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

Jun Liu1 and Robert L. Last1,2,*1Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA, and2Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA

Received 12 December 2014; revised 4 March 2015; accepted 31 March 2015; published online 6 April 2015.

*For correspondence (e-mail lastr@msu.edu).

SUMMARY

The structure and function of photosystem II (PSII) are highly susceptible to photo-oxidative damage

induced by high-fluence or fluctuating light. However, many of the mechanistic details of how PSII homeo-

stasis is maintained under photoinhibitory light remain to be determined. We describe an analysis of the

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 it is named ‘Mainte-

nance of PSII under High light 1’ (MPH1). The MPH1 protein co-purifies with PSII core complexes and co-

immunoprecipitates core proteins. Consistent with a role in PSII structure, PSII complexes (supercomplexes,

dimers and monomers) of the mph1 mutant 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 the MPH1 protein in grana and

stroma thylakoids, and its interaction with PSII core complexes. Taken together, these results strongly sug-

gest a role for MPH1 in the protection and/or stabilization of PSII under high-light stress in land plants.

Keywords: high-light stress, photosystem II, photoinhibition, thylakoid membranes, PSII damage/repair

cycle, Arabidopsis thaliana.

INTRODUCTION

The energy of light is essential for photosynthesis, but

harnessing this power may damage the extensive and well-

tuned photosynthetic machinery, especially the oxygen-

evolving photosystem II (PSII) complex (Prasil et al., 1992;

Aro et al., 1993). Photoinhibition occurs when the light-

induced photo-oxidative 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 constitu-

tive and inducible photoprotective measures have evolved

to avoid or minimize photoinhibition in plants. These

include avoidance mechanisms such as light screening and

movement of leaves and chloroplasts, and dissipation of

excess absorbed light energy as heat (non-photochemical

quenching) (Niyogi, 1999; Takahashi and Badger, 2011),

and protective mechanisms such as antioxidant scavenging

systems, alternative electron transport pathways, retro-

grade signals, and systemic acquired acclimation (Eberhard

et al., 2008; Li et al., 2009). Despite operation of these

multi-faceted photoprotective mechanisms, photo-oxida-

tive 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

multi-step process that includes movement of damaged

proteins from grana to stroma thylakoids, proteolytic deg-

radation and replacement of damaged protein subunits,

and movement of the repaired PSII machinery back to per-

form 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 much progress has been achieved in elucidating

the PSII photoinhibition/repair mechanism in recent years

(Mulo et al., 2008; Nath et al., 2013; Nickelsen and Rengstl,

2013; J€arvi et al., 2015), the details of this intricate process

and the proteins that help stabilize PSII under photoda-

maging conditions remain to be elucidated. Moreover,

auxiliary protein factors associated with PSII that partici-

pate in its turnover, assembly and organization continue to

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd

731

The Plant Journal (2015) 82, 731–743 doi: 10.1111/tpj.12845

be discovered, some of which are not conserved among all

oxygenic organisms (Nixon et al., 2010). To identify novel

chloroplast proteins with roles in the regulation of the pho-

toinhibition/repair cycle of PSII, we took advantage of

results from the Chloroplast 2010 Project (http://www.plas-

tid.msu.edu/). In this project, over 5200 Arabidopsis thali-

ana T–DNA mutants with insertions in nuclear-encoded

genes that were predicted to be plastid-targeted were

screened and analyzed for a variety of mutant phenotypes.

Assessment of in vivo chlorophyll fluorescence was used

in this screen because it is a non-invasive 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 that was

shown to be involved in repair and reassembly of PSII (Lu,

2011; Lu et al., 2011a). The screen also identified PSB33, a

recently described PSII-associated thylakoid membrane

protein that ensures proper maintenance of PSII/light-har-

vesting complex II (LHCII) supercomplexes (Fristedt et al.,

2015).

This phenomics project also identified SALK_143436, a

T-DNA insertion mutant of At5g07020, which showed an

altered chlorophyll fluorescence phenotype under photoin-

hibitory light conditions. Here, we report biochemical

analysis of this protein MPH1 (Maintenance of PSII under

High light 1) and phenotypic characterization of two inde-

pendent 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 associ-

ated with PSII core proteins in A. thaliana. Loss-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 pho-

toinhibitory stress.

RESULTS

MPH1 is annotated as a proline-rich protein and has

homologs across land plants

To determine whether MPH1 is evolutionarily conserved

among oxygenic photosynthetic organisms, BLAST

searches were performed using the MPH1 full-length pro-

tein 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 sor-

ghum (Sorghum bicolor), and the bryophyte Physcomitrel-

la patens (Figure 1 and Figure S1). In contrast, MPH1

homologs were not detected in algae or prokaryotes, sug-

gesting that MPH1 is conserved in land plants. The homo-

logs in land plants share all domains predicted for the

Arabidopsis MPH1: an N–terminal chloroplast transit pep-

tide, a single-pass transmembrane domain, and conserved

interspersed proline residues.

MPH1 is a chloroplast intrinsic thylakoid membrane

protein

Arabidopsis thaliana MPH1 is annotated as a 235 amino

acid protein of unknown function on TAIR (https://www.

arabidopsis.org). In silico analyses (http://suba.plantenergy.

uwa.edu.au/ and http://aramemnon.uni-koeln.de/) predict

that MPH1 harbors an N–terminal chloroplast signal

sequence of 38 amino acids and has a single transmem-

brane domain (Schwacke et al., 2003; Tanz et al., 2013).

These predictions are supported by published proteomic

studies that detected MPH1 in chloroplast thylakoids (Pel-

tier et al., 2004; Giacomelli et al., 2006). To experimentally

test the localization, proteins prepared from whole leaves,

chloroplasts and sub-chloroplast compartments were

immunodetected using an MPH1 polyclonal antibody. As

shown in Figure 2(a), MPH1 protein was found in total cell

extracts, intact chloroplasts and thylakoid membranes, but

not in the stroma-enriched fraction, similar to the thyla-

koid membrane light-harvesting chlorophyll-binding pro-

tein b2 (Lhcb2). To determine whether MPH1 is an

intrinsic or extrinsic membrane protein, sonicated thyla-

koid membranes were incubated with chaotropic salts or

under alkaline conditions that are known to release periph-

eral proteins from thylakoids. As shown in Figure 2(b),

MPH1 showed co-fractionation similar to the integral

membrane protein D2 but not like the peripheral protein

PsbO, supporting the hypothesis that MPH1 is an intrinsic

thylakoid membrane protein. Further sub-fractionation of

thylakoid membranes into grana core- and grana margin-

membranes and stroma lamellae showed that MPH1 was

distributed among all three thylakoid fractions (Figure 2c),

consistent with the hypothesis that the 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 the photosynthetic func-

tion of two independent mutants. In the Chloroplast 2010

Project (Ajjawi et al., 2010; Lu et al., 2011b), the

SALK_143436 line (now named mph1–1), containing a

T–DNA in the first intron of MPH1 (Figure 3a), was found

to have a very low Fv/Fm (an indicator of the maximum

photochemical efficiency of PSII), but showed no non-

photochemical quenching phenotype after 3 h of photoin-

hibitory light treatment (1000 lmol m�2 sec�1). A second

homozygous mutant, SAIL_1284_E04 (now named mph1–2), has a T–DNA insertion in the second intron of MPH1

(Figure 3a). Neither MPH1 transcript nor MPH1 protein was

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

732 Jun Liu and Robert L. Last

detectable in either mutant (Figure S2), showing that these

are strong loss-of-function mutants, and demonstrating the

specificity of the a–MPH1 antiserum.

