Accepted Article

Received Date : 12-Dec-2014 Accepted Date : 31-Mar-2015 Article type : Original Article

A land plant-specific thylakoid membrane protein contributes to photosystem II maintenance in Arabidopsis thaliana

Jun Liu1 and Robert L. Last1,2

1

Departments of Biochemistry and Molecular Biology and 2Plant Biology, Michigan State

University, East Lansing, Michigan 48824, United States of America

*For correspondence ([email protected].)

Corresponding author: Dr. Robert L. Last

Mailing address: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, 603 Wilson Road, 301 Biochemistry Building, 48824-1319

Phone number: 001-517-432-3278 Fax number: 001-517-353-9334 E-mail: [email protected].

Running head: A PSII-associated thylakoid membrane protein MPH1

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/tpj.12845 This article is protected by copyright. All rights reserved.

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SUMMARY The structure and function of photosystem II (PSII) is highly susceptible to photooxidative damage, which is induced by high fluence or fluctuating light. However, many of the mechanistic details of how PSII homeostasis is maintained under photoinhibitory light remain to be defined. We describe analysis of Arabidopsis thaliana gene At5g07020, which encodes an unannotated integral thylakoid membrane protein. Loss of the protein causes altered PSII function under high irradiance light, and hence is named Maintenance of PSII under High light1 (MPH1). The MPH1 protein co-purifies with PSII core complexes and coimmunoprecipitates core proteins. Consistent with a role in PSII structure, mph1 mutant PSII complexes (supercomplexes, dimers, and monomers) are less stable in plants subjected to photoinhibitory light. Accumulation of PSII core proteins is compromised under these conditions in the presence of translational inhibitors. This is consistent with the hypothesis that the mutant has enhanced PSII protein damage rather than defective repair. These data are consistent with the distribution of MPH1 protein in grana and stroma thylakoids and its interaction with PSII core complexes. Taken together, these results strongly suggest a role for MPH1 in the protection and/or stabilization of PSII under high-light stress in land plants.

Keywords: high-light stress, Photosystem PSII, photoinhibition, thylakoid membranes, PSII damage-repair cycle, Arabidopsis thaliana

INTRODUCTION

The energy of light is essential for photosynthesis; yet harnessing this power can damage the large and well tuned photosynthetic machinery, especially the oxygen-evolving photosystem II (PSII) complex (Prasil et al., 1992; Aro et al., 1993). Photoinhibition results when the light-induced photooxidative damage to the PSII reaction center is irreversible, reducing photosynthetic efficiency, and reducing plant growth and productivity (Eberhard et al., 2008; Nishiyama and Murata, 2014). A variety of constitutive and inducible photoprotective measures evolved to avoid or minimize photoinhibition in plants. These include avoidance This article is protected by copyright. All rights reserved.

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mechanisms such as light screening and movement of leaves and chloroplasts, dissipation of excess absorbed light energy as heat (non-photochemical quenching [NPQ]) (Niyogi, 1999; Takahashi and Badger, 2011), protective mechanisms such as antioxidant scavenging systems, alternative electron transport pathways, retrograde signals, and systemic acquired acclimation (Eberhard et al., 2008; Li et al., 2009). Despite the operation of these multifaceted photoprotective mechanisms, photooxidative damage to PSII inevitably occurs and is exacerbated by a variety of environmental conditions, including rapid changes in light levels (Komenda et al., 2012; Nath et.al., 2013; Nickelsen and Rengstl, 2013; Nishiyama and Murata, 2014). Damage to PSII core proteins is repaired through a multistep process that includes movement of damaged proteins from grana- to stroma-thylakoids, proteolytic degradation and replacement of damaged protein subunits and movement of the repaired PSII machinery back to perform photosynthesis in grana-thylakoid membranes (Aro et al., 2005; Herbstova et al., 2012; Komenda et al., 2012; Nath et.al., 2013; Puthiyaveetil et al., 2014).

While great progress has been achieved regarding the PSII photoinhibition-repair cycle mechanism in recent years (Mulo et al., 2008; Nath et al, 2013; Nickelsen and Rengstl, 2013; Järvi et al., 2015), the details of this intricate process, and the proteins that help stabilize PSII under photodamaging conditions remain to be elucidated. Moreover, new auxiliary protein factors associated with PSII participating in the turnover, assembly and organization continue to be discovered, and some are not conserved among all oxygenic organisms (Nixon et al., 2010). To identify novel chloroplast proteins with roles in the regulation of photoinhibitionrepair cycle of PSII, we took advantage of results from the Chloroplast 2010 Project (http://www.plastid.msu.edu/). In this project over 5200 Arabidopsis thaliana T-DNA mutants with insertions in nuclear-encoded genes predicted to be plastid targeted were screened and analyzed for a variety of mutant phenotypes. In vivo chlorophyll fluorescence was used in this screen because it is a noninvasive technique to assess the status of photosynthetic processes (Lu et al., 2008, 2011b; Ajjawi et al., 2010; Savage et al., 2013). This screen led to identification of LQY1 (LOW QUANTUM YIELD OF PSII 1), a small thylakoid zinc finger protein, which was revealed to be involved in repair and reassembly of PSII (Lu et al., 2011a; Lu, 2011). The screen identified PSB33, a recently described PSII-associated thylakoid This article is protected by copyright. All rights reserved.

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membrane protein that ensures proper maintenance of the PSII-LHCII supercomplexes (Fristedt et al., 2015).

This phenomics project also identified SALK_143436, a mutant of At5g07020, which showed an altered chlorophyll fluorescence phenotype under photoinhibitory light conditions. Here, we report biochemical analysis of this protein (MPH1, Maintenance of PSII under High light 1) and phenotypic characterization of two independent mutants defective in the MPH1 gene. These results indicate that MPH1 is found across land plants, and is an intrinsic thylakoid membrane protein that is associated with PSII core proteins in A. thaliana. Losses of function mutants of MPH1 have a variety of PSII-associated defects, which collectively suggest that the protein acts to protect PSII against photodamage and/or stabilize PSII under photoinhibitory stress.

RESULTS

MPH1 is annotated as a proline rich protein and has homologs across land plants To ask whether MPH1 is evolutionarily conserved among oxygenic photosynthetic organisms, BLAST searches were performed using the MPH1 full-length protein sequence (http://www.phytozome.net). Homologs were found in the sequenced genomes of phylogenetically diverse land plants, including the eudicots poplar (Populus trichocarpa), castor bean (Ricinus communis), tomato (Solanum lycopersicum), and soybean (Glycine max); the monocots rice (Oryza sativa), maize (Zea mays), and sorghum (Sorghum bicolor); and the bryophyte Physcomitrella patens (Figures 1 and S1). In contrast, MPH1 homologs were not detected in algae or prokaryotes, suggesting that MPH1 is conserved in land plants. The homologs in other land plants share all domains predicted for the Arabidopsis MPH1: an N-terminal chloroplast transit peptide, single-pass transmembrane domain and conserved interspersed proline residues.

