastically reduced CP47 protein levels and the accu-mulation of redox-sensitive CP47 oligomers (Figs. 3, 8,and 9), we tentatively propose that these three m-typeTRX proteins play a role in assisting the proper assemblyof CP47 into the PSII core complex.

Are There Other Relevant Targets of m-Type TRXDisulfide-Reducing Activity in PSII Biogenesis?

Despite evidence supporting the disulfide-reducingactivity of TRX m1, TRX m2, and TRX m4 in modu-lating the redox state of PSII subunits, which may inturn correlate with the reduced accumulation of PSIIcore subunits and PSII complexes (Figs. 3, 4, 7, and 9),the physiological significance of redox-sensitive CP47oligomerization seems rather unclear. In principle, threescenarios for CP47 oligomerization can be envisioned:(1) several CP47 proteins can form CP47-CP47 homo-oligomers through intermolecular disulfide bridges;(2) CP47 combines with other PSII subunits such as D1to form CP47-D1 heteroligomers; and (3) CP47 tran-siently associates with PSII assembly factors by means ofintermolecular disulfide bridges. Based on crystal-line structure analyses of the PSII complex and the lowamounts of PSII protein oligomers in comparison withtheir monomers (Fig. 9; Iwata and Barber, 2004; Nelsonand Ben-Shem, 2004; Dekker and Boekema, 2005), thefirst two possibilities seem unlikely. Interestingly, asshown in Table III, many PSII auxiliary proteins thatare involved in the early steps of PSII biogenesis, suchas Albino3 (Alb3), C-Terminal Processing Protease forD1 Protein of PSII, HCF243, LPA1, PAM68, andLPA3, all have conserved Cys residues. Strikingly,Degradation of Periplasmic Proteins1 and FK-506BINDING PROTEIN20-2 have been suggested to betarget proteins of chloroplastic TRXs (Lima et al., 2006;Ströher and Dietz, 2008). Therefore, in order to clarifythe m-type TRX-mediated redox regulation of PSII bio-genesis, we will further identify the potential reducingacceptors that are essential for PSII biogenesis andassembly of the PSII complex in a future study.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana ecotype Columbia) was grown in soil in agrowth chamber (110 mmol photons m22 s21, 16-h photoperiod, 21°C, and 60%relative humidity). The T-DNA insertion mutant lines for two m-type TRX genes(trx m1 [Salk_087118C] and trx m4 [Salk_032538]) were obtained from the Arabi-dopsis Biological Resource Center as described by Courteille et al. (2013). Thehomozygous T-DNA insertion lines were confirmed by PCR using the gene-specific and T-DNA-specific primers described in Supplemental Table S3. Thetrx m1trx m4 double mutant was obtained by crossing single mutants of trx m1 andtrx m4 and screening the F2 population by PCR using the primers described above.

VIGS Assay

Plasmids pTRV1 and pTRV2 are VIGS vectors based on Tobacco rattle virusas described by Liu et al. (2002). To construct the pTRV2-TRX m vectors, TRXm1, TRX m2, and TRX m4 complementary DNAs (cDNAs) were generated byRT-PCR with the primers described in Supplemental Table S3 and insertedinto the pTRV2 vector. The pTRV2-GFP vector was used as a negative control.pTRV1 and pTRV2 derivatives were introduced into Arabidopsis plants byAgrobacterium tumefaciens infiltration as described by Burch-Smith et al. (2006).

Transmission Electron Microscopy

Transmission electron microscopy was performed as described by Yao andGreenberg (2006). Fully expanded leaves from the VIGS-TRX m and VIGS-GFP plants were collected 3 weeks after infection. Micrographs were takenusing a transmission electron microscope (JEM1400; JEOL).

Pigment Analysis and ChlorophyllFluorescence Measurements

Chlorophyll was extracted from whole leaves with 80% acetone in 25 mM

HEPES-KOH (pH 7.5), and the chlorophyll content was determined as de-scribed by Wellburn (1994).

Chlorophyll fluorescence was measured using Maxi-Imaging PAM andDual-PAM 100 (Walz; http://www.walz.com). Arabidopsis leaves were darkadapted for 15 min prior to fluorescence measurements. The minimum fluorescenceyield was measured under a weak measuring light. The maximum fluor-escence yield was determined by applying a saturating light pulse (33 103 mmolphotons m22 s21). Leaves were illuminated for 6 min under actinic light illumi-nation (110 mmol photons m22 s21). Thereafter, the maximal fluorescence of light-adapted leaves was measured after applying a second saturating pulse. The FPSII,excitation pressure (1 – qP), and NPQ were recorded. Measurement of light-induced P700 absorbance changes at 820 nm was performed as described byMeurer et al. (1996). Absorbance changes induced by saturating far-red lightrepresent the relative amounts of photooxidizable P700.