To test whether inactivation of the MPH1 gene is respon-

sible for the high-light phenotype originally observed in

the mph1–1 mutant (Ajjawi et al., 2010; Lu et al., 2011b),

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 (Figure 3 and Figure S3). In contrast,

the growth phenotype and Fv/Fm of the mutants were iden-

tical to those of the wild type propagated under normal

growth light conditions (100 lmol m�2 sec�1), suggesting

that MPH1 is dispensable for optimal PSII function under

‘normal’ laboratory growth light conditions. In contrast to

the wild type, Fv/Fm did not return to the pre-photoinhibito-

ry levels in either mutant following a 2 day recovery period

under normal growth light conditions (Figure 3c). More-

over, both mutants exhibited a higher qI (photoinhibitory

quenching) and Fo (minimum fluorescence with PSII reac-

tion centers open) but lower qE (energy-dependent

quenching) after 3 h high-light treatment (Figure 3c), sug-

gesting that mph1 mutants may be impaired in photopro-

tection of PSII under photoinhibitory light. Taken together,

these results show that the mph1 photosynthetic pheno-

type is due to the loss of At5g07020.

Association of MPH1 protein with PSII

The observations that MPH1 protein is a thylakoid mem-

brane protein, and that lack of MPH1 results in a marked

defect in PSII activity under photoinhibitory light condi-

tions, suggest that MPH1 may 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 BN (blue

native)/SDS–PAGE and immunodetected using antibodies

against PSII core proteins and MPH1. Additionally, to

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 (http://www.jalview.org/). Amino acids with similar physicochemical properties are shaded

with the same colors, and proline residues are shaded in yellow. The predicted chloroplast signal peptide and the potential transmembrane (TM) domain are

indicated. The accession numbers (http://www.phytozome.net/) for MPH1 and its homologs are: Arabidopsis thaliana, AT5G07020.1.ath.19665710; Ricinus

communis, 29686.m000859.rco.16805770; Glycine max, Glyma04 g16410.1.gma.26304243; Solanum lycopersicum, Solyc01 g096660.2.1.sly.27303893; Populus

trichocarpa, Potri.003G192800.1.ptr.26998839; Sorghum bicolor, Sb04 g022740.1.sbi.1966831; Oryza sativa, LOC_Os02 g35090.1.osa.24130686; Zea mays,

GRMZM2G147279_T01.zma.20858364; Physcomitrella patens, Pp1s198_75V6.1.ppa.18067847.

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

A PSII-associated thylakoid protein MPH1 733

examine the specificity of possible interaction of MPH1 with

PSII, a psbw mutant was used as a control. PsbW is a low-

molecular-weight protein that is exclusive to PSII of photo-

synthetic eukaryotes, and loss of PsbW destabilizes the

supramolecular organization of PSII (Shi and Schr€oder,

2004; Garc�ıa-Cerd�an et al., 2011). As shown in Figure 4(a),

under normal growth light conditions, a substantial

proportion of MPH1 was found to co-migrate with PSII

monomers and a small proportion co-migrated with unas-

sembled proteins in the wild type and psbw mutant; this

indicates that MPH1 associates with PSII monomeric com-

plexes. In thylakoids isolated from leaves of the wild type

following high-light treatment, most of the MPH1 protein

was found in the region of the gel corresponding to PSII

supercomplexes, with a small amount co-migrating with

PSII monomers (Figure 4a). In contrast, in the psbw mutant

samples, more of the MPH1 protein co-migrated with unas-

sembled proteins and a small fraction migrated in the

region of PSII monomers (Figure 4a). The results suggest

that MPH1 co-purifies with PSII supercomplexes under pho-

toinhibitory 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, co-immunoprecipitation assays using

a–MPH1 antibody were performed on thylakoid membranes

solubilized using the non-ionic detergent dodecyl-b–D–maltopyranoside (DM). Consistent with the hypothesis that

MPH1 protein associates with the PSII core proteins, D1,

D2, CP43 and CP47 co-precipitated using anti-MPH1 anti-

body, but the PSI core protein PsaA did not (Figure 4b). The

co-immunoprecipitation 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 is of interest because it has been shown to play

a role in PSII repair and reassembly following photodam-

age, and also interacts with CP43 and CP47 (Lu et al.,

2011a). We found no evidence for LQY1 co-precipitation

with MPH1 using anti-MPH1 antibody (Figure 4b).

Loss of MPH1 affects PSII complex abundance under high

light

The observations that the MPH1 protein co-purifies with

PSII complexes and core proteins, and that the mutant has

altered Fv/Fm under high light, led us to examine the accu-

mulation of PSII proteins in mph1 mutants. Immunoblot

analysis of a variety of photosynthetic proteins was per-

formed on total leaf protein extracts from the wild type

and mph1 mutants under normal growth light and after a

3 h high-light treatment. Under normal growth light condi-

tions, 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 (mean

reductions of 40, 27, 29 and 42%, respectively; Figure 5

and Figure S4). In contrast, MPH1 abundance was

increased by approximately 30% in wild-type leaves after a

3 h high-light treatment, suggesting that MPH1 synthesis

or stability may increase under photoinhibitory conditions

(Figure 5 and Figure S4).

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

the PSII oxygen-evolving complex protein PsbO, the PSII

light-harvesting complex protein Lhcb2, the PSI reaction

center protein PsaA and antenna protein Lhca2, the ATP

synthase subunit CF1b, the cytochrome b6f complex

(a)

(b)

(c)

Figure 2. Subcellular localization of MPH1 protein.

(a) Fractionation of MPH1 is consistent with thylakoid membrane localiza-

tion. Total leaf extracts and chloroplasts were isolated. The latter were fur-

ther fractionated into thylakoid membrane and stromal fractions, and

proteins were detected by SDS–PAGE and immunoblotting. Polyclonal anti-

bodies against reference proteins Lhcb2 and RbcL were used as controls for

thylakoid and stromal proteins (a–Lhcb2 and a–RbcL, respectively).(b) MPH1 behaves as an intrinsic membrane protein. Sonicated thylakoids

were extracted with various chaotropic agents as indicated. Thylakoid mem-

branes 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. Proteins from sub-fractions were immunodetected on

SDS–PAGE.

