The Arabidopsis Tellurite resistance C protein together with ALB3 is involved in photosystem II protein synthesis.

The Plant Journal (2014) 78, 344–356

doi: 10.1111/tpj.12474

The Arabidopsis Tellurite resistance C protein together with ALB3 is involved in photosystem II protein synthesis Anja Schneider1,†,*, Iris Steinberger1,†, Henning Strissel1, Hans-Henning Kunz2,‡, Nikolay Manavski1, Jo€ rg Meurer1, € nemann3, Stefan Geimer4, Ulf-Ingo Flu € gge2 and Dario Leister1 Gabi Burkhard1, Sabine Jarzombski1, Danja Schu 1 Molekularbiologie der Pflanzen (Botanik), Department Biologie I, Ludwig Maximilians Universität München, 82152 Martinsried, Germany, 2 Biozentrum Ko€ ln, Botanisches Institut der Universität zu Köln, Lehrstuhl II, 50674 Köln, Germany, 3 Arbeitsgruppe Molekularbiologie Pflanzlicher Organellen, Ruhr Universität Bochum, 44801 Bochum, Germany, and 4 Zellbiologie/Elektronenmikroskopie, Universita€ t Bayreuth, 95447 Bayreuth, Germany Received 14 August 2013; accepted 4 February 2014; published online 25 February 2014. *For correspondence (email [email protected]). † These authors contributed equally to this work. ‡ Present address: Division of Biological Science and Center for Molecular Genetics, University of California, San Diego, CA 92093-0116, USA.

SUMMARY Assembly of photosystem II (PSII) occurs sequentially and requires several auxiliary proteins, such as ALB3 (ALBINO3). Here, we describe the role of the Arabidopsis thaliana thylakoid membrane protein Tellurite resistance C (AtTerC) in this process. Knockout of AtTerC was previously shown to be seedling-lethal. This phenotype was rescued by expressing TerC fused C–terminally to GFP in the terc–1 background, and the resulting terc–1TerC–GFP line and an artificial miRNA-based knockdown allele (amiR-TerC) were used to analyze the TerC function. The alterations in chlorophyll fluorescence and thylakoid ultrastructure observed in amiR-TerC plants and terc–1TerC–GFP were attributed to defects in PSII. We show that this phenotype resulted from a reduction in the rate of de novo synthesis of PSII core proteins, but later steps in PSII biogenesis appeared to be less affected. Yeast two-hybrid assays showed that TerC interacts with PSII proteins. In particular, its interaction with the PSII assembly factor ALB3 has been demonstrated by co-immunoprecipitation. ALB3 is thought to assist in incorporation of CP43 into PSII via interaction with Low PSII Accumulation2 (LPA2) Low PSII Accumulation3 (LPA3). Homozygous lpa2 mutants expressing amiR-TerC displayed markedly exacerbated phenotypes, leading to seedling lethality, indicating an additive effect. We propose a model in which TerC, together with ALB3, facilitates de novo synthesis of thylakoid membrane proteins, for instance CP43, at the membrane insertion step. Keywords: Arabidopsis thaliana, photosystem II, assembly, thylakoid membrane, AtTerC, ALB3.

INTRODUCTION Photosystem II (PSII) is a multi-protein complex that is located in the thylakoid membrane, which catalyzes lightinduced transfer of electrons from water to plastoquinone, thereby producing oxygen. In the chloroplasts of higher plants, PSII consists of more than 20 subunits (Wollman et al., 1999; Iwata and Barber, 2004; Nelson and Yocum, 2006). Significant progress has been achieved in recent years in understanding the complex process of PSII assembly (Komenda et al., 2012). PSII biogenesis starts with addition of a pre-complex containing the D1 precursor (pD1) and the subunit PsbI to a pre-complex consisting of subunits D2, PsbE and PsbF, to form the PSII reaction center € ller and Eichacker, 1999; complex (van Wijk et al., 1997; Mu Zhang et al., 1999; Rokka et al., 2005). Integration of 344

subunit CP47 into the reaction center generates the next PSII sub-assembly complex. Then subunit PsbH and three other low-molecular-mass subunits are incorporated (Rokka et al., 2005) to form a CP43-free PSII monomer. The subunit CP43 is synthesized independently and inserted into the PSII complex concomitantly with subunit PsbK. This step appears to be essential for dimerization of PSII monomers, and assimilation of CP43 is also a prerequisite for stable association of the extrinsic subunit PsbO (Rokka et al., 2005). PsbO, together with PsbP, PsbQ and PsbR at the lumenal side of PSII, is required for O2 evolution (Roose et al., 2007; Enami et al., 2008, Allahverdiyeva et al., 2013). Finally, PSII super-complexes are formed by attachment of Light Harvesting Complex II (LHCII) trimers © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd

TerC function in thylakoid membrane protein synthesis 345 to PSII dimers (Boekema et al., 1995; Hankamer et al., 1997), probably via Lhcb4, Lhcb5 and Lhcb6, which serve as linkers (Nelson and Yocum, 2006). Over recent years, almost a dozen PSII assembly factors have been identified in Arabidopsis thaliana and other species (Mulo et al., 2008; Komenda et al., 2012; Chi et al., 2012). These include several lumenal immunophilins, and knockout alleles of some of the corresponding genes result € et al., in severe growth defects (Lima et al., 2006; Sirpio 2008). However, the most dramatic phenotype yet seen upon inactivation of an assembly factor is observed in Arabidopsis alb3 mutants (Sundberg et al., 1997). These mutants are albinotic and fail to survive beyond the seedling stage. Inactivation of ALB3 genes in Chlamydomonas reinhardtii and Synechocystis sp. PCC6803 also results in severe phenotypes (Bellafiore et al., 2002; Spence et al., 2004). ALB3 has been shown to interact with PSII subunits D1, D2 and CP43, as well as with the photosystem I (PSI) reaction center protein PsaA and the ATPase III subunit (Pasch et al., 2005). The functions of ALB3 orthologs in € hre et al., 2006) € hl et al., 2004, Go C. reinhardtii (Ossenbu and Synechocystis sp. PCC6803 have also been characterized, and the results suggest that they play a role in inte€ hl gration of pD1 into the thylakoid membrane (Ossenbu et al., 2006). In addition to its role in D1 incorporation, there is considerable evidence that ALB3 is also involved in insertion of LHCII and formation of PSII–LHCII supercomplexes (Moore et al., 2000; Woolhead et al., 2001). The role of ALB3 in PSII assembly processes is probably mediated via its interactions with Low PSII Accumulation2 (LPA2) and Low PSII Accumulation3 (LPA3) (Ma et al., 2007; Cai et al., 2010), which are thought to promote assembly of CP43 into CP43-free PSII to form PSII monomers in Arabidopsis (Ma et al., 2007; Cai et al., 2010). The emerging picture of PSII assembly indicates that some assembly factors may operate at several stages during PSII biogenesis. This view is supported by analyses of the Arabidopsis Photosynthesis Affected Mutant68 (PAM68) protein (Armbruster et al., 2010). PAM68 is required for efficient maturation of pD1 and for conversion of the reaction center into larger PSII intermediates. Likewise, it has been shown to interact with ALB3, LPA2, LPA1 and High Chlorophyll Flourescence136 (HCF136) in yeast two-hybrid assays. HCF136 is necessary for early PSII biogenesis (Meurer et al., 1998), and predominantly associates with the PSII pre€ cken et al., 2002). LPA1 binds to D1 during complexes (Plu de novo biogenesis of PSII (Peng et al., 2006), and may act as a chaperone, facilitating correct folding and integration of D1 into the receptor complex. In the present study, we reassessed the function of the integral thylakoid membrane protein TerC in Arabidopsis. AtTerC is nuclear-encoded (At5g12130), and shares sequence similarity with bacterial TerC, which is the product of a member of an operon associated with tellurite

resistance (Jobling and Ritchie, 1987, 1988). The terc lossof-function phenotype resembles that of alb3, in that the mutant is pigment-deficient and seedling-lethal. In this mutant, levels of the thylakoid membrane proteins D1, PsaA and cytochrome f are severely reduced, although transcription of the respective genes is unaffected (Kwon and Cho, 2008). We assessed the terc phenotype using a combination of genetic and biochemical approaches, with particular focus on the molecular mechanism underlying the assembly of PSII. Here we show that TerC is essential for the de novo synthesis of PSII core proteins, presumably via interaction with ALB3. RESULTS Phenotype of terc-1 under dim light It has been shown previously that TerC is not directly involved in chlorophyll biosynthesis (Kwon and Cho, 2008), although terc-1 homozygotes formed pale-green cotyledons when grown for 6 days at 4 lmol photons m2 sec1 (Figure 1). Under these conditions, the chloroplasts of wild-type (WT) leaves retained the typical lensshaped structure, with stacked grana membranes, non-appresed stroma lamellae and starch granules (Figure 1b). In contrast, terc–1 chloroplasts lacked starch granules and organized thylakoid membranes. Instead, they contained tightly to loosely appressed thylakoid lamellae, but no typical grana (Figure 1d–f), indicating that the TerC protein is essential for stacking of thylakoid membranes into grana. When the terc–1 mutant was grown under normal light conditions (100 lmol photons m2 sec1), proteins representative of each of the four major thylakoid membrane protein complexes were almost undetectable (Kwon and Cho, 2008). When we reassessed the levels of thylakoid proteins in the terc–1 mutant grown in dim light, we were unable to detect any of the plastome-encoded PSII core subunits D1, D2, CP43 and CP47, although the Lhcb2 protein of the LHCII accumulated to some extent, but less than 10% of WT levels. However, the PSII assembly factors ALB3, PAM68, LPA1 and LPA2 were clearly detectable in terc–1, with the levels of ALB3 and PAM68 being slightly decreased compared with the WT (Figure 2a). Any involvement of TerC in the transcription of plastome-localized genes and mRNA association with ribosomes has already been ruled out (Kwon and Cho, 2008). To investigate the rates of synthesis of thylakoid membrane proteins in the terc–1 mutant, in vivo pulse-labeling experiments were performed. To block synthesis of nucleus-encoded proteins, labeling with [35S]Met was performed in the presence of cycloheximide. Synthesis of the PSII core proteins CP47, CP43, D2, D1, the PSI core proteins PsaA/B, and the ATPase a/b subunits was clearly observed in WT leaves, but the amounts of newly translated thylakoid membrane proteins were below the detection limit in