Thylakoid Membrane and Total Protein Preparations

Thylakoid membranes were prepared according to Robinson et al. (1980).Isolated thylakoid membranes were quantified based on total chlorophyll asdescribed by Porra et al. (1989). Total proteins extracted from leaves of VIGSplants or from thylakoid membrane preparations were prepared according toMartinez-Garcia et al. (1999). Protein concentrations were determined usingthe Bio-Rad detergent-compatible colorimetric protein assay according to themanufacturer’s protocol.

BN/SDS-PAGE and Immunoblot Analyses

BN-PAGE was performed essentially according to Schägger et al. (1994) withthe modifications described by Peng et al. (2006). For two-dimensional analysis,the excised BN-PAGE lanes were soaked in SDS sample buffer (100 mM Tris-HCl[pH 6.8], 2% [w/v] SDS, 15% [v/v] glycerol, and 2.5% [v/v] b-mercaptoethanol)for 15 min at room temperature and then layered onto 1.5-mm-thick 12% SDS-PAGE gels containing 6 M urea. The gels were stained with Coomassie BrilliantBlue G250 and scanned using a GS-800 densitometer (Bio-Rad).

For immunoblot analysis, total proteins extracted from VIGS leaves orthylakoid membrane preparations were separated on 12% SDS-PAGE gels


Wang et al.




http://www.walz.com


containing 6 M urea. After electrophoresis, proteins were transferred to poly-vinylidene difluoride membranes (Millipore) and probed using antibodiesspecifically directed against individual PSII subunits (D1, D2, CP43, CP47, andPsbO), PSI subunits (PsaA and PsaO), the light-harvesting antenna proteins(Lhcb1 and Lhca1), the Cyt b6/f complex (cytochrome f), and the ATP syn-thase b-subunit (CF1 b; Agrisera). Signals were detected with the SuperSignalWest Pico Chemiluminescent Substrate (Thermo Scientific).

Gene Expression Analysis

Total RNA was extracted from Arabidopsis plants with the Plant RNeasyMini kit (Omega). Five-microgram samples of total RNA were used for cDNAsynthesis using the PrimeScript RT reagent kit (Takara) according to themanufacturer’s instructions. Quantitative real-time RT-PCR was performedwith SYBR Premix Ex Taq (Takara), and real-time amplification was moni-tored on the LightCycler480 system (Roche). Expression of ACTIN2 (At3g18780)and UBIQUITIN4 (At5g20620) was relatively stable in VIGS plants, and thegenes were amplified as internal controls in this study. Primer sequences arelisted in Supplemental Table S3.

Determination of ROS

In situ detection of H2O2 and O22 was performed mainly according to Lu

et al. (2011) with minor modifications. For H2O2 visualization, leaves fromVIGS plants were vacuum infiltrated with a solution containing 1 mg mL21 3,39-diaminobenzidine (pH 3.8) at room temperature for 1 h and incubated in thesame solution under growth light illumination for 1 h. The stained leaves werethen boiled in a solution of ethanol:acetic acid:glycerol (3:1:1, v/v/v) for 10 min.For O2

2 detection, leaves were vacuum infiltrated for 30 min with 50 mM

phosphate buffer (pH 7.5) containing 1 mg mL21 nitroblue tetrazolium andsubsequently boiled in 90% (v/v) ethanol for 10 min.

Transient Expression of Mutant TRX m Proteins inArabidopsis Protoplasts

The full-length cDNAs for TRX m1, TRX m2, TRX m3, and TRX m4 wereamplified from Arabidopsis cDNA by RT-PCR. Site-directed mutagenesis ofthe C-terminal Cys residues in the TRX m active-site motif (WCGPC) wereintroduced by overlap-extension PCR. Full-length cDNA fragments of TRXm1C107S, TRX m2C113S, TRX m3C100S, and TRX m4C119S were then individuallycloned into the pUC19-FLAG transient expression vector. Primer sequences arelisted in Supplemental Table S3. Protoplasts were isolated from 14-d-oldArabidopsis seedlings according to Zhai et al. (2009) and transformed withthe pUC19-TRX m1C107S, pUC19-TRX m2C113S, pUC19-TRX m3C100S, and pUC19-TRX m4C119S-FLAG plasmids according to Yoo et al. (2007). Transformedprotoplasts were collected by centrifugation for 2 min at 100g, resuspended inW5 solution, and kept in the dark for 16 h prior to further analysis.