(c) Distribution of MPH1 in thylakoid membrane sub-fractions. Intact thylak-

oids (Thy) were sub-fractionated into grana core-enriched (GC), grana mar-

gin-enriched and stroma lamellae-enriched (SL) thylakoids. Proteins from

sub-fractions were immunodetected on SDS–PAGE using polyclonal anti-

bodies.

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

734 Jun Liu and Robert L. Last

protein Cyt.f or the ribulose bisphosphate carboxylase

large-subunit protein RbcL (Figure 5 and Figure S4). In

addition, the decrease in PSII core proteins detected in the

mph1 mutant under high light was not caused by a limita-

tion in the abundance of corresponding transcripts of PSII

as analyzed by both RT–PCR 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 may be involved in pro-

tecting the 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 for-

mation. BN-PAGE fractionation was performed on thyla-

koid membrane protein complexes obtained using

dodecyl-b–D–maltopyranoside (Figure 6a), and the com-

plexes 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 and PSII dimers

and monomers in thylakoids in mph1 high light-treated

(a) (c)

(b)

a

b

c

d

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 normal growth light conditions. Panels B–D: false-colorimages representing Fv/Fm under normal growth light (B), after 3 h high-light treatment (C), and after a 2 day recovery period following 3 h high-light 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 (mini-

mum fluorescence with PSII reaction centers open) before and after 3 h high-light treatment, and after a 2 day recovery period following 3 h high-light treat-

ment. Values are means � SE (n = 5). Asterisks indicate statistically significant differences between the wild type and the mutant (*P < 0.05, **P < 0.01,

***P < 0.001; Student’s t test).

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

A PSII-associated thylakoid protein MPH1 735

mutants compared with their wild-type counterparts (Fig-

ure 6), suggesting that lack of MPH1 disrupts 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 rele-

vant 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

proteins (CP43, D2, and D1) and LHCII was detected in dark-

adapted wild-type plant samples, and the levels of phos-

phorylation were enhanced under growth light (Figure S6).

No difference in the amount of phosphorylation was

observed when comparing the wild type and the mph1

mutant either in the dark or under growth light. In contrast,

after high-light treatment, the phosphorylation levels of PSII

core proteins but not LHCII were significantly reduced in the

mph1mutant compared to the wild type (Figure S6).

Recovery and turnover of PSII in mph1 mutants under

high light

The above analyses indicated that loss of MPH1 caused

decreased PSII activity and abundance of PSII complexes

following a shift to photoinhibitory light conditions. The

reduction may be due to accelerated rates of photodamage

or suppressed rates of repair. To distinguish between these

two mechanisms, we tracked the recovery process after

high-light treatment. Both wild-type and mph1 mutant

plants were exposed to a 3 h high-light treatment, and

subsequently transferred to continuous dim light (20

lmol m�2 sec�1) to allow recovery, and Fv/Fm was moni-

tored during the process. As shown in Figure 7(a), Fv/Fmdecreased dramatically after high-light treatment in both

the wild type and the mph1 mutants, but the decrease was

more pronounced in the mutant, indicating that mph1

mutants suffer from more extensive PSII photoinactivation.

In contrast, Fv/Fm recovered rapidly under continuous low

light in both the wild type and the mph1 mutants, but the

rate of increase in the mph1 mutant appears to be slightly

higher than that in the wild type. However, after 10 h

recovery Fv/Fm was 97% of its original level in the mutant

but fully restored in the wild type (Figure 7a). These results

suggest that the slight reduction in the final recovery of

PSII activity in the mph1 mutant is attributable to more

severe PSII photoinhibition during high-light treatment.

To test the hypothesis that photosensitivity of mph1

mutants results from increased photodamage to PSII rather

than decreased repair, we performed in vitro photoinhibi-

tion assays in the presence or absence of lincomycin. This

compound blocks the repair of PSII by inhibiting de novo

synthesis of chloroplast-encoded proteins, thus allowing

direct evaluation of net damage to PSII. Total leaf proteins

were extracted and subjected to immunoblot analysis to

(a) (b)

Figure 4. Association of MPH1 protein with PSII.

(a) Immunoblot analyses of two-dimensional BN/SDS–PAGE gels for wild-type and psbw mutant plants. BN-PAGE-separated thylakoid protein complexes in a

single lane were separated in the second dimension by 12.5% SDS–PAGE and subsequently probed with antibodies as indicated on the right. GL, growth light;

HL, high light.

(b) Analysis of interactions between MPH1 and PSII core proteins by co-immunoprecipitation. Thylakoid membrane proteins from leaves of 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

on the right.

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

736 Jun Liu and Robert L. Last

examine the rates of protein degradation caused by photo-

damage during the course of high-light treatment. Without

lincomycin, levels of PSII core proteins D1 and CP47 gradu-

ally decreased in both the wild type and the mph1 mutant

during 4 h high-light treatment, although the reduction

was higher in the mph1 mutant (Figure 7b and Figure S7).

In contrast, the rate of D1 protein degradation was espe-

cially pronounced in the presence of lincomycin in the

mph1 mutant relative to the wild type, and CP47 degrada-

tion also increased (Figure 7b and Figure S7). However,

markers for PSI, ATPase and Cyt.b6f complexes were unaf-

fected in the mph1 mutant, as was the large subunit of

ribulose bisphosphate carboxylase (Figure S7). These data

are consistent with the hypothesis that accelerated degra-

dation of photodamaged PSII core subunits results in the

photoinhibition observed in mph1 mutants under high

light.

To simultaneously assess PSII stability, photodamage

and repair, we performed two-dimensional BN/SDS–PAGE

followed by Coomassie Brilliant Blue staining or immuno-

blot analyses after high-light treatment. As shown in Fig-

ure 8, the levels of PSI complex, ATP synthase and Cyt.b6f

complex in mph1 mutants were comparable to those in

wild-type plants. In contrast, the proportions of the PSII

core subunits D1, D2, CP43 and CP47 in various PSII

complexes (PSII supercomplexes, and PSII dimers and

monomers) were generally reduced in mph1 mutants rela-

tive to the wild-type plants, and no smaller PSII complexes,

such as RC47 or PSII monomers, accumulated in the

mutant after high-light treatment (Figure 8). These results

are consistent with the hypothesis that disassembly and/or

reassembly of PSII complexes during the 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 pho-

tosynthetic eukaryotes and cyanobacteria (Chi et al., 2012;

Komenda et al., 2012). The damage and rapid degradation

of the PSII reaction center D1 protein in response to pho-

toinhibitory 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 other

PSII subunits (Jansen et al., 1996; Bergantino et al., 2003;

Rokka et al., 2005). Much remains to be learned about the

molecular mechanisms influencing maintenance and turn-

over of PSII reaction center proteins and complexes under

high-irradiance light and the regulation of the PSII photoin-

hibition/repair cycle, although it is clear that a suite of aux-

iliary protein factors are required to assist 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; J€arvi et al., 2015).