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 344–356

346 Anja Schneider et al.

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Figure 1. Phenotype of terc–1 grown under dim light. Wild-type and terc–1 were grown under a photon flux density of 4 lmol photons m2 sec1 for 6 days. (a,b) Cotyledons (a) and a chloroplast (b) from WT. GT, grana thylakoid stacks; ST, stromal thylakoids; S, starch. (c,d) Pale-green cotyledons (c) and a chloroplast (d) with appressed thylakoid stacks from terc–1. (e,f) Two abnormal chloroplasts from terc–1 in which grana thylakoids are loosely appressed and interspersed with small vesicles. T, thylakoid stacks; V, small vesicles; L, lipids.

terc–1 (Figure 2b). The trace amounts of some translated proteins seen in terc–1 may be of mitochondrial origin, as they do not correspond to any labeled product in WT. These results suggests that newly synthesized membrane proteins are very rapidly degraded and/or that their synthesis and co-translational insertion into the thylakoid membrane is impaired in terc–1. Partial rescue of terc–1 using a TerC–GFP fusion protein Because all attempts to generate an antibody that specifically recognized TerC failed, plants over-expressing a

Figure 2. Accumulation of thylakoid membrane proteins in terc–1. (a) Total leaf proteins of wild-type and terc–1, grown under photon flux density of 4 lmol photons m2 sec1 for 6 days, were subjected to immunoblot analysis. Total protein samples (approximately 3.5 mg fresh weight) were loaded in the lanes marked WT and terc–1. Decreasing levels of wild-type proteins were loaded in the lanes marked WT 1/2, WT 1/4 and WT 1/10. Immunoblots were probed with antibodies raised against individual subunits of PSII core proteins, Lhcb2 and assembly factors. Actin served as loading control. (b) Thylakoid membrane proteins of WT and terc–1 seedlings were pulselabeled for 20 min. Samples containing 106 cpm (counts per minute) were loaded in lanes marked WT and terc–1. Decreasing levels of wild-type proteins were loaded in the lanes marked WT 1/2, WT 1/4 and WT 1/10.

TerC–GFP fusion protein were constructed in the WT (WTTerC–GFP) and terc–1 (terc–1TerC–GFP) backgrounds. The phenotype of WTTerC–GFP was indistinguishable from that of the WT with respect to growth and photosynthetic performance, but the TerC transcript level was increased approximately 20-fold (Figure 3 and Table 1). On the other hand, the terc–1TerC–GFP plant was able to grow photoautotrophically but retained a variegated leaf phenotype (Figure 3a). In the variegated leaves, the chlorophyll a fluorescence revealed an increase in the minimum fluorescence (F0) and a decrease in the maximum quantum yield of PSII (FV/FM) (Figure 3a). Consequently, a lower effective quantum yield for PSII in terc–1TerC–GFP was recorded (Table 1). Closer


TerC function in thylakoid membrane protein synthesis 347 Figure 3. Characterization of WTTerC–GFP, terc– 1TerC–GFP and amiR-TerC lines. (a) Four-week-old plants were grown under a photon flux density of 100 photons m2 sec1, and the photosynthetic parameters F0 (middle panel) and FV/FM (bottom panel) were recorded. The color scale at the bottom indicates the signal intensities. (b) Chlorophyll a fluorescence curve for WT and transformed lines. The arrows indicate application of saturating light pulses. The black bars indicate dark incubation, and the white bar indicates exposure to actinic light. F0 and FM are indicated. (c) Relative expression levels of TerC in WT, WTTerC–GFP, terc–1TerC–GFP and amiR-TerC, as determined by quantitative real-time PCR. Values are means SD derived from measurements of three individuals (three technical replicates each).