Expression and Purification of Recombinant His-TaggedMutant TRX m Proteins

The cDNA fragments encoding the mature TRX m mutant proteins wereamplified from pUC19-TRX m1C107S, pUC19-TRX m2C113S, pUC19-TRX m3C100S,and pUC19-TRX m4C119S-FLAG plasmids by RT-PCR and individually clonedinto the pET28a expression vector (Novagen). Primer sequences are listed inSupplemental Table S3. The pET28a-TRX m1C107S, pET28a-TRX m2C113S,pET28a-TRX m3C100S, and pET28a-TRX m4C119S plasmids were transformed intoEscherichia coli BL21 (DE3; Stratagene) and induced to express with 0.6 mM

isopropyl b-D-thiogalactoside. Purification of four mutant His-tagged TRX mproteins was carried out with Ni+ affinity chromatography according to themanufacturer’s protocol (GE Healthcare). The imidazole in the elution bufferwas removed by fast ultrafiltration using 3-kD-limit Amicon Ultra-15 cen-trifugal filter units (Millipore).

Pull-Down Assay

The pull-down assay was performed essentially according to the method ofTada et al. (2008) with some modifications. Purified His-TRX m1C107S, His-TRXm2C113S, His-TRX m3C100S, and His-TRX m4C119S recombination proteins wereimmobilized on Sepharose 4B resin according to the manufacturer’s protocol

(GE Healthcare) and incubated with thylakoid membrane proteins solubilizedin 1% (w/v) DM overnight at 4°C. The empty resin without TRX m proteinwas used as the negative control. The resins were then extensively washedwith washing buffer containing 0.5 M NaCl until the A280 reached almost zero.The TRX m target proteins linked by the newly formed heterodisulfide bondswere eluted with buffer containing 10 mM DTT and 0.5 M NaCl and analyzedby immunoblotting.

Determination of the in Vivo Redox State of PSII Proteins

The in vivo redox state of PSII proteins was determined as in previousstudies (Ikegami et al., 2007; Tada et al., 2008) with some modifications. Totalleaf proteins were extracted from VIGS plants 3 weeks after infiltrationwithout reducing agents. The extracts were divided into three equal parts andincubated with the oxidizing agent (1 mM CuCl2), the reducing agent (10 mM

DTT), or the corresponding amount of distilled, deionized water at 23°C for30 min, and the proteins were then precipitated with 5% (w/v) TCA. Thedenatured protein pellets were washed twice with 100% acetone and resus-pended in 1 volume of 1% (w/v) SDS buffer and 1 volume of SDS-PAGE gelloading buffer without DTT. One part of each sample was incubated withAMS (Invitrogen) for 20 min at room temperature. Both AMS-treated anduntreated samples were separated by nonreducing SDS-PAGE and immuno-blotted with antibodies against PSII subunits. For a loading control, the thirdpart of the leaf protein extracts was boiled in reducing SDS-PAGE gel loadingbuffer containing DTT and probed with antibodies against PSII subunits andthe large subunit of Rubisco.

Supplemental Data

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

Supplemental Figure S1. Phylogenetic tree and amino acid sequence align-ment of TRX m proteins in Arabidopsis and rice.

Supplemental Figure S2. Phenotype and identification of trx m1trx m4double mutant plants.

Supplemental Figure S3. Expression of TRX m-FLAG proteins in Arabi-dopsis protoplasts and purification of His-TRX m proteins.

Supplemental Figure S4. Visualization of the redox status of intramolec-ular disulfide bonds in PSII subunits from VIGS plants.

Supplemental Table S1. Summary of TRX m silencing plants and TRX mmutant plants in this study.

Supplemental Table S2. Chlorophyll content and chlorophyll fluorescenceparameters in TRX m mutant plants.

Supplemental Table S3. List of primers used for vector construction in thisstudy.

ACKNOWLEDGMENTS

We thank Yule Liu (Tsinghua University) for the kind gift of TRV-basedVIGS vectors, Lianwei Peng (Institute of Botany, Chinese Academy of Sciences)for assistance in BN/SDS-PAGE and for discussing the manuscript, BernhardGrimm (Humboldt University Berlin) for critical reading of the manuscript, andXia Feng (Plant Protection Research Institute, Guangdong Academy of Agricul-tural Science) for supporting Dual-PAM 100.

Received September 11, 2013; accepted October 19, 2013; published October22, 2013.