Here 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.

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 func-

tion 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; Figure 3 and Figure S3), and photo-

synthetic proteins accumulated to wild-type levels (Figure 5

and Figure 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 (Figure 3 and Figure S3). Further biochemi-

cal assays using BN-PAGE, immunoblotting of BN gels

and two-dimensional BN/SDS–PAGE using antibodies

directed against representative subunits of photosynthetic

membrane complexes showed that the amounts of PSII

Figure 5. Analysis of chloroplast photosynthetic protein abundance.

Total leaf proteins were isolated from rosette leaves of wild-type and mph1

mutant plants grown under normal growth light or after 3 h high-light treat-

ment. Samples with equal amounts of total proteins were separated by

12.5% SDS–PAGE, blotted and immunodetected using specific antibodies as

indicated. Assignments of each protein complex and their diagnostic com-

ponents are shown on the left. Dilutions of extracts are shown at the top (‘1/

2’ refers to a twofold dilution, ‘1/4’ refers to a fourfold dilution). GL, growth

light; HL, high light.

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

A PSII-associated thylakoid protein MPH1 737

supercomplexes, PSII dimers and monomers were mod-

estly reduced in the mph1 mutant shifted to high-light

conditions (Figures 6 and 8). The effect appears to be spe-

cific to PSII, as the levels of PSI complexes, the Cyt.b6f

complex and ATP synthase were largely unaltered (Fig-

ures 6 and 8). The observation that steady-state levels of

the PSII core proteins D1, D2, CP43 and CP47 were

reduced in mph1 mutants further supports the hypothesis

that the PSII reaction center is defective in mph1 mutants

under high light. In contrast, no decreases were observed

for PsbO or LHCII (Figure 5 and Figure S4). A decrease of

all PSII core proteins was also observed in high light-sen-

sitive lqy1, hhl1 and cyp38 mutants (Fu et al., 2007; Sirpi€o

et al., 2008; Lu et al., 2011a; Jin et al., 2014). These data

provide evidence that MPH1 protein is required for main-

taining normal levels of the PSII reaction center complex

under high-light irradiance, leading us to hypothesize that

MPH1 plays a role in maintenance of the PSII reaction

center under high-light stress.

PSII core phosphorylation has been proposed to regu-

late photoprotection under high light intensities (Dannehl

et al., 1995; Koivuniemi et al., 1995; Georgakopoulos

and Argyroudi-Akoyunoglou, 1998; Kanervo et al., 2005;

Yamamoto et al., 2008; Tikkanen and Aro, 2012; Kato and

Sakamoto, 2014). We found reduced phosphorylation of

PSII core proteins in mph1 mutants compared with the

wild type under high light (Figure S6). This phenotype is

shared with the high light-sensitive psb33, psbw and cyp38

mutants (Sirpi€o et al., 2008; Garc�ıa-Cerd�an 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 Bian-

chi et al., 2008), was significantly increased in mph1

mutants compared to wild type after high-light treatment

(Figure 3c). This may be the result of a reduction in the

(a)

(b)

Figure 6. Accumulation of chlorophyll-protein com-

plexes in the wild-type and mph1 mutant plants.

(a) BN-PAGE analysis of thylakoid membrane pro-

tein complexes solubilized using 2% dodecyl-b–D–maltopyranoside. Samples were loaded on the

basis of equivalent chlorophyll content in each lane:

left gel, 2 lg; right gel, 6 lg.(b) Immunoblot analysis of CP43-and D1-containing

complexes of PSII as indicated. Thylakoid samples

were loaded on the basis of equivalent chlorophyll

content (1.5 lg) in each lane.

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

738 Jun Liu and Robert L. Last

trapping efficiency of PSII reaction centers in mph1

mutants (Kov�acs et al., 2006; Johnson and Ruban, 2010),

as the mutants showed reduced levels of PSII reaction cen-

ter proteins and no change in LHCII protein levels (Figure 5

and Figure S4). This increase in Fo is consistent with a sig-

nificantly decreased Fv/Fm in mph1 mutants after high-light

treatment (Figure 3b,c and Figure S3). In addition, qI, the

slowly forming and relaxing component of non-photoche-

mical quenching, was significantly enhanced in mph1

mutants compared to the wild type after high-light treat-

ment (Figure 3c). It should be noted that the term qI was

originally ascribed solely to 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

qI 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 com-

pared with the wild-type (Figures 3c and 5 and Figure S4),

the increase in qI may be primarily due to the higher

requirement for degradation and replacement of photo-

damaged D1 protein in the mutants under high light (Aro

et al., 1993; M€uller et al., 2001; Murata et al., 2007). Con-

versely, qE, the major and reversible component of non-

photochemical quenching, was significantly reduced in

mph1 mutants relative to the wild type after high-light

treatment (Figure 3c). Collectively, the increase of qI and Foand the decrease in 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 D1, was more rapid in the pres-

ence of lincomycin than in its absence in the mph1

mutants compared with the wild type (Figure 7b and Fig-

ure S7). These results strongly suggest that acceleration of

photodamage, rather than deceleration of repair, is respon-

sible for the severe photoinhibition observed in mph1

mutants. We observed that these mutants suffered more

damage to PSII compared to the wild type during photoin-

hibitory light treatment (Figures 3c and 7); despite a faster

recovery rate during the first 4 h, the Fv/Fm levels in the

mutants were not restored to wild-type levels after a shift

to continuous dim light conditions for 10 h (Figure 7a).

Taken together, these data suggest that loss of MPH1 ren-

ders PSII susceptible to 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

thylakoid membrane sub-fractions. Under normal growth

light conditions, the bulk of MPH1 co-migrated with PSII

monomers in two-dimensional BN/SDS–PAGE in both

the wild type and psbw mutant, in positions corre-

sponding to higher molecular masses than free MPH1

(Figure 4a). Much of the detectable MPH1 protein

co-migrated with PSII supercomplexes under photoinhib-

itory light in the wild type (Figure 4a). This increase in

MPH1-associated PSII supercomplexes in response to

high light was not seen in the psbw mutant, which was

severely defective in accumulation of PSII supercomplex-

es (Figure 4a). MPH1 was detected at the position of

PSII monomers irrespective of light fluence. This sug-

gests that MPH1 is generally associated with PSII mono-

mers, and becomes more stably associated with PSII

supercomplexes under high light. These immunoblot

results correlate with an overall increase in MPH1 pro-

tein in the wild type following a shift to high light (Fig-

ure 5 and Figure S4).