MPH1 is a chloroplast thylakoid intrinsic membrane protein A. thaliana MPH1 is annotated as a 235-amino acid protein of unknown function. In silico This article is protected by copyright. All rights reserved.

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analyses (http://suba.plantenergy.uwa.edu.au/; http://aramemnon.uni-koeln.de/) predict that MPH1 harbors an N-terminal chloroplast signal sequence of 38 amino acids and has a single transmembrane domain (Schwacke et al., 2003; Tanz et al., 2013). These predictions are supported by published proteomic studies that detected MPH1 in chloroplast thylakoids (Peltier et al., 2004; Giacomelli et al., 2006). To experimentally test the localization, proteins prepared from whole leaves, chloroplasts, and sub-chloroplast compartments were immunodetected by an MPH1 polyclonal antibody. As shown in Figure 2a, MPH1 protein was found in total cell extracts, intact chloroplasts and thylakoid membranes but not in the stromal-enriched fraction, similar to the thylakoid membrane protein Lhcb2. To determine whether MPH1 is an intrinsic or extrinsic membrane protein, sonicated thylakoid membranes were incubated with chaotropic salts or under alkaline conditions known to release peripheral proteins from thylakoids. As shown in Figure 2b, MPH1 fractionated similarly to the integral membrane protein D2 but not like the peripheral protein PsbO, supporting the hypothesis that MPH1 is an intrinsic-membrane thylakoid protein. Further subfractionation of thylakoid membranes into fractions of grana core- and grana margin-membranes and stroma lamellae revealed MPH1 distributed among all the three thylakoid fractions (Figure 2c); consistent with the hypothesis that MPH1 protein has roles in all of these locations.

mph1 mutant plants are susceptible to high-light treatment The location of MPH1 protein in photosynthetic membranes suggests a potential role for MPH1 in photosynthesis. To test this hypothesis, we analyzed photosynthetic function of two independent mutants. The SALK_143436 line (now named mph1-1), containing a T-DNA inserted in the first intron of MPH1 (Figure 3a), was found in the Chloroplast 2010 Project (Lu et al., 2011b; Ajjawi et al., 2010) to have very low Fv/Fm (a indicator of maximum photochemical efficiency of PSII), but has no NPQ phenotype following 3-h of photoinhibitory light (1000 μmol m-2s-1) treatment. A second homozygous mutant, SAIL_1284_E04 (now named as mph1-2), has a T-DNA insertion in the second intron of MPH1 (Figure 3a). Neither MPH1 transcript nor MPH1 protein was detectable in either mutant (Figure S2), showing that these are strong loss of function mutants, and demonstrating the specificity of the α-MPH1 antiserum. This article is protected by copyright. All rights reserved.

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To test whether inactivation of the MPH1 gene is responsible for the high-light phenotype originally observed in the mph1-1 mutant (Lu et al., 2011b; Ajjawi et al., 2010), both mutants were subjected to 3-h high-light treatment (Figure 3b). Fv/Fm values were statistically significantly reduced in both mutants compared to the wild type after high-light treatment (Figures 3 and S3). In contrast, the growth phenotype and Fv/Fm of the mutants were identical to the wild type propagated under growth light conditions (100 μmol m-2 s-1), suggesting that MPH1 is dispensable for optimal PSII function under 'normal' lab growth light conditions. In contrast to the wild type, Fv/Fm did not return to the pre-photoinhibitory levels in either mutant following a 2-d recovery under growth light conditions (Figures 3c). Moreover, both mutants exhibited higher qI (photoinhibitory quenching) and Fo (minimal fluorescence with PSII reaction centers open) but lower qE (energy-dependent quenching) after 3-h high-light treatment (Figure 3c), suggesting that mph1 mutants might be impaired in photoprotection of PSII under photoinhibitory light. Taken together, these results show that the mph1 photosynthetic phenotype is due to the loss of At5g07020.

Association of MPH1 protein with PSII The observation that MPH1 protein is a thylakoid membrane protein, and that lack of MPH1 confers a marked defect in PSII activity under photoinhibitory light conditions, suggests that MPH1 might play a photoprotective role in photosynthesis by interaction with PSII proteins. To test this hypothesis, thylakoid samples from leaves of plants grown either under normal growth light or shifted to high-light treatment were resolved by two-dimensional (2D) BN/SDS-PAGE and immunodetected with antibodies against PSII core proteins and MPH1. Additionally, to examine the specificity of possible interaction of MPH1 with PSII, a psbw mutant was employed as a control. PsbW is low-molecular-weight protein exclusive to PSII of photosynthetic eukaryotes, and loss of PsbW destabilizes the supramolecular organization of PSII (Shi and Schröder, 2004; García-Cerdán et al., 2011). As shown in Figure 4a, under growth light conditions a substantial fraction of MPH1 was found to comigrate with PSII monomers and a small portion with unassembled proteins in the wild type and psbw mutant; this indicates that MPH1 associates with PSII monomeric complexes. In thylakoids isolated from leaves of wild type following high-light treatment, most of the MPH1 protein was found This article is protected by copyright. All rights reserved.

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in the region of the gel corresponding to PSII supercomplexes, with a small amount comigrating with PSII monomers (Figure 4a). In contrast, the psbw mutant samples had more of the MPH1 protein comigrating with unassembled proteins and a small fraction in the region of PSII monomers (Figure 4a). The results suggest that MPH1 copurifies with PSII supercomplexes under photoinhibitory light and this binding is disrupted in the psbw mutant due to drastic reduction of PSII supercomplexes.

To

cross-validate

the

association

of

MPH1

with

PSII

protein

complexes,

coimmunoprecipitation (Co-IP) assays were performed on non-ionic detergent (dodecyl-β-Dmaltopyranoside; DM) solubilized thylakoid membranes using α-MPH1 antibody. Consistent with the hypothesis that MPH1 protein associates with the PSII core proteins, D1, D2, CP43, and CP47 co-precipitated using anti-MPH1 antibody, while PSI core protein PsaA did not (Figure 4b). The co-IP assay was also used to test MPH1 interaction with the thylakoid membrane protein LQY1, which is another land plant-specific protein associated with PSII. LQY1 was of interest because of evidence that it plays a role in PSII repair and reassembly following photodamage and also interacts with CP43 and CP47 (Lu et al., 2011a). We found no evidence for LQY1 co-precipitation with anti-MPH1 antibody (Figure 4b).