LITERATURE CITED

Anbudurai PR, Mor TS, Ohad I, Shestakov SV, Pakrasi HB (1994) ThectpA gene encodes the C-terminal processing protease for the D1 proteinof the photosystem II reaction center complex. Proc Natl Acad Sci USA91: 8082–8086

Armbruster U, Zühlke J, Rengstl B, Kreller R, Makarenko E, Rühle T,Schünemann D, Jahns P, Weisshaar B, Nickelsen J, et al (2010) TheArabidopsis thylakoid protein PAM68 is required for efficient D1 bio-genesis and photosystem II assembly. Plant Cell 22: 3439–3460















Arsova B, Hoja U, Wimmelbacher M, Greiner E, Ustün S, Melzer M,Petersen K, Lein W, Börnke F (2010) Plastidial thioredoxin z interactswith two fructokinase-like proteins in a thiol-dependent manner: evi-dence for an essential role in chloroplast development in Arabidopsis andNicotiana benthamiana. Plant Cell 22: 1498–1515

Baker NR (2008) Chlorophyll fluorescence: a probe of photosynthesis invivo. Annu Rev Plant Biol 59: 89–113

Balmer Y, Koller A, del Val G, Manieri W, Schürmann P, Buchanan BB(2003) Proteomics gives insight into the regulatory function of chloro-plast thioredoxins. Proc Natl Acad Sci USA 100: 370–375

Bellafiore S, Ferris P, Naver H, Göhre V, Rochaix JD (2002) Loss ofAlbino3 leads to the specific depletion of the light-harvesting system.Plant Cell 14: 2303–2314

Benitez-Alfonso Y, Cilia M, San Roman A, Thomas C, Maule A, Hearn S,Jackson D (2009) Control of Arabidopsis meristem development bythioredoxin-dependent regulation of intercellular transport. Proc NatlAcad Sci USA 106: 3615–3620

Buchanan BB, Balmer Y (2005) Redox regulation: a broadening horizon.Annu Rev Plant Biol 56: 187–220

Burch-Smith TM, Schiff M, Liu Y, Dinesh-Kumar SP (2006) Efficientvirus-induced gene silencing in Arabidopsis. Plant Physiol 142: 21–27

Burnap RL, Qian M, Shen JR, Inoue Y, Sherman LA (1994) Role of di-sulfide linkage and putative intermolecular binding residues in thestability and binding of the extrinsic manganese-stabilizing protein tothe photosystem II reaction center. Biochemistry 33: 13712–13718

Cai W, Ma J, Chi W, Zou M, Guo J, Lu C, Zhang L (2010) Cooperation ofLPA3 and LPA2 is essential for photosystem II assembly in Arabidopsis.Plant Physiol 154: 109–120

Chen H, Zhang D, Guo J, Wu H, Jin M, Lu Q, Lu C, Zhang L (2006) APsb27 homologue in Arabidopsis thaliana is required for efficient repairof photodamaged photosystem II. Plant Mol Biol 61: 567–575

Chi YH, Moon JC, Park JH, Kim HS, Zulfugarov IS, Fanata WI, Jang HH,Lee JR, Lee YM, Kim ST, et al (2008) Abnormal chloroplast develop-ment and growth inhibition in rice thioredoxin m knock-down plants.Plant Physiol 148: 808–817

Chibani K, Tarrago L, Schürmann P, Jacquot JP, Rouhier N (2011) Bio-chemical properties of poplar thioredoxin z. FEBS Lett 585: 1077–1081

Chibani K, Wingsle G, Jacquot JP, Gelhaye E, Rouhier N (2009) Com-parative genomic study of the thioredoxin family in photosynthetic or-ganisms with emphasis on Populus trichocarpa. Mol Plant 2: 308–322

Collin V, Issakidis-Bourguet E, Marchand C, Hirasawa M, Lancelin JM,Knaff DB, Miginiac-Maslow M (2003) The Arabidopsis plastidial thio-redoxins: new functions and new insights into specificity. J Biol Chem 278:23747–23752

Collin V, Lamkemeyer P, Miginiac-Maslow M, Hirasawa M, Knaff DB,Dietz KJ, Issakidis-Bourguet E (2004) Characterization of plastidialthioredoxins from Arabidopsis belonging to the new y-type. PlantPhysiol 136: 4088–4095

Courteille A, Vesa S, Sanz-Barrio R, Cazalé AC, Becuwe-Linka N, Farran I,Havaux M, Rey P, Rumeau D (2013) Thioredoxin m4 controls photosyntheticalternative electron pathways in Arabidopsis. Plant Physiol 161: 508–520

Danielsson R, Suorsa M, Paakkarinen V, Albertsson PA, Styring S, AroEM, Mamedov F (2006) Dimeric and monomeric organization of pho-tosystem II. Distribution of five distinct complexes in the different do-mains of the thylakoid membrane. J Biol Chem 281: 14241–14249

Dekker JP, Boekema EJ (2005) Supramolecular organization of thylakoidmembrane proteins in green plants. Biochim Biophys Acta 1706: 12–39

Dietz KJ, Pfannschmidt T (2011) Novel regulators in photosynthetic redoxcontrol of plant metabolism and gene expression. Plant Physiol 155: 1477–1485

Dobakova M, Sobotka R, Tichy M, Komenda J (2009) Psb28 protein isinvolved in the biogenesis of the photosystem II inner antenna CP47(PsbB) in the cyanobacterium Synechocystis sp. PCC 6803. Plant Physiol149: 1076–1086