(a)

(b)

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 lmol m�2 sec�1)

were transferred to high light (1000 lmol m�2 sec�1) for 3 h, subsequently

shifted to continuous dim light (20 lmol m�2 sec�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 lin-

comycin solution (1 mM) in the dark overnight and subsequently illumi-

nated under high light (1000 lmol m�2 sec�1) for 2 or 4 h. The levels of D1

were measured by SDS–PAGE and immunoblotting using D1 antibody. Val-

ues are means � SD (n = 3).

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

A PSII-associated thylakoid protein MPH1 739

Co-immunoprecipitation assays further demonstrated

that MPH1 interacted specifically with core subunits of PSII

(D1, D2, CP43 and CP47) but not PSI (Figure 4b). Co-immu-

noprecipitation assays also showed that MPH1 did not

interact with LQY1, although MPH1 has similar phyloge-

netic distribution to LQY1 and HHL1, and loss–of-functionmutants display similar high light-sensitive phenotypes (Lu

et al., 2011a; Jin et al., 2014). This suggests that MPH1

may have evolved a role distinct from 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 the distribution of MPH1 among grana

core- and grana margin-membranes and the stroma lamel-

lae of thylakoids (Figure 2c). However, the pattern of MPH1

distribution is quite different from that of 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; Puthiyavee-

til et al., 2014). This observation strengthens the hypothe-

sis that MPH1 is involved in protection of PSII against

damage rather than the repair of PSII (Figures 3c and 7

and Figure S7).

The hypothesis that MPH1 plays a role in PSII mainte-

nance is also supported by the observation that the levels

of PSII core protein subunits in various 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

in the absence of MPH1 the overall stability of PSII com-

plexes is compromised under high light. This hypothesis

is supported by the observation that the rates of degrada-

tion of PSII core proteins under high light were faster in

either the presence or absence of lincomycin in the mph1

mutant relative to the wild type (Figure 7b and Figure S7).

(a)

(b)

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 two-dimensional BN/SDS–PAGE. Lanes of the BN gels were sliced and denatured, and subsequently subjected to SDS–PAGE in the second dimension fol-

lowed by Coomassie Brilliant Blue staining. The major proteins are indicated by arrows.

(b) Immunoblot analysis of two-dimensional BN/SDS–PAGE. Assignments of each protein complex and their diagnostic components are shown on the left.

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

740 Jun Liu and Robert L. Last

It is noteworthy that MPH1 has a similar distribution pat-

tern to CYP38, which functions in the stability and biogen-

esis of PSII (Fu et al., 2007; Sirpi€o et al., 2008). Both

MPH1 and CYP38 associate mainly with the monomeric

PSII complex in BN-PAGE (Sirpi€o et al., 2008). Taken

together, these data are consistent with the hypothesis

that MPH1 is involved in protection of PSII against photo-

damage and the stable accumulation of PSII, presumably

via interaction with protein complexes of PSII and/or with

other regulatory factors required for sustaining normal

PSII activity such as CYP38.

The results of this study expand our understanding of

the dynamic regulation of the fine-tuned photodamage/

repair cycle of PSII. Phylogenetic analysis of MPH1 homo-

logs indicates that MPH1 is found in land plant genomes,

but is not conserved among other oxygenic organisms

(Figure 1 and Figure S1), similar to the previously

described phylogenetic distribution of LQY1 and HHL1 (Lu

et al., 2011a; Jin et al., 2014). The existence 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 that play 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

The Arabidopsis thaliana T–DNA insertion line mph1–1(SALK_143436) in ecotype Columbia (Col–0) was identified as hav-ing altered Fv/Fm in the Chloroplast 2010 Project (http://www.plas-tid.msu.edu/), while mph1–2 (SAIL_1284_E04) was obtained fromthe Arabidopsis Biological Resource Center (http://www.arabidop-sis.org/), and homozygous mph1–2 plants were identified bygenomic PCR using primers specific for MPH1 (Table S2). Thehomozygous mph1–1 and mph1–2 mutant lines used in this studywere deposited in the Arabidopsis Biological Resource Centerunder accession numbers CS68902 and CS68903, respectively.The psbw mutant (SAIL_885_A03) was also identified in the Chlo-roplast 2010 Project. Seeds sown on soil were stratified in the darkat 4°C for 3 days, and then grown in a controlled growth chamberas previously described, except that the photoperiod was 16/8 hrather than 12/12 h (Lu et al., 2008, 2011b; Ajjawi et al., 2010).Unless otherwise noted, all plants used in this study were 4 weeksold. For high-light treatment, plants were placed in an imagingchamber under illumination of 1000 lmol m�2 sec�1 for 3 h whilemaintaining the temperature at 21°C.

Chlorophyll fluorescence measurements

Chlorophyll fluorescence images of intact plants were obtainedusing a custom-designed plant imaging chamber, and imagingwas performed as described by Attaran et al. (2014). After adapta-tion of plants in the dark for 30 min, minimum fluorescence (Fo)was determined under weak red light. Fluorescence quenchingwas induced by 10 min of actinic illumination with white light.

The maximal fluorescence in the dark-adapted state (Fm) and inthe light-adapted state (F 0

m) and after 10 min of dark relaxation fol-lowing actinic illumination (F 00

m) were determined using a saturat-ing pulse of light applied at 2 min intervals (Damkjær et al., 2009;de Bianchi et al., 2011). Fv/Fm, qI and qE were calculated accordingto the equations Fv/Fm = (Fm – Fo)/Fm, qI = (Fm – F 00

m)/F 00m, and

qE = Fm/F0m – Fm/F

00m (Farber et al., 1997; Kov�acs et al., 2006). Maxi-

mum fluorescence images were analyzed using ImageJ software(Schneider et al., 2012).

Production of anti-MPH1 polyclonal antibodies

Affinity-purified anti-MPH1 polyclonal antibodies were generatedby Pierce Biotechnology (http://www.pierce-antibodies.com). A 18amino acid peptide (corresponding to amino acids 175–192 ofMPH1) with an additional N–terminal Cys residue (CLPETMA-SEAQPEASSVPT) was synthesized, conjugated with keyhole lim-pet hemocyanin, and used to raise antibody against MPH1 proteinin rabbits.