Loss of MPH1 affects PSII complex abundance under high light The observation that the MPH1 protein co-purifies with PSII complexes and core proteins and the mutant has altered Fv/Fm under high light led us to examine PSII in mph1 mutants. Immunoblot analysis of a variety of photosynthetic proteins was conducted on total leaf protein extracts from the wild type and mph1 mutants under growth light and after a 3-h highlight treatment. In contrast to the growth light conditions, mph1 mutants showed modest but reproducible decreases in the amounts of the PSII core subunit proteins D1, D2, CP43, and CP47 compared with wild type (average reductions of 40, 27, 29 and 42%, respectively; Figures 5 and S4). In contrast, MPH1 abundance was increased by ~ 30% in wild-type leaves after a 3-h high-light treatment, suggesting that MPH1 synthesis or stability might increase under photoinhibitory conditions (Figures 5 and S4).

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The reductions seen for PSII core proteins were not observed for proteins in other photosynthetic complexes. For example, no high light-induced decreases were observed in mph1 mutants compared with wild type for PSII oxygen evolving complex protein PsbO, PSII light-harvesting complex Lhcb2, PSI reaction center protein PsaA and antenna protein Lhca2, ATP synthase subunit CF1β, Cytochrome b6f complex protein Cyt.f or ribulose biphosphate carboxylase large subunit protein RbcL (Figures 5 and S4). In addition, the decrease of PSII core proteins detected in the mph1 mutant under high light was not caused by limitation in the abundance of corresponding transcripts of PSII as analyzed by both RTPCR and quantitative real-time RT-PCR (Figure S5 and Table S1). Collectively, these results demonstrate that the absence of MPH1 specifically causes reduction in the levels of PSII core subunits after high-light treatment, suggesting that MPH1 might be involved in protecting PSII reaction center against high-light stress.

We tested the hypothesis that these reductions in PSII core protein concentrations in mph1 mutants under high light are associated with altered PSII protein complex formation. BNPAGE fractionation was performed on thylakoid membrane protein complexes obtained using DM (Figure 6a), and the complexes were analyzed by immunoblotting with antibodies specific for the PSII core subunits D1 and CP43 (Figure 6b). These experiments revealed a modest but consistent reduction in PSII-LHCII supercomplexes, PSII dimer and monomer in thylakoids in mph1 high-light treated mutants relative to their wild-type counterparts (Figure 6), suggesting that lack of MPH1 disturbs the stability of PSII complexes under high-light stress.

PSII core proteins are highly phosphorylated under high light conditions, and there is evidence that this light-induced phosphorylation especially of D1 protein is of relevance to the regulation of PSII core protein turnover upon photodamage (Dannehl et al., 1995; Koivuniemi et al., 1995; Georgakopoulos and Argyroudi-Akoyunoglou, 1998; Yamamoto et al., 2008; Tikkanen and Aro, 2012; Kato and Sakamoto, 2014). This led us to examine whether the defects observed in PSII in mph1 mutants are correlated with the in vivo phosphorylation status of PSII. As expected relatively low phosphorylation of PSII core This article is protected by copyright. All rights reserved.

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Figure 7

Figure 7. Recovery from photoinactivation and turnover of PSII proteins in photoinhibition assays. (a) Time-course of recovery following photoinactivation of PSII. Wild-type and mph1 mutant plants grown under normal light (100 μmol m-2 s-1) were transferred to high light (1000 μmol m-2 s-1) for 3 h, and subsequently shifted to continuous dim light (20 μmol m-2 s-1) to allow recovery, and Fv/Fm values were monitored. (b) Degradation of the reaction center D1 protein of PSII in the presence or absence of the chloroplast protein synthesis inhibitor lincomycin under photoinhibitory conditions. Detached leaves were incubated in water or lincomycin solution (1 mM) in dark overnight and subsequently illuminated under high light (1000 μmol m-2 s-1) for 2, or 4 h. The levels of D1 were measured by SDS-PAGE and immunoblotting with D1 antibody. Data are presented as means ± SD (n = 3).

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during the course of high-light illumination. Without lincomycin, levels of PSII core proteins D1 and CP47 gradually declined both in the wild type and the mph1 mutant during 4-h highlight treatment, though the reduction was higher in the mph1 mutant (Figures 7b and S7). In contrast, the rate of D1 protein degradation was especially pronounced in the presence of lincomycin in the mph1 mutant relative to the wild type, and CP47 degradation also increased (Figures 7b and S7). In contrast, markers for PSI, ATPase and Cyt.b6f complexes were unaffected in mph1, as was the large subunit of RuBisCo (Figure S7). These data are consistent with the hypothesis that accelerated degradation of photodamaged PSII core subunits results in the photoinhibition displayed in mph1 mutants under high light.

To simultaneously assess PSII stability, photodamage and repair, we performed 2D BN/SDSPAGE followed by either Coomassie blue staining or immunoblot analyses after high-light treatment. As shown in Figure 8, the levels of PSI complex, ATP synthase, or Cyt.b6f complex in mph1 mutants were comparable to those in wild-type plants. In contrast, proportions of the PSII core subunits D1, D2, CP43, and CP47 in various PSII complexes (PSII supercomplexes, PSII dimer and monomer) were generally reduced in mph1 mutants relative to the wild-type plants, and no lower order of PSII complexes, such as RC47 or PSII monomer, accumulated in the mutant after high light (Figure 8). These results are consistent with the hypothesis that disassembly and/or reassembly of PSII complexes during PSII repair cycle are normal, but that photodamaged PSII complexes are less stable in mph1 mutants.

DISCUSSION

In recent years considerable progress has been made in understanding the biogenesis and assembly of PSII in photosynthetic eukaryotes and cyanobacteria (Chi, et al., 2012; Komenda et al., 2012). The damage and rapid degradation of PSII reaction center D1 protein in response to photoinhibitory light has been intensively studied (Aro et al., 1993; Yamamoto et al., 2008; Nixon et al., 2010; Kato and Sakamoto, 2014). In addition to D1, the D2, CP43 and PsbH subunits also show faster degradation compared to the other PSII subunits (Jansen et This article is protected by copyright. All rights reserved.