Eberhard S, Finazzi G, Wollman FA (2008) The dynamics of photosyn-thesis. Annu Rev Genet 42: 463–515

Erickson JM, Rahire M, Malnoe P, Girard-Bascou J, Pierre Y, Bennoun P,Rochaix JD (1986) Lack of the D2 protein in a Chlamydomonas rein-hardtii psbD mutant affects photosystem II stability and D1 expression.EMBO J 5: 1745–1754

Friso G, Giacomelli L, Ytterberg AJ, Peltier JB, Rudella A, Sun Q, Wijk KJ(2004) In-depth analysis of the thylakoid membrane proteome of Arabidopsisthaliana chloroplasts: new proteins, new functions, and a plastid proteomedatabase. Plant Cell 16: 478–499

Fu A, He Z, Cho HS, Lima A, Buchanan BB, Luan S (2007) A chloroplastcyclophilin functions in the assembly and maintenance of photosystemII in Arabidopsis thaliana. Proc Natl Acad Sci USA 104: 15947–15952

Göhre V, Ossenbühl F, Crèvecoeur M, Eichacker LA, Rochaix JD (2006)One of two alb3 proteins is essential for the assembly of the photo-systems and for cell survival in Chlamydomonas. Plant Cell 18: 1454–1466

Hajdukiewicz PT, Allison LA, Maliga P (1997) The two RNA polymerasesencoded by the nuclear and the plastid compartments transcribe distinctgroups of genes in tobacco plastids. EMBO J 16: 4041–4048

Holmgren A (1989) Thioredoxin and glutaredoxin systems. J Biol Chem264: 13963–13966

Ikegami A, Yoshimura N, Motohashi K, Takahashi S, Romano PG,Hisabori T, Takamiya K, Masuda T (2007) The CHLI1 subunit ofArabidopsis thaliana magnesium chelatase is a target protein of thechloroplast thioredoxin. J Biol Chem 282: 19282–19291

Ishihara S, Takabayashi A, Ido K, Endo T, Ifuku K, Sato F (2007) Distinctfunctions for the two PsbP-like proteins PPL1 and PPL2 in the chloro-plast thylakoid lumen of Arabidopsis. Plant Physiol 145: 668–679

Issakidis-Bourguet E, Mouaheb N, Meyer Y, Miginiac-Maslow M (2001)Heterologous complementation of yeast reveals a new putative functionfor chloroplast m-type thioredoxin. Plant J 25: 127–135

Iwata S, Barber J (2004) Structure of photosystem II and molecular ar-chitecture of the oxygen-evolving centre. Curr Opin Struct Biol 14:447–453

Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B,Cavet G, Linsley PS (2003) Expression profiling reveals off-target generegulation by RNAi. Nat Biotechnol 21: 635–637

Kapri-Pardes E, Naveh L, Adam Z (2007) The thylakoid lumen proteaseDeg1 is involved in the repair of photosystem II from photoinhibition inArabidopsis. Plant Cell 19: 1039–1047

Karamoko M, Cline S, Redding K, Ruiz N, Hamel PP (2011) Lumen ThiolOxidoreductase1, a disulfide bond-forming catalyst, is required for theassembly of photosystem II in Arabidopsis. Plant Cell 23: 4462–4475

Keren N, Ohkawa H, Welsh EA, Liberton M, Pakrasi HB (2005) Psb29, aconserved 22-kD protein, functions in the biogenesis of photosystem IIcomplexes in Synechocystis and Arabidopsis. Plant Cell 17: 2768–2781

Kieselbach T (2013) Oxidative folding in chloroplasts. Antioxid RedoxSignal 19: 72–82

Kirchsteiger K, Pulido P, González M, Cejudo FJ (2009) NADPH thiore-doxin reductase C controls the redox status of chloroplast 2-Cys per-oxiredoxins in Arabidopsis thaliana. Mol Plant 2: 298–307

Komenda J, Nickelsen J, Tichý M, Prásil O, Eichacker LA, Nixon PJ (2008)The cyanobacterial homologue of HCF136/YCF48 is a component of anearly photosystem II assembly complex and is important for both theefficient assembly and repair of photosystem II in Synechocystis sp. PCC6803. J Biol Chem 283: 22390–22399

Komenda J, Sobotka R, Nixon PJ (2012) Assembling and maintaining thephotosystem II complex in chloroplasts and cyanobacteria. Curr OpinPlant Biol 15: 245–251

König J, Baier M, Horling F, Kahmann U, Harris G, Schürmann P, DietzKJ (2002) The plant-specific function of 2-Cys peroxiredoxin-mediateddetoxification of peroxides in the redox-hierarchy of photosyntheticelectron flux. Proc Natl Acad Sci USA 99: 5738–5743