Immunoblot and BN-PAGE analyses

For immunoblot analysis, total protein samples of Arabidopsisrosette leaves were prepared as described previously (Mart�ınez-Garc�ıa et al., 1999). Rabbit primary antibodies were purchasedfrom Agrisera (http://www.agrisera.com). Immunoblotting wasperformed according to standard techniques by probing withspecific antibodies after electroblotting of SDS–PAGE gels ontonitrocellulose membranes (GE Healthcare, http://www3.gehealthcare.com) (Liu et al., 2012). Primary antibodies were diluted20 000-fold (antibodies against D1 and D2), 10 000-fold (PsbO andLhcb2), 5000-fold (CP47, Lhca2, cytochrome f, CF1b and RbcL),2500-fold (CP43 and PsaA) or 200-fold (MPH1), and signals fromhorseradish peroxidase-conjugated goat anti-rabbit IgG (H+L)were visualized using Clarity Western ECL substrate (http://www.bio-rad.com/en-us/product/clarity-ecl-western-blotting-substrate) andanalyzed using software Image LabTM (version 5.0) (Bio–Rad,http://www.bio-rad.com). Protein accumulation was normalized tothe amount of Coomassie Brilliant Blue-stained LHCII of PSII as aninternal standard, and, when required, was quantified fromChemiDocTM XRS+ scans of the membrane using Image LabTM soft-ware (http://www.bio-rad.com/en-mx/sku/170-8265-chemidoc-xrs-system-with-image-lab-software).

BN-PAGE was performed by modification of a previouslydescribed protocol (Liu et al., 2012), and thylakoid membraneswere solubilized using 2% dodecyl-b–D–maltopyranoside. Electro-phoresis was performed using a Native PAGETM Novex� 4–16%Bis/Tris mini gel and an XCellSureLock mini-cell (Life Technolo-gies, https://www.lifetechnologies.com) at 4°C according to themanufacturer’s instructions. For two-dimensional analysis,excised BN-PAGE lanes were soaked in SDS sample buffer con-taining 5% b–mercaptoethanol for 30 min, and layered onto 12.5%SDS–PAGE gels.

Recovery and in vitro photoinhibition assays

For photoinactivation and subsequent recovery treatments, plantswere illuminated at an irradiance of 1000 lmol m�2 sec�1 for 3 h,and the restoration of maximal photochemical efficiency (Fv/Fm)was then followed at an irradiance of 20 lmol m�2 sec�1.

To estimate the contribution of the translation-dependent repairprocesses, detached leaves from plants grown under growth lightwere first soaked in 1 mM lincomycin solution or water in thedark overnight, followed by exposure to an irradiance of1000 lmol m�2 sec�1 for 2 or 4 h. The temperature was main-tained at 21°C during photoinhibitory treatments.

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

A PSII-associated thylakoid protein MPH1 741

Immunolocalization of MPH1 and co-immunoprecipitation

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

In vivo phosphorylation assays

For phosphorylation analyses, thylakoid membrane proteins wereextracted from leaves of plants under growth light, plants keptovernight in the dark, or plants exposed to high light(1000 lmol m�2 sec�1) for 3 h, and isolated with solutions con-taining 10 mM NaF (Ser/Thr phosphatase inhibitor). Phosphory-lated proteins were immunodetected using a phospho-threonineantibody (Cell Signaling Technology, http://www.cellsignal.com).

Quantitative real-time RT–PCR

Arabidopsis total RNA was isolated using a Plant RNeasy kitaccording to the manufacturer’s instructions (Qiagen, https://www.qiagen.com), and treated with DNase I. cDNA was synthe-sized with random primers using a reverse transcription system(Promega, https://www.promega.com). cDNA concentrationswere normalized to ACTIN2 (At3 g18780). Quantitative real-timePCR was performed using a 7500 Fast Real-Time PCR Systemwith Fast SYBR Green Master Mix (Applied Biosystems, http://www.appliedbiosystems.com). Expression was determined intriplicate biological measurements.

ACKNOWLEDGMENTS

We gratefully acknowledge David M. Kramer, Jeffrey A. Cruz,Linda J. Savage and David A. Hall (Center for Advanced Algal andPlant Phenotyping, Michigan State University, East Lansing, MI)for access to equipment for high-light treatments and assistancein acquiring the chlorophyll fluorescence data. We are grateful tomembers of the Last group and to Yan Lu and her research groupat Western Michigan University (Kalamazoo, MI) for valuable sug-gestions. We greatly appreciate the three anonymous reviewersfor their constructive suggestions and comments. This work wassupported by Arabidopsis 2010 Project grants MCB-0519740 andMCB-124400, both from the US National Science Foundation.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online ver-sion of this article.Figure S1. Phylogeny of protein sequences of MPH1 and its homo-logs in other land plants.Figure S2. Molecular characterization of homozygous mph1–1 andmph1–2 mutants.Figure S3. Lack of MPH1 resulted in susceptibility of PSII to pho-toinhibition under high light.Figure S4. Relative abundance of chloroplast photosynthetic pro-teins in wild-type plants and mph1 mutants.Figure S5. Analysis of photosynthesis-asociated transcript abun-dance by RT–PCR in high light-treated wild-type and mph1 mutantplants.Figure S6. In vivo phosphorylation levels of the wild type andmph1 mutant thylakoid proteins examined using a phospho-threo-nine antibody.Figure S7. Analysis of the stability of chloroplast photosyntheticproteins in photoinhibitory assays.

Table S1. Transcript analysis of the wild-type and mph1 mutantplants by quantitative real-time RT–PCR.Table S2. List of primers used in the study.

REFERENCES

Ajjawi, I., Lu, Y., Savage, L.J., Bell, S.M. and Last, R.L. (2010) Large-scale

reverse genetics in Arabidopsis: case studies from the Chloroplast 2010

Project. Plant Physiol. 15, 529–540.Aro, E.-M., Virgin, I. and Andersson, B. (1993) Photoinhibition of photosys-

tem II. Inactivation, protein damage and turnover. Biochim. Biophys.

Acta, 1143, 113–134.Aro, E.-M., Suorsa, M., Rokka, A., Allahverdiyeva, Y., Paakkarinen, V., Sa-

leem, A., Battchikova, N. and Rintam€aki, E. (2005) Dynamics of photosys-

tem II: a proteomic approach to thylakoid protein complexes. J. Exp. Bot.

56, 347–356.Attaran, E., Major, I.T., Cruz, J.A., Rosa, B.A., Koo, A.J., Chen, J., Kramer,

D.M., He, S. and Howe, G.A. (2014) Temporal dynamics of growth and

photosynthesis suppression in response to jasmonate signaling. Plant

Physiol. 165, 1302–1314.Bergantino, E., Brunetta, A., Touloupakis, E., Segalla, A., Szabo, I. and Gia-

cometti, G.M. (2003) Role of the PSII-H subunit in photoprotection: novel

aspects of D1 turnover in Synechocystis 6803. J. Biol. Chem. 278, 41820–41829.

Bhuiyana, N.H., Frisoa, G., Poliakova, A., Ponnalab, L. and van Wijk, K.J.

(2015) MET1 is a thylakoid-associated TPR protein involved in photosys-

tem II supercomplex formation and repair in Arabidopsis. Plant Cell, 27,

262–285.de Bianchi, S., Dall’Osto, L., Tognon, G., Morosinotto, T. and Bassi, R.