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al., 1996; Bergantino et al., 2003; Rokka et al., 2005). Much remains to be learned about the molecular mechanisms influencing maintenance and turnover of PSII reaction center proteins and complexes under high irradiance light and the regulation of the PSII photoinhibitionrepair cycle, though it is clear that a suite of auxiliary protein factors are needed to aid in this seemingly elaborate process (Mulo et al., 2008; Goral et al., 2010; Nixon et al., 2010; Nath et.al., 2013; Nickelsen and Rengstl, 2013; Bhuiyana et al., 2015; Järvi et al., 2015). In this report, we describe analysis of the nuclear-encoded thylakoid transmembrane protein MPH1, and provide evidence that it contributes to the maintenance of PSII homeostasis under photoinhibitory stress in land plants.

The mph1 mutants are defective in PSII maintenance under high light Multiple lines of evidence presented in this study reveal that the mph1 mutants are defective in maintaining proper function of PSII under photoinhibitory light. Under permissive growth light conditions, loss of function of MPH1 protein did not affect the development and maximal photochemical efficiency of PSII (Fv/Fm; Figures 3 and S3), and photosynthetic proteins accumulated to wild-type levels (Figures 5 and S4) and assembled into functional membrane complexes (Figures 6 and 8). These results suggest that MPH1 is dispensable for optimal PSII function under 'normal' laboratory growth light conditions. In contrast, maximal PSII activity was significantly decreased in the mph1 mutant compared to the wild type under high light conditions (Figures 3 and S3). Further biochemical assays with BN-PAGE, immunoblotting of BN gels and 2D BN/SDS gels using antibodies directed against representative subunits of photosynthetic membrane complexes showed that the amounts of PSII supercomplexes, PSII dimers and monomers were modestly reduced in the mph1 mutant shifted to high-light conditions (Figures 6 and 8). The effect appears to be specific to PSII since the levels of PSI complexes, Cyt.b6f complex, and ATP synthase were largely unaltered (Figures 6 and 8). That the PSII reaction center is defective in mph1 mutants under high light is further supported by the observation that steady state levels of the PSII core proteins D1, D2, CP43, and CP47 were reduced in mph1 mutants. In contrast, no decreases were observed for PsbO or LHCII (Figures 5 and S4). The concerted decrease of PSII core proteins was also observed in high light-sensitive lqy1, hhl1 and cyp38 mutants (Fu et al., 2007; Sirpiö et al., This article is protected by copyright. All rights reserved.

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2008; Lu et al., 2011a; Jin et al., 2014). These data provide evidence that MPH1 protein is required for maintaining normal levels of PSII reaction center complex under high-light irradiance, leading us to hypothesize that MPH1 plays a role in the maintenance of PSII reaction center under high-light stress.

PSII core phosphorylation has been proposed to regulate photoprotection at high light intensities (Dannehl et al., 1995; Koivuniemi et al., 1995; Georgakopoulos and ArgyroudiAkoyunoglou, 1998; Kanervo et al., 2005; Yamamoto et al., 2008; Tikkanen and Aro, 2012; Kato and Sakamoto, 2014). We found reduced total PSII core protein phosphorylation in mph1 mutants relative to the wild type under high light (Figure S6). This phenotype is shared with the high light sensitive psb33, psbw and cyp38 mutants (Sirpiö et al., 2008; GarcíaCerdán et al., 2011; Fristedt et al., 2015).

Other lines of evidence support a role for MPH1 in the maintenance of PSII reaction centers under photoinhibitory light conditions. For example, the initial fluorescence level Fo, a parameter inversely related to the efficiency of energy transfer from LHCII to open PSII reaction centers (de Bianchi et al., 2008), was significantly increased in mph1 mutants compared to wild type after high-light treatment (Figure 3c). This could be the result of reduction in the trapping efficiency of PSII reaction centers in mph1 (Kovács et al., 2006; Johnson and Ruban, 2010) since the mutants showed reduced PSII reaction center proteins and no change in LHCII protein levels (Figures 5 and S4). This elevation of Fo is consistent with significantly decreased Fv/Fm in mph1 mutants after high light-treatment (Figure 3b, c and S3). In addition, the slowly forming and relaxing NPQ component qI was significantly enhanced in mph1 mutants compared to wild type after high-light treatment (Figure 3c). It should be noted that the term qI was originally ascribed solely to the photoinhibitory damage of PSII reaction centers (Krause, 1988; Aro et al., 1993; Long et al., 1994; Murata et al., 2007). It is increasingly clear that this sustained quenching encompasses many processes that lead to long-lasting damage, inactivation, or down regulation of PSII (Jahns and Holzwarth, 2012; Ruban et al., 2012). Because Fo was significantly higher and the steady state levels of PSII reaction center proteins were lower in mph1 mutants (Figures 3c, 5 and S4), the increase This article is protected by copyright. All rights reserved.

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in qI could be primarily due to the higher requirement for the degradation and replacement of photodamaged D1 protein in the mutants under high light (Aro et al., 1993; Müller et al., 2001; Murata et al., 2007). Conversely, the major and reversible NPQ component qE was significantly reduced in mph1 mutants relative to wild type after high-light treatment (Figure 3c). Collectively, the concomitant elevation of qI and Fo and reduction of qE are consistent with the hypothesis that mph1 mutants are defective in PSII photoprotection under high-light stress.

In vitro photoinhibitory assays coupled with immunoblot analyses further demonstrated that degradation of PSII core proteins especially of D1 was more rapid in the presence than in the absence of lincomycin in the mph1 mutants relative to the wild type (Figures 7b and S7). These results strongly suggest that acceleration of photodamage, rather than deceleration of repair, is responsible for the severe photoinhibition observed in mph1 mutants. We observed that these mutants suffered more damage to PSII compared to wild type during photoinhibitory light treatment (Figures 3c and 7); despite a faster recovery rate during the first four hours, the mutants were not restored to wild-type Fv/Fm levels after shift to continuous dim light conditions for ten hours (Figure 7a). Taken together, these data suggest that loss of MPH1 renders plants suffer from severe damage to PSII under high-light stress.

MPH1 protein interacts with PSII core proteins The role of MPH1 in PSII maintenance is reinforced by its interaction with PSII complexes and distribution in different thylakoid membrane subfractions. Under normal growth light conditions, the bulk of MPH1 comigrated with PSII monomers in 2D BN/SDS PAGE both in the wild type and psbw mutant, in a position corresponding to higher molecular masses than free MPH1 (Figure 4a). Much of the detectable MPH1 protein comigrated with PSII supercomplexes under photoinhibitory light in the wild type (Figure 4a). This high light increase in MPH1-associated PSII supercomplexes was not seen in the psbw mutant, which was severely defective in accumulation of PSII supercomplexes (Figure 4a). MPH1 was detected in the position of PSII monomers irrespective of light fluence. This suggests that MPH1 is generally associated with PSII monomers, and becomes more stably associated with This article is protected by copyright. All rights reserved.