Lang JD, Haselkorn R (1989) Isolation, sequence and transcription of thegene encoding the photosystem II chlorophyll-binding protein, CP-47, inthe cyanobacterium Anabaena 7120. Plant Mol Biol 13: 441–457

Laugier E, Tarrago L, Courteille A, Innocenti G, Eymery F, Rumeau D,Issakidis-Bourguet E, Rey P (2013) Involvement of thioredoxin y2 in thepreservation of leaf methionine sulfoxide reductase capacity and growthunder high light. Plant Cell Environ 36: 670–682

Lemaire SD, Michelet L, Zaffagnini M, Massot V, Issakidis-Bourguet E(2007) Thioredoxins in chloroplasts. Curr Genet 51: 343–365

Lennartz K, Plücken H, Seidler A, Westhoff P, Bechtold N, Meierhoff K (2001)HCF164 encodes a thioredoxin-like protein involved in the biogenesis of thecytochrome b6f complex in Arabidopsis. Plant Cell 13: 2539–2551

Lima A, Lima S, Wong JH, Phillips RS, Buchanan BB, Luan S (2006) Aredox-active FKBP-type immunophilin functions in accumulation of thephotosystem II supercomplex in Arabidopsis thaliana. Proc Natl AcadSci USA 103: 12631–12636

Lindahl M, Mata-Cabana A, Kieselbach T (2011) The disulfide proteomeand other reactive cysteine proteomes: analysis and functional signifi-cance. Antioxid Redox Signal 14: 2581–2642


Wang et al.



Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP (2002) Tobacco Rar1, EDS1and NPR1/NIM1 like genes are required for N-mediated resistance totobacco mosaic virus. Plant J 30: 415–429

Lu Y, Hall DA, Last RL (2011) A small zinc finger thylakoid protein plays a role inmaintenance of photosystem II in Arabidopsis thaliana. Plant Cell 23: 1861–1875

Luo T, Fan T, Liu Y, Rothbart M, Yu J, Zhou S, Grimm B, Luo M (2012)Thioredoxin redox regulates ATPase activity of magnesium chelataseCHLI subunit and modulates redox-mediated signaling in tetrapyrrolebiosynthesis and homeostasis of reactive oxygen species in pea plants.Plant Physiol 159: 118–130

Ma J, Peng L, Guo J, Lu Q, Lu C, Zhang L (2007) LPA2 is required for efficientassembly of photosystem II in Arabidopsis thaliana. Plant Cell 19: 1980–1993

Martinez-Garcia JF, Monte E, Quail PH (1999) A simple, rapid andquantitative method for preparing Arabidopsis protein extracts for im-munoblot analysis. Plant J 20: 251–257

Meurer J, Meierhoff K, Westhoff P (1996) Isolation of high-chlorophyll-fluorescence mutants of Arabidopsis thaliana and their characterisationby spectroscopy, immunoblotting and northern hybridisation. Planta198: 385–396

Meyer Y, Reichheld JP, Vignols F (2005) Thioredoxins in Arabidopsis andother plants. Photosynth Res 86: 419–433

Montrichard F, Alkhalfioui F, Yano H, Vensel WH, Hurkman WJ,Buchanan BB (2009) Thioredoxin targets in plants: the first 30 years. JProteomics 72: 452–474

Motohashi K, Hisabori T (2006) HCF164 receives reducing equivalentsfrom stromal thioredoxin across the thylakoid membrane and mediatesreduction of target proteins in the thylakoid lumen. J Biol Chem 281:35039–35047

Motohashi K, Hisabori T (2010) CcdA is a thylakoid membrane protein re-quired for the transfer of reducing equivalents from stroma to thylakoidlumen in the higher plant chloroplast. Antioxid Redox Signal 13: 1169–1176

Motohashi K, Kondoh A, Stumpp MT, Hisabori T (2001) Comprehensivesurvey of proteins targeted by chloroplast thioredoxin. Proc Natl AcadSci USA 98: 11224–11229

Mulo P, Sirpio S, Suorsa M, Aro EM (2008) Auxiliary proteins involvedin the assembly and sustenance of photosystem II. Photosynth Res 98:489–501

Nelson N, Ben-Shem A (2004) The complex architecture of oxygenicphotosynthesis. Nat Rev Mol Cell Biol 5: 971–982

Nelson N, Yocum CF (2006) Structure and function of photosystems I andII. Annu Rev Plant Biol 57: 521–565

Nickelsen J, Rengstl B (2013) Photosystem II assembly: from cyanobacteriato plants. Annu Rev Plant Biol 64: 609–635

Nixon PJ, Michoux F, Yu J, Boehm M, Komenda J (2010) Recent advancesin understanding the assembly and repair of photosystem II. Ann Bot106: 1–16