(2008) Minor antenna proteins CP24 and CP26 affect the interactions

between photosystem II subunits and the electron transport rate in grana

membranes of Arabidopsis. Plant Cell, 20, 1012–1028.de Bianchi, S., Betterle, N., Kouril, R., Cazzaniga, S., Boekema, E., Bassi, R.

and Dall’Osto, L. (2011) Arabidopsis mutants depleted in the light har-

vesting protein Lhcb4 have a disrupted photosystem II macrostructure

and are defective in photoprotection. Plant Cell, 23, 2659–2679.Chi, W., Ma, J. and Zhang, L. (2012) Regulatory factors for the assembly of

thylakoid membrane protein complexes. Philos. Trans. R. Soc. Lond. B

Biol. Sci. 367, 3420–3429.Damkjær, J., Kere€ıche, S., Johnson, M., Kov�acs, L., Kiss, A., Boekema, E.,

Ruban, A., Horton, P. and Jansson, S. (2009) The photosystem II light--

harvesting protein Lhcb3 affects the macrostructure of photosystem II

and the rate of state transitions in Arabidopsis. Plant Cell, 21, 3245–3256.Dannehl, H., Herbik, A. and Godde, D. (1995) Stress-induced degradation of

the photosynthetic apparatus is accompanied by changes in thylakoid

protein turnover and phosphorylation. Physiol. Plant. 93, 179–186.Eberhard, S., Finazzi, G. and Wollman, F.A. (2008) The dynamics of photo-

synthesis. Annu. Rev. Genet. 42, 463–515.Farber, A., Young, A.J., Ruban, A.V., Horton, P. and Jahns, P. (1997) Dynam-

ics of xanthophyll-cycle activity in different antenna subcomplexes in the

photosynthetic membranes of higher plants – the relationship between

zeaxanthin conversion and nonphotochemical fluorescence quenching.

Plant Physiol. 115, 1609–1618.Fristedt, R., Herdean, A., Blaby-Haas, C.E., Mamedov, F., Merchant, S.S.,

Last, R.L. and Lundin, B. (2015) PSB33, a protein conserved in the plastid

lineage, is associated with the chloroplast thylakoid membrane and pro-

vides stability to photosystem II supercomplexes in Arabidopsis. Plant

Physiol. 167, 481–492.Fu, A., He, Z., Cho, H.S., Lima, A., Buchanan, B.B. and Luan, S. (2007) A

chloroplast cyclophilin functions in the assembly and maintenance of

photosystem II in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA, 104,

15947–15952.Garc�ıa-Cerd�an, J.G., Kov�acs, L., T�oth, T., Kere€ıche, S., Aseeva, E., Boekema,

E.J., Mamedov, F., Funk, C. and Schr€oder, W.P. (2011) The PsbW protein

stabilizes the supramolecular organization of photosystem II in higher

plants. Plant J. 65, 368–381.Georgakopoulos, J.H. and Argyroudi-Akoyunoglou, J.H. (1998) Thylakoid

protein phosphorylation is suppressed by ‘free radical scavengers’. Cor-

relation between PSII core protein degradation and thylakoid protein

phosphorylation. Photosynth. Res. 58, 269–280.

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

742 Jun Liu and Robert L. Last

Giacomelli, L., Rudella, A. and van Wijk, K.J. (2006) High light response of

the thylakoid proteome in Arabidopsis wild type and the ascorbate-defi-

cient mutant vtc2–2. A comparative proteomics study. Plant Physiol. 141,

685–701.Goral, T.K., Johnson, M.P., Kirchhoff, H., Ruban, A.V. and Mullineaux, C.W.

(2010) Visualizing the diffusion of chlorophyll-proteins in higher plant

thylakoid membranes: effects of photoinhibition and protein phosphory-

lation. Plant J. 62, 948–959.Herbstova, M., Tietz, S., Kinzel, C., Turkina, M.V. and Kirchhoff, H. (2012)

Architectural switch in plant photosynthetic membranes induced by light

stress. Proc. Natl Acad. Sci. USA, 109, 20130–20135.Jahns, P. and Holzwarth, A.R. (2012) The role of the xanthophyll cycle and

of lutein in photoprotection of photosystem II. Biochim. Biophys. Acta,

1817, 182–193.Jansen, M.A.K., Gaba, V., Greenberg, B.M., Mattoo, A.K. and Edelman, M.

(1996) Low threshold levels of UV–B in a background of photosyntheti-

cally acive radiation trigger rapid degradation of the D2 protein of photo-

system-II. Plant J. 9, 693–699.J€arvi, S., Suorsa, M. and Aro, E.-M. (2015) Photosystem II repair in plant

chloroplasts — regulation, assisting proteins and shared components

with photosystem II biogenesis. Biochim. Biophys. Acta, doi 10.1016/

j.bbabio.2015.01.006 [Epub ahead of Print].

Jin, H., Liu, B., Luo, L. et al. (2014) Hypersensitive to high light1 interacts

with low quantum yield of photosystem II1 and functions in protection

of photosystem II from photodamage in Arabidopsis. Plant Cell, 26,

1213–1229.Johnson, M.P. and Ruban, A.V. (2010) Arabidopsis plants lacking PsbS

protein possess photoprotective energy dissipation. Plant J. 61, 283–289.Kanervo, E., Suorsa, M. and Aro, E.-M. (2005) Functional flexibility and accli-

mation of the thylakoid membrane. Photochem. Photobiol. Sci. 4, 1072–1080.

Kato, Y. and Sakamoto, W. (2014) Phosphorylation of photosystem II core

proteins prevents undesirable cleavage of D1 and contributes to the fine-

tuned repair of photosystem II. Plant J. 79, 312–321.Koivuniemi, A., Aro, E.-M. and Andersson, B. (1995) Degradation of the D1-

and D2-proteins of photosystem II in higher plants is regulated by

reversible phosphorylation. Biochemistry, 34, 16022–16029.Komenda, J., Sobotka, R. and Nixon, P.J. (2012) Assembling and maintain-

ing the photosystem II complex in chloroplasts and cyanobacteria. Curr.

Opin. Plant Biol. 15, 245–251.Kov�acs, L., Damkjaer, J., Kere€ıche, S., Ilioaia, C., Ruban, A.V., Boekema,

E.J., Jansson, S. and Horton, P. (2006) Lack of the light-harvesting com-

plex CP24 affects the structure and function of the grana membranes of

higher plant chloroplasts. Plant Cell, 18, 3106–3120.Krause, G.H. (1988) Photoinhibiton of photosynthesis: an evaluation of dam-

aging and protective mechanisms. Physiol. Plant. 74, 566–574.Li, Z., Wakao, S., Fischer, B.B. and Niyogi, K.K. (2009) Sensing and respond-

ing to excess light. Annu. Rev. Plant Biol. 60, 239–260.Liu, J., Yang, H., Lu, Q., Wen, X., Chen, F., Peng, L., Zhang, L. and Lu, C.