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PSII supercomplexes under high light. These blue native gel immunoblot results correlate with an overall increase in MPH1 protein in wild type following shift to high light (Figures 5 and S4).

Co-IP assays further demonstrated that MPH1 interacted specifically with core subunits of PSII (D1, D2, CP43, and CP47) but not with PSI (Figure 4b). Co-IP assays also showed that MPH1 didn’t interact with LQY1, although MPH1 has similar phylogenetic distribution to LQY1 and HHL1 and losses of function mutants display similar high light-sensitive phenotypes (Lu et al., 2011a; Jin et al., 2014). This suggests that MPH1 could have evolved a role distinct with LQY1 and HHL1 in maintaining normal PSII function under high-light stress in land plants. The association of MPH1 with PSII complexes is compatible with distribution of MPH1 among grana core- and grana margin-membranes and stroma lamellae of thylakoids (Figure 2c). However, the pattern of MPH1 distribution is quite different from both LQY1 and HHL1; MPH1 does not show the enrichment in stroma lamellae found in proteins involved in repair (Mulo et al., 2008; Nixon et al., 2010; Komenda et al., 2012; Puthiyaveetil et al., 2014). This observation strengthens the hypothesis that MPH1 is involved in protection of PSII against damage rather than in the repair of PSII (Figures 3c, 7 and S7).

The hypothesis that MPH1 plays a role in PSII maintenance is also supported by the observation that the levels of PSII core protein subunits in different PSII complexes (supercomplexes, dimers, and monomers) were generally reduced in the mph1 mutant compared to the wild type under photoinhibitory light (Figure 8). This suggests that the overall stability of PSII complexes is compromised upon lack of MPH1 under high light. This hypothesis is supported by the observation that the rates of degradation of PSII core proteins under high light were faster either in the presence or absence of lincomycin in the mph1 mutant relative to the wild type (Figures 7b and S7). It is noteworthy that MPH1 has a similar distribution pattern as CYP38, which functions in the stability and biogenesis of PSII (Fu et al., 2007; Sirpiö et al., 2008). Both MPH1 and CYP38 associate mainly with the monomeric PSII complex in BN-PAGE (Sirpiö et al., 2008). Taken together, these data are consistent This article is protected by copyright. All rights reserved.

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with the hypothesis that MPH1 is involved in the protection of PSII against photodamage and in the stable accumulation of PSII, presumably via interaction with protein complexes of PSII and/or with other regulatory factors for sustaining normal PSII activity such as CYP38. The results of this study expand our understanding of the dynamic regulation of the finetuned photodamage-repair cycle of PSII. Phylogenetic analysis of MPH1 homologs indicates that MPH1 is found in land plant genomes, but not conserved among other oxygenic organisms (Figures 1 and S1), which is similar to previously described phylogenetic distribution of LQY1 and HHL1 (Lu et al., 2011a; Jin et al., 2014). Occurrence of a suite of auxiliary factors specific to land plants may result from the higher requirement for photoprotection of damage-prone PSII against excess light during the transition from aquatic to terrestrial conditions (Jin et al., 2014). The continued identification of auxiliary protein factors playing roles in the maintenance of PSII activity suggests that much remains to learn about the photodamage-repair cycle of PSII.

EXPERIMENTAL PROCEDURES Plant Material and Growth Conditions Arabidopsis thaliana ecotype Columbia T-DNA insertion line mph1-1 (SALK_143436) was identified

as

having

altered

Fv/Fm

in

the

Chloroplast

2010

Project

(http://www.plastid.msu.edu/) while mph1-2 (SAIL_1284_E04) was obtained from the Arabidopsis Biological Resource Center (ABRC; http://www.arabidopsis.org/ ) and homozygous mph1-2 was identified by genomic PCR with primers specific for MPH1 (Table S2). The homozygous mutant lines of mph1-1 and mph1-2 used in this study were deposited in the ABRC and have accession numbers CS68902 and CS68903, respectively. Homozygous psbw mutant (SAIL_885_A03) was also identified in the Chloroplast 2010 Project. Seeds sown on soil were stratified in the dark at 4ºC for 3-dand then grown in a controlled growth chamber as previously described, except that the photoperiod was 16-h/8-h rather than 12h/12-h (Lu et al., 2008, 2011b; Ajjawi et al., 2010). Unless otherwise noted, all plants used in this study were 4 weeks old. For high-light treatment, plants were placed in an imaging chamber under an illumination of 1000 μmol m-2 s-1 for 3 h while maintaining the temperature at 21ºC. This article is protected by copyright. All rights reserved.

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Chlorophyll fluorescence measurements Chlorophyll fluorescence images of intact plants were obtained from a custom-designed plant imager chamber, and imaging was performed as described by Attaran et al., (2014). After an adaptation of plants in the dark for 30 min, minimum fluorescence (Fo) was determined by a weak red light. Fluorescence quenching was induced by 10 min of actinic illumination with white light. The maximal fluorescence in the dark-adapted state (Fm) and in the light-adapted state (Fm′) and after 10 min of dark relaxation following the actinic illumination (Fm′′) were determined by a saturating pulse of light applied at 2-min intervals (Damkjær et al., 2009; de Bianchi et al., 2011). Fv/Fm, qI, and qE were calculated according to the following equations (Farber et al., 1997; Kovács et al., 2006): Fv/Fm = (Fm-Fo)/Fm, qI = (Fm-Fm′′)/Fm′′, qE=Fm/Fm′Fm/Fm′′. Maximum fluorescence images were analyzed by ImageJ software (Schneider et al., 2012).