Nott A, Jung HS, Koussevitzky S, Chory J (2006) Plastid-to-nucleus ret-rograde signaling. Annu Rev Plant Biol 57: 739–759

Ossenbühl F, Göhre V, Meurer J, Krieger-Liszkay A, Rochaix JD,Eichacker LA (2004) Efficient assembly of photosystem II in Chlamydo-monas reinhardtii requires Alb3.1p, a homolog of Arabidopsis ALBINO3.Plant Cell 16: 1790–1800

Peltier JB, Emanuelsson O, Kalume DE, Ytterberg J, Friso G, Rudella A,Liberles DA, Söderberg L, Roepstorff P, von Heijne G, et al (2002)Central functions of the lumenal and peripheral thylakoid proteome ofArabidopsis determined by experimentation and genome-wide predic-tion. Plant Cell 14: 211–236

Peng L, Ma J, Chi W, Guo J, Zhu S, Lu Q, Lu C, Zhang L (2006) LOW PSIIACCUMULATION1 is involved in efficient assembly of photosystem IIin Arabidopsis thaliana. Plant Cell 18: 955–969

Plücken H, Müller B, Grohmann D, Westhoff P, Eichacker LA (2002) TheHCF136 protein is essential for assembly of the photosystem II reactioncenter in Arabidopsis thaliana. FEBS Lett 532: 85–90

Porra R, Thompson WA, Kriedemann PE (1989) Determination of accurateextinction coefficients and simultaneous equations for assaying chloro-phylls a and b extracted with four different solvents: verification of theconcentration of chlorophyll standards by atomic absorption spectros-copy. Biochim Biophys Acta 975: 384–394

Rascher U, Nedbal L (2006) Dynamics of photosynthesis in fluctuatinglight. Curr Opin Plant Biol 9: 671–678

Rengstl B, Oster U, Stengel A, Nickelsen J (2011) An intermediate mem-brane subfraction in cyanobacteria is involved in an assembly networkfor photosystem II biogenesis. J Biol Chem 286: 21944–21951

Richter AS, Peter E, Rothbart M, Schlicke H, Toivola J, Rintamäki E,Grimm B (2013) Posttranslational influence of NADPH-dependent thio-redoxin reductase C on enzymes in tetrapyrrole synthesis. Plant Physiol162: 63–73

Robinson HH, Yocum CF (1980) Cyclic photophosphorylation reactionscatalyzed by ferredoxin, methyl viologen and anthraquinone sulfonate:use of photochemical reactions to optimize redox poising. Biochim BiophysActa 590: 97–106

Rochaix JD (2011) Assembly of the photosynthetic apparatus. Plant Physiol155: 1493–1500

Ruckle ME, DeMarco SM, Larkin RM (2007) Plastid signals remodel lightsignaling networks and are essential for efficient chloroplast biogenesisin Arabidopsis. Plant Cell 19: 3944–3960

Schägger H, Cramer WA, von Jagow G (1994) Analysis of molecularmasses and oligomeric states of protein complexes by blue native elec-trophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem 217: 220–230

Schürmann P, Buchanan BB (2008) The ferredoxin/thioredoxin system ofoxygenic photosynthesis. Antioxid Redox Signal 10: 1235–1274

Schürmann P, Jacquot JP (2000) Plant thioredoxin systems revisited. AnnuRev Plant Physiol Plant Mol Biol 51: 371–400

Serrato AJ, Pérez-Ruiz JM, Spínola MC, Cejudo FJ (2004) A novelNADPH thioredoxin reductase, localized in the chloroplast, which de-ficiency causes hypersensitivity to abiotic stress in Arabidopsis thaliana.J Biol Chem 279: 43821–43827

Shiina T, Tsunoyama Y, Nakahira Y, Khan MS (2005) Plastid RNA poly-merases, promoters, and transcription regulators in higher plants. Int RevCytol 244: 1–68

Shimada H, Mochizuki M, Ogura K, Froehlich JE, Osteryoung KW,Shirano Y, Shibata D, Masuda S, Mori K, Takamiya K (2007) Arabi-dopsis cotyledon-specific chloroplast biogenesis factor CYO1 is a proteindisulfide isomerase. Plant Cell 19: 3157–3169

Sirpiö S, Allahverdiyeva Y, Suorsa M, Paakkarinen V, Vainonen J,Battchikova N, Aro EM (2007) TLP18.3, a novel thylakoid lumen proteinregulating photosystem II repair cycle. Biochem J 406: 415–425

Sirpiö S, Khrouchtchova A, Allahverdiyeva Y, Hansson M, Fristedt R,Vener AV, Scheller HV, Jensen PE, Haldrup A, Aro EM (2008) At-CYP38 ensures early biogenesis, correct assembly and sustenance ofphotosystem II. Plant J 55: 639–651