(2012) PsbP-domain protein1, a nuclear-encoded thylakoid lumenal pro-

tein, is essential for photosystem I assembly in Arabidopsis. Plant Cell,

24, 4992–5006.Long, S.P., Humphries, S. and Falkowski, P.G. (1994) Photoinhibition of pho-

tosynthesis in nature. Plant Physiol. Plant. Mol. Biol. 45, 633–662.Lu, Y. (2011) The occurrence of a thylakoid-localized small zinc finger pro-

tein in land plants. Plant Signal. Behav. 6, 1881–1885.Lu, Y., Savage, L.J., Ajjawi, I. et al. (2008) New connections across path-

ways and cellular processes: industrialized mutant screening reveals

novel associations between diverse phenotypes in Arabidopsis. Plant

Physiol. 146, 1482–1500.Lu, Y., Hall, D.A. and Last, R.L. (2011a) A small zinc finger thylakoid protein

plays a role in maintenance of photosystem II in Arabidopsis thaliana.

Plant Cell, 23, 1861–1875.Lu, Y., Savage, L.J., Larson, M.D., Wilkerson, C.G. and Last, R.L. (2011b)

Chloroplast 2010: a database for large-scale phenotypic screening of Ara-

bidopsis mutants. Plant Physiol. 155, 1589–1600.Mart�ınez-Garc�ıa, J.F., Monte, E. and Quail, P.H. (1999) A rapid and quantita-

tive method for preparing Arabidopsis protein extracts for immunoblot

analysis. Plant J. 20, 251–257.

M€uller, P., Li, X.P. and Niyogi, K.K. (2001) Non-photochemical quenching. A

response to excess light energy. Plant Physiol. 125, 1558–1566.Mulo, P., Sirpi€o, S., Suorsa, M. and Aro, E.-M. (2008) Auxiliary proteins

involved in the assembly and sustenance of photosystem II. Photosynth.

Res. 98, 489–501.Murata, N., Takahashi, S., Nishiyama, Y. and Allakhverdiev, S.I. (2007) Pho-

toinhibition of photosystem II under environmental stress. Biochim. Bio-

phys. Acta, 1767, 414–421.Nath, K., Jajoo, A., Poudyal, R.S., Timilsina, R., Park, Y.S., Aro, E.-M., Nam,

H.G. and Lee, C.H. (2013) Towards a critical understanding of the photo-

system II repair mechanism and its regulation during stress conditions.

FEBS Lett. 587, 3372–3381.Nickelsen, J. and Rengstl, B. (2013) Photosystem II assembly: from cyano-

bacteria to plants. Annu. Rev. Plant Biol. 64, 609–635.Nishiyama, Y. and Murata, N. (2014) Revised scheme for the mechanism of

photoinhibition and its application to enhance the abiotic stress toler-

ance of the photosynthetic machinery. Appl. Microbiol. Biotechnol. 98,

8777–8796.Nixon, P.J., Michoux, F., Yu, J., Boehm, M. and Komenda, J. (2010) Recent

advances in understanding the assembly and repair of photosystem II.

Ann. Bot. 106, 1–16.Niyogi, K.K. (1999) Photoprotection revisited: genetic and molecular

approaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 333–359.Peltier, J.B., Ytterberg, A.J., Sun, Q. and van Wijk, K.J. (2004) New functions

of the thylakoid membrane proteome of Arabidopsis thaliana revealed

by a simple, fast, and versatile fractionation strategy. J. Biol. Chem. 279,

49367–49383.Prasil, O., Adir, N. and Ohad, I. (1992) Dynamics of photosystem II: mecha-

nisms of photoinhibition and recovery process. In The Photosystems:

Structure, Function and Molecular Biology, Vol. 11 (Barber, J., ed.).

Amsterdam: Elsevier Science Publishers, pp. 295–348.Puthiyaveetil, S., Tsabari, O., Lowry, T., Lenhert, S., Lewis, R.R., Reich, Z.

and Kirchhoff, H. (2014) Compartmentalization of the protein repair

machinery in photosynthetic membranes. Proc. Natl Acad. Sci. USA, 111,

15839–15844.Rokka, A., Suora, M., Saleem, A., Battchikova, N. and Aro, E.-M. (2005) Syn-

thesis and assembly of thylakoid protein complexes: multiple assembly

steps of photosystem II. Biochem. J. 388, 159–168.Ruban, A.V., Johnson, M.P. and Duffy, C.D.P. (2012) The photoprotective

molecular switch in the photosystem II antenna. Biochim. Biophys. Acta,

1817, 167–181.Savage, L.J., Imre, K.M., Hall, D.A. and Last, R.L. (2013) Analysis of essential

Arabidopsis nuclear genes encoding plastid-targeted proteins. PLoS

ONE, 8, e73291.

Schneider, C.A., Rasband, W.S. and Eliceiri, K.W. (2012) NIH Image to Ima-

geJ: 25 years of image analysis. Nat. Methods, 9, 671–679.Schwacke, R., Schneider, A., van der Graaff, E., Fischer, K., Catoni, E., Desi-

mone, M., Frommer, W.B., Flugge, U.I. and Kunze, R. (2003) ARAMEM-

NON, a novel database for Arabidopsis integral membrane proteins.

Plant Physiol. 131, 16–26.Shi, L.X. and Schr€oder, W.P. (2004) The low molecular mass subunits of the

photosynthetic supracomplex, photosystem II. Biochim. Biophys. Acta,

1608, 75–96.Sirpi€o, S., Khrouchtchova, A., Allahverdiyeva, Y., Hansson, M., Fristedt, R.,

Vener, A.V., Scheller, H.V., Jensen, P.E., Haldrup, A. and Aro, E.-M.

(2008) AtCYP38 ensures early biogenesis, correct assembly and suste-

nance of photosystem II. Plant J. 55, 639–651.Takahashi, S. and Badger, M.R. (2011) Photoprotection in plants: a new light

on photosystem II damage. Trends Plant Sci. 16, 53–60.Tanz, S.K., Castleden, I., Hooper, C.M., Vacher, M., Small, I. and Millar, H.A.

(2013) SUBA3: a database for integrating experimentation and prediction

to define the SUBcellular location of proteins in Arabidopsis. Nucleic

Acids Res. 41, D1185–D1191.Tikkanen, M. and Aro, E.M. (2012) Thylakoid protein phosphorylation in

dynamic regulation of photosystem II in higher plants. Biochim. Biophys.

Acta, 1817, 232–238.Yamamoto, Y., Aminaka, R., Yoshioka, M. et al. (2008) Quality control of

photosystem II: impact of light and heat stresses. Photosynth. Res. 98,

589–608.

© 2015 The AuthorsThe Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 731–743

A PSII-associated thylakoid protein MPH1 743