Production of anti-MPH1 polyclonal antibodies Affinity-purified anti-MPH1 polyclonal antibodies were generated by Pierce Biotechnology. A 18-amino acid peptide (corresponding to amino acids 175–192 of MPH1) with additional N-terminus Cys residue, CLPETMASEAQPEASSVPT, was synthesized, conjugated with keyhole limpet hemocyanin, and used to raise antibody against MPH1 protein in rabbits. Immunoblot and blue native (BN)-PAGE Analyses For immunoblot analysis, total protein samples of Arabidopsis rosette leaves were prepared as described (Martínez-García et al., 1999). Rabbit primary antibodies were purchased from Agrisera (Vännäs, Sweden). Immunoblotting was performed according to standard techniques by probing with specific antibodies after electroblotting SDS-PAGE onto Amersham nitrocellulose membranes (GE Healthcare). Primary antibodies were diluted 20,000-fold (antibodies against D1 and D2), 10,000-fold (PsbO and Lhcb2), 5000-fold (CP47, Lhca2, cytochrome f, CF1β and RbcL), 2500-fold (CP43 and PsaA), or 200-fold (MPH1), and signals from Goat Anti-Rabbit IgG (H+L)-HRP Conjugate were visualized using Clarity Western ECL Substrate and analyzed by Image LabTM (Bio-Rad). Protein accumulation was normalized to the amount of CBB-stained LHCII of PSII as internal standard and was, when required, quantified from ChemiDocTMXRS+scans of the membrane using the ImagelabTM (v5.0) software (Bio-Rad). This article is protected by copyright. All rights reserved.

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BN-PAGE was carried out by modification of a previously described protocol (Liu et al., 2012), and thylakoid membranes were solubilized with 2% DM. Electrophoresis was performed using Native PAGE™ Novex®4-16% Bis-Tris mini gel and XCellSureLock minicell (Life Technology) according to the manufacturer’s protocols at 4ºC. For two-dimensional analysis, excised BN-PAGE lanes were soaked in SDS sample buffer containing 5% βmercaptoethanol for 30 min and layered onto 12.5% SDS-PAGE gels.

Recovery and in vitro photoinhibition assays For photoinactivation and subsequent recovery treatments, plants were illuminated at an irradiance of 1000 μmol m-2 s-1 for 3 h, and the restoration of maximal photochemical efficiency (Fv/Fm) was then followed at an irradiance of 20 μmol m-2 s-1 for indicated amounts of time.

To estimate the contribution of the translation-dependent repair processes, detached leaves from plants grown under growth light were first soaked in 1mM lincomycin solution or water in the dark overnight, followed by exposure to an irradiance of 1000 μmol m-2 s-1 for 2 or 4 h. The temperature was maintained at 21ºC during photoinhibitory treatments.

Immunolocalization of MPH1 and co-immunoprecipitation Subcellular

localization

treatment

of

thylakoids

with

chaotropic

agents

and

immunoprecipitation of PSII core subunits by anti-MPH1 antibody were performed as described by Liu et al. (2012). Subfractionation of grana core- and grana margin-, and stroma lamellae-enriched thylakoids was performed as described by Lu et al., 2011a.

In vivo phosphorylation assays For phosphorylation analyses, thylakoid membrane proteins were extracted from leaves of plants at growth light, or were kept overnight in the dark or exposed to high light (1000 μmol m-2 s-1) for 3 h and isolated with solutions containing 10 mM NaF (Ser/Thr phosphatase inhibitor). Phosphorylated proteins were immunodetected with Phospho-Threonine Antibody (P-Thr-Polyclonal) (Cell Signaling Technology).

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Accepted Article

Quantitative real-time RT-PCR Arabidopsis total RNA was isolated with the Plant RNeasy Kit (Qiagen) and treated with DNaseI according to the manufacturer’s instructions. cDNA was synthesized with random primers using the Reverse Transcription System (Promega). cDNA concentrations were normalized to ACTIN2 (At3g18780). Quantitative real-time PCR was performed using the 7500 Fast Real-Time PCR System with Fast SYBR Green Master Mix (Applied Biosystems). Expression was determined in triplicate biological measurements.

ACKNOWLEDGEMENTS We gratefully acknowledge David M. Kramer, Jeffrey A. Cruz, Linda J. Savage and David A. Hall of the Center for Advanced Algal and Plant Phenotyping at MSU for access to equipment for high-light treatments and the assistance in acquiring the chlorophyll fluorescence data. We are grateful to members of the Last group and Yan Lu and her research group at Western Michigan University for valuable suggestions. We greatly appreciate the three anonymous reviewers for their constructive suggestions and comments. This work was supported by Arabidopsis 2010 Project Grant MCB-0519740 and MCB-124400, both from the U.S. National Science Foundation.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article.

Figure S1. Phylogeny of protein sequences of MPH1 and its homologs in other land plants.

Figure S2. Molecular characterization of homozygous mph1-1 and mph1-2 mutants.

Figure S3. Lack of MPH1 caused consistent susceptibility of PSII to photoinhibition under high light.

Figure S4. Relative abundance of chloroplast photosynthetic proteins in the wild-type plants This article is protected by copyright. All rights reserved.

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and mph1 mutants.

Figure S5. Analysis of photosynthesis-associated transcript abundance by RT-PCR in the high light-treated wild type and mph1 mutant plants.

Figure S6. In vivo phosphorylation levels of the wild-type and mph1 mutant thylakoid proteins were examined by phospho-threonine antibody.

Figure S7. Analysis of stability of chloroplast photosynthetic proteins in photoinhibitory assays.

Table S1. Transcript analysis of the wild-type and mph1 mutant plants by quantitative realtime RT-PCR.

Table S2. List of primers used in the study.

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regulation of photosystem II in higher plants. Biochim. Biophys. Acta, 1817, 232–238. Yamamoto, Y., Aminaka, R., Yoshioka, M., Khatoon, M., Komayama, K., Takenaka, D., Yamashita, A., Nijo, N., Inagawa, K., Morita, N., Sasaki, T. and Yamamoto, Y. (2008) Quality control of photosystem II: Impact of light and heat stresses. Photosynth. Res. 98, 589–608.

FIGURE LEGENDS

Figure 1. Alignment of amino acid sequences of MPH1 and its homologs in other land plants. Full length protein sequences were aligned in Jalview using MUSCLE program (http://www.jalview.org/). Amino acids with similar physicochemical properties are shaded with the same colors, and proline residues are shown with yellow color. Predicted chloroplast signal peptide and the potential transmembrane (TM) domain are indicated. Accession numbers retrieved from Phytozome v9.1 (http://www.phytozome.net/) are as follows: Arabidopsis

thaliana

(AT5G07020.1.ath.19665710),

Ricinus

communis

(29686.m000859.rco.16805770), Glycine max (Glyma04g16410.1.gma.26304243), Solanum lycopersicum

(Solyc01g096660.2.1.sly.27303893),

Populus

trichocarpa

(Potri.003G192800.1.ptr.26998839), Sorghum bicolor (Sb04g022740.1.sbi.1966831), Oryza sativa

(LOC_Os02g35090.1.osa.24130686),

(GRMZM2G147279_T01.zma.20858364),

and

Zea Physcomitrella

mays patens

(Pp1s198_75V6.1.ppa.18067847).