Ströher E, Dietz KJ (2008) The dynamic thiol-disulphide redox proteome ofthe Arabidopsis thaliana chloroplast as revealed by differential elec-trophoretic mobility. Physiol Plant 133: 566–583

Sun X, Ouyang M, Guo J, Ma J, Lu C, Adam Z, Zhang L (2010) The thy-lakoid protease Deg1 is involved in photosystem-II assembly in Arabi-dopsis thaliana. Plant J 62: 240–249

Tada Y, Spoel SH, Pajerowska-Mukhtar K, Mou Z, Song J, Wang C, Zuo J,Dong X (2008) Plant immunity requires conformational changes [cor-rected] of NPR1 via S-nitrosylation and thioredoxins. Science 321:952–956

Takahashi S, Badger MR (2011) Photoprotection in plants: a new light onphotosystem II damage. Trends Plant Sci 16: 53–60

Tanz SK, Kilian J, Johnsson C, Apel K, Small I, Harter K, Wanke D,Pogson B, Albrecht V (2012) The SCO2 protein disulphide isomerase isrequired for thylakoid biogenesis and interacts with LHCB1 chlorophylla/b binding proteins which affects chlorophyll biosynthesis in Arabi-dopsis seedlings. Plant J 69: 743–754

Thormählen I, Ruber J, von Roepenack-Lahaye E, Ehrlich SM, Massot V,Hümmer C, Tezycka J, Issakidis-Bourguet E, Geigenberger P (2013)Inactivation of thioredoxin f1 leads to decreased light activation of ADP-glucose pyrophosphorylase and altered diurnal starch turnover inleaves of Arabidopsis plants. Plant Cell Environ 36: 16–29

Tovar-Méndez A, Matamoros MA, Bustos-Sanmamed P, Dietz KJ,Cejudo FJ, Rouhier N, Sato S, Tabata S, Becana M (2011) Peroxi-redoxins and NADPH-dependent thioredoxin systems in the model le-gume Lotus japonicus. Plant Physiol 156: 1535–1547

Vasudevan D, Fu A, Luan S, Swaminathan K (2012) Crystal structure ofArabidopsis cyclophilin38 reveals a previously uncharacterized im-munophilin fold and a possible autoinhibitory mechanism. Plant Cell 24:2666–2674

Vermaas WF, Williams JG, Rutherford AW, Mathis P, Arntzen CJ (1986)Genetically engineered mutant of the cyanobacterium Synechocystis6803 lacks the photosystem II chlorophyll-binding protein CP-47. ProcNatl Acad Sci USA 83: 9474–9477





Wang Q, Sullivan RW, Kight A, Henry RL, Huang J, Jones AM, Korth KL(2004) Deletion of the chloroplast-localized Thylakoid formation1 geneproduct in Arabidopsis leads to deficient thylakoid formation and var-iegated leaves. Plant Physiol 136: 3594–3604

Wei L, Guo J, Ouyang M, Sun X, Ma J, Chi W, Lu C, Zhang L (2010)LPA19, a Psb27 homolog in Arabidopsis thaliana, facilitates D1 proteinprecursor processing during PSII biogenesis. J Biol Chem 285: 21391–21398

Wellburn AR (1994) The spectral determination of chlorophyll a andchlorophyll b, as well as total carotenoids, using various solvents withspectrophotometers of different resolution. J Plant Physiol 144: 307–313

Wollman FA, Minai L, Nechushtai R (1999) The biogenesis and assemblyof photosynthetic proteins in thylakoid membranes1. Biochim BiophysActa 1411: 21–85

Yao N, Greenberg JT (2006) Arabidopsis ACCELERATED CELL DEATH2modulates programmed cell death. Plant Cell 18: 397–411

Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatilecell system for transient gene expression analysis. Nat Protoc 2: 1565–1572

Zhai Z, Sooksa-nguan T, Vatamaniuk OK (2009) Establishing RNA in-terference as a reverse-genetic approach for gene functional analysis inprotoplasts. Plant Physiol 149: 642–652

Zhang D, Zhou G, Liu B, Kong Y, Chen N, Qiu Q, Yin H, An J, Zhang F,Chen F (2011) HCF243 encodes a chloroplast-localized protein involvedin the D1 protein stability of the Arabidopsis photosystem II complex.Plant Physiol 157: 608–619

Zhang L, Paakkarinen V, van Wijk KJ, Aro EM (2000) Biogenesis of thechloroplast-encoded D1 protein: regulation of translation elongation,insertion, and assembly into photosystem II. Plant Cell 12: 1769–1782


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Evidence for a Role of Chloroplastic m-Type … for a Role of Chloroplastic m-Type Thioredoxins ......

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