Figure 2. Subcellular localization of MPH1 protein. (a) Fractionation of MPH1 is consistent with thylakoid membrane localization. Total leaf extracts and chloroplasts were isolated. The latter were further fractionated into thylakoid membrane and stromal fractions, and proteins were detected by SDS-PAGE and immunoblotting. Polyclonal antibodies against reference proteins Lhcb2 and RbcL were employed as controls for thylakoid and stromal proteins (α-Lhcb2 and α-RbcL, respectively). (b) MPH1 behaves as an intrinsic membrane protein. Sonicated thylakoids were extracted This article is protected by copyright. All rights reserved.

Accepted Article

with various chaotropic agents as indicated. Thylakoid membranes that had not been subjected to salt treatment were used as controls (CK). D2 and PsbO served as controls for intrinsic and extrinsic membrane proteins, respectively. Subfraction proteins were immunodetected on SDS-PAGE. (c) Distribution of MPH1 in thylakoid membrane subfractions. Intact thylakoids (Thy) were subfractionated into grana core (GC)- and grana margin (GM)-, and stroma lamellae (SL)enriched thylakoids. Subfraction proteins were immunodetected on SDS-PAGE using polyclonal antibodies.

Figure 3. Disruption of the MPH1 gene leads to altered chlorophyll fluorescence parameters following photoinhibitory light treatment. (a) T-DNA insertions in MPH1/At5g07020. The confirmed location and orientation of T-DNA insertions corresponding to mph1-1 (SALK_143436) and mph1-2 (SAIL_1284_E04) are indicated. Note that the T-DNAs are not drawn to scale. (b) Panel (A) Phenotypes of 4-week-old wild-type (Col-0), mph1-1, and mph1-2 plants grown under growth light conditions. Panels (B-D) False-color images representing Fv/Fm under growth light (B), after 3-h high-light treatment (C), after 2-d recovery following 3-h highlight treatment (D). GL, growth light; HL, high light. (c)

Quantification

of

fluorescence-detected

photosynthetic

parameters

Fv/Fm,

qI

(photoinhibitory quenching), qE (energy-dependent quenching ) and Fo (minimal fluorescence with PSII reaction centers open) before and after 3-h high-light treatment, and after 2-d recovery following 3-h high-light treatment. Data are presented as means ± SE (n = 5). The asterisks indicates a significant difference between the wild type and mutant (Student’s t test; *, P < 0.05;**, P< 0.01;***, P< 0.001).

Figure 4. Association of MPH1 protein with PSII. (a) Immunoblot analyses of 2D BN/SDS-PAGE gels in the wild-type and psbw mutant plants. BN-PAGE-separated thylakoid protein complexes in a single lane from BN gels were separated in a second dimension by 12.5% SDS-PAGE and subsequently probed with antibodies as indicated to the right. GL, growth light; HL, high light. This article is protected by copyright. All rights reserved.

Accepted Article

(b) Assaying interactions between MPH1 and PSII core proteins by Co-IP. Thylakoid membrane proteins from leaves of the wild type and mph1 mutant plants were incubated with anti-MPH1 antiserum coupled to Protein A/G agarose. The immunoprecipitates were detected with antibodies as indicated to the right. Figure 5. Analysis of chloroplast photosynthetic protein abundance. Total leaf proteins were isolated from rosette leaves of the wild-type and mph1 mutant plants grown under growth light or after 3-h high-light treatment. Samples with equal amounts of total proteins were separated by 12.5% SDS-PAGE, blotted and immunodetected by specific antibodies as indicated. Assignments of each protein complex (in larger typeface) and their diagnostic components (in smaller typeface) are shown to the left. Dilutions of extracts are shown on the top (e.g. '1/2' refers to a two-fold dilution, '1/4' a four-fold dilution. GL, growth light; HL, high light.

Figure 6. Accumulation of chlorophyll-protein complexes in the wild type and mph1 mutant plants. (a) BN-PAGE analysis of thylakoid membrane protein complexes solubilized with 2% DM. Samples were loaded on an equal chlorophyll basis: left gel, 2μg; right gel, 6μg. (b) Immunoblot analysis of CP43- and D1-containing complexes of PSII as indicated (αCP43, left; α-D1, right). Thylakoid samples were loaded on the basis of equivalent chlorophyll content (1.5μg) in each lane.

Figure 7. Recovery from photoinactivation and turnover of PSII proteins in photoinhibition assays. (a) Time-course of recovery following photoinactivation of PSII. Wild-type and mph1 mutant plants grown under normal light (100 μmol m-2 s-1) were transferred to high light (1000 μmol m-2 s-1) for 3 h, and subsequently shifted to continuous dim light (20 μmol m-2 s-1) to allow recovery, and Fv/Fm values were monitored. (b) Degradation of the reaction center D1 protein of PSII in the presence or absence of the chloroplast protein synthesis inhibitor lincomycin under photoinhibitory conditions. Detached leaves were incubated in water or lincomycin solution (1 mM) in dark overnight and This article is protected by copyright. All rights reserved.

Accepted Article

subsequently illuminated under high light (1000 μmol m-2 s-1) for 2, or 4 h. The levels of D1 were measured by SDS-PAGE and immunoblotting with D1 antibody. Data are presented as means ± SD (n = 3).

Figure 8. Assays of subunit components in thylakoid membrane protein complexes after high-light treatment. (a) Chlorophyll-protein complexes were isolated from thylakoid membranes of leaves of high-light treated wild-type and mph1 mutant plants and fractionated by 2D BN/SDS-PAGE. Lanes of the BN gels were sliced and denatured, and subsequently subjected to SDS-PAGE in the second dimension followed by Coomassie blue staining, and the prominent proteins are highlighted with arrows. (b) Immunoblot analysis of 2D BN/SDS-PAGE. Designations of protein complexes (in larger typeface) identified by component proteins (in smaller typeface) are shown to the left.

This article is protected by copyright. All rights reserved.

Accepted Article

Figure 1

Figure 1. Alignment of amino acid sequences of MPH1 and its homologs in other land plants. Full length protein sequences were aligned in Jalview using MUSCLE program (http://www.jalview.org/). Amino acids with similar physicochemical properties are shaded with the same colors, and proline residues are shown with yellow color. Predicted chloroplast signal peptide and the potential transmembrane (TM) domain are indicated. Accession numbers retrieved from Phytozome v9.1 (http://www.phytozome.net/) are as follows: Arabidopsis

thaliana

(AT5G07020.1.ath.19665710),

Ricinus

communis

(29686.m000859.rco.16805770), Glycine max (Glyma04g16410.1.gma.26304243), Solanum lycopersicum

(Solyc01g096660.2.1.sly.27303893),

Populus

trichocarpa

(Potri.003G192800.1.ptr.26998839), Sorghum bicolor (Sb04g022740.1.sbi.1966831), Oryza sativa

(LOC_Os02g35090.1.osa.24130686),

(GRMZM2G147279_T01.zma.20858364),

and

(Pp1s198_75V6.1.ppa.18067847).

This article is protected by copyright. All rights reserved.

Zea Physcomitrella

mays patens

Accepted Article

Figure 2

Figure 2. Subcellular localization of MPH1 protein. (a) Fractionation of MPH1 is consistent with thylakoid membrane localization. Total leaf extracts and chloroplasts were isolated. The latter were further fractionated into thylakoid membrane and stromal fractions, and proteins were detected by SDS-PAGE and immunoblotting. Polyclonal antibodies against reference proteins Lhcb2 and RbcL were employed as controls for thylakoid and stromal proteins (α-Lhcb2 and α-RbcL, respectively). (b) MPH1 behaves as an intrinsic membrane protein. Sonicated thylakoids were extracted with various chaotropic agents as indicated. Thylakoid membranes that had not been subjected to salt treatment were used as controls (CK). D2 and PsbO served as controls for intrinsic and extrinsic membrane proteins, respectively. Subfraction proteins were immunodetected on SDS-PAGE. (c) Distribution of MPH1 in thylakoid membrane subfractions. Intact thylakoids (Thy) were subfractionated into grana core (GC)- and grana margin (GM)-, and stroma lamellae (SL)enriched thylakoids. Subfraction proteins were immunodetected on SDS-PAGE using polyclonal antibodies.

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Accepted Article

Figure 3

Figure 3. Disruption of the MPH1 gene leads to altered chlorophyll fluorescence parameters following photoinhibitory light treatment. (a) T-DNA insertions in MPH1/At5g07020. The confirmed location and orientation of T-DNA insertions corresponding to mph1-1 (SALK_143436) and mph1-2 (SAIL_1284_E04) are indicated. Note that the T-DNAs are not drawn to scale. (b) Panel (A) Phenotypes of 4-week-old wild-type (Col-0), mph1-1, and mph1-2 plants grown under growth light conditions. Panels (B-D) False-color images representing Fv/Fm under growth light (B), after 3-h high-light treatment (C), after 2-d recovery following 3-h highlight treatment (D). GL, growth light; HL, high light. (c)

Quantification

of

fluorescence-detected

photosynthetic

parameters

Fv/Fm,

qI

(photoinhibitory quenching), qE (energy-dependent quenching ) and Fo (minimal fluorescence with PSII reaction centers open) before and after 3-h high-light treatment, and after 2-d recovery following 3-h high-light treatment. Data are presented as means ± SE (n = 5). The asterisks indicates a significant difference between the wild type and mutant (Student’s t test; *, P < 0.05;**, P< 0.01;***, P< 0.001).

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Accepted Article

Figure 4

Figure 4. Association of MPH1 protein with PSII. (a) Immunoblot analyses of 2D BN/SDS-PAGE gels in the wild-type and psbw mutant plants. BN-PAGE-separated thylakoid protein complexes in a single lane from BN gels were separated in a second dimension by 12.5% SDS-PAGE and subsequently probed with antibodies as indicated to the right. GL, growth light; HL, high light. (b) Assaying interactions between MPH1 and PSII core proteins by Co-IP. Thylakoid membrane proteins from leaves of the wild type and mph1 mutant plants were incubated with anti-MPH1 antiserum coupled to Protein A/G agarose. The immunoprecipitates were detected with antibodies as indicated to the right.

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Accepted Article

Figure 5

Figure 5. Analysis of chloroplast photosynthetic protein abundance. Total leaf proteins were isolated from rosette leaves of the wild-type and mph1 mutant plants grown under growth light or after 3-h high-light treatment. Samples with equal amounts of total proteins were separated by 12.5% SDS-PAGE, blotted and immunodetected by specific antibodies as indicated. Assignments of each protein complex (in larger typeface) and their diagnostic components (in smaller typeface) are shown to the left. Dilutions of extracts are shown on the top (e.g. '1/2' refers to a two-fold dilution, '1/4' a four-fold dilution. GL, growth light; HL, high light.

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Accepted Article

Figure 6

Figure 6. Accumulation of chlorophyll-protein complexes in the wild type and mph1 mutant plants. (a) BN-PAGE analysis of thylakoid membrane protein complexes solubilized with 2% DM. Samples were loaded on an equal chlorophyll basis: left gel, 2μg; right gel, 6μg. (b) Immunoblot analysis of CP43- and D1-containing complexes of PSII as indicated (αCP43, left; α-D1, right). Thylakoid samples were loaded on the basis of equivalent chlorophyll content (1.5μg) in each lane.

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Accepted Article

Figure 7

Figure 7. Recovery from photoinactivation and turnover of PSII proteins in photoinhibition assays. (a) Time-course of recovery following photoinactivation of PSII. Wild-type and mph1 mutant plants grown under normal light (100 μmol m-2 s-1) were transferred to high light (1000 μmol m-2 s-1) for 3 h, and subsequently shifted to continuous dim light (20 μmol m-2 s-1) to allow recovery, and Fv/Fm values were monitored. (b) Degradation of the reaction center D1 protein of PSII in the presence or absence of the chloroplast protein synthesis inhibitor lincomycin under photoinhibitory conditions. Detached leaves were incubated in water or lincomycin solution (1 mM) in dark overnight and subsequently illuminated under high light (1000 μmol m-2 s-1) for 2, or 4 h. The levels of D1 were measured by SDS-PAGE and immunoblotting with D1 antibody. Data are presented as means ± SD (n = 3).

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Accepted Article

Figure 8

Figure 8. Assays of subunit components in thylakoid membrane protein complexes after high-light treatment. (a) Chlorophyll-protein complexes were isolated from thylakoid membranes of leaves of high-light treated wild-type and mph1 mutant plants and fractionated by 2D BN/SDS-PAGE. Lanes of the BN gels were sliced and denatured, and subsequently subjected to SDS-PAGE in the second dimension followed by Coomassie blue staining, and the prominent proteins are highlighted with arrows. (b) Immunoblot analysis of 2D BN/SDS-PAGE. Designations of protein complexes (in larger typeface) identified by component proteins (in smaller typeface) are shown to the left.

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