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Table 1 Parameters of chlorophyll a fluorescence in wild-type and transgenic lines ΦII

FV/FM WT WTTerC–GFP terc–1TerC–GFP amiR-TerC

0.82 0.82 0.73 0.69

0.03 0.01 0.04 0.08

0.74 0.68 0.58 0.52

the GFP-tagged version of the Synechocystis ALB3/Oxa1 € hl et al., 2006). homolog (Ossenbu amiR-TerC lines phenocopy terc–1Ter–GFP lines

0.04 0.01 0.04 0.08

Values are means SD for five different plants. The actinic light intensity was 62 lmol photons m2 sec1. FV/FM, maximum quantum yield of PSII; ΦII, effective quantum yield of PSII.

inspection of the fluorescence curves revealed that the chlorophyll a fluorescence decreases to close to the F0 level after the initial rise induced by exposure to actinic light (Figure 3b). These observations suggest that PSII activity is not fully restored in terc–1TerC–GFP. Moreover, the phenotype of terc–1TerC–GFP did not result from poor expression of the TerC–GFP transcript, as the level of TerC RNA was approximately twice as high as in WT (Figure 3c). Furthermore, the TerC–GFP fusion protein was correctly targeted to chloroplasts and behaved as an integral membrane protein in terc–1TerC–GFP (Figure S1, Methods S1 and S2). Taken together, these results demonstrate that GFP-tagged TerC only partially substitutes for the WT protein. This is probably because the C–terminally fused GFP tag interferes with the physiological function of TerC, as has been observed for

To further assess the TerC function, a knockdown terc allele was generated. An artificial miRNA specifically targeting nucleotides 657–677 of the endogenous TerC transcript (Figure S2) was introduced into WT, and five T1 plants were further characterized. All five exhibited a variegated phenotype. One of these lines, which carries a single-copy homozygous insertion, was selected for further use in the T3 generation, and is designated as amiR-TerC. The variegated phenotype of amiR-TerC resembles that of terc–1TerC–GFP (Figure 3a). Further similarities were found between amiR-TerC and terc–1TerC–GFP with respect to photosynthetic performance. In the palegreen sectors of amiR-TerC leaves, the minimum chlorophyll a fluorescence (F0) was increased (Figure 3a), while FV/FM and the effective quantum yield of PSII were reduced (Table 1). In amiR-TerC, chlorophyll a fluorescence decreased to levels just below F0 after actinic light induction (Figure 3b), indicating imbalanced photosynthesis. We also examined the degree of down-regulation of TerC expression in amiR-TerC using quantitative RT–PCR. The data indicated that amiR-TerC plants expressed only approximately 17% as much TerC RNA as WT plants (Figure 3c).


348 Anja Schneider et al. Formation of grana stacks is perturbed in amiR-TerC and terc–1TerC–GFP plants To investigate the ultrastructure of thylakoid organization, WT, WTTerC–GFP, terc–1TerC–GFP and amiR-TerC plants were analyzed by transmission electron microscopy. Chloroplasts from 14-day-old leaves of WTTerC–GFP were indistinguishable from their WT counterparts with respect to plastid structure, thylakoid formation and starch accumulation (Figure 4a–c). The variegated phenotype of terc– 1TerC–GFP and amiR-TerC was reflected in the ultrastructure of their chloroplasts. In the green parts of terc–1TerC–GFP leaves, the majority of plastids displayed an ordered thylakoid structure with grana stacks and stroma thylakoids

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(Figure 4d). In contrast, the vast majority of plastids in the pale-green sectors of terc–1TerC–GFP leaves had disordered thylakoids. Membrane stacks were very loosely appressed, and there was no clear distinction between grana and stroma lamellae (Figure 4e,f). Nevertheless, stroma-localized metabolic pathways remained active, as indicated by the accumulation of starch. A similar situation was found in leaves of amiR-TerC. In green tissue, plastids with an ordered thylakoid structure characterized by grana stacks and stroma thylakoids were observed (Figure 4 g). In palegreen tissues, grana stacks of thylakoids were only loosely appressed, and connections between adjacent stacks appeared to be missing (Figure 4 h,i). Thus, the chloroplast ultrastructure is in good agreement with the observed photosynthetic parameters, indicating that the low FV/FM ratio (Figure 3a) is due to a disordered thylakoid structure. Levels of PSII core proteins are reduced in terc–1TerC–GFP and amiR-TerC

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The results obtained so far led us to focus on the impact of TerC on PSII. Total protein extracts were prepared from the four genotypes (WT, WTTerC–GFP, terc–1TerC–GFP and amiRTerC) (Figure 5a). The levels of plastome-encoded PSII core subunits CP47, CP43, D2 and D1 were reduced to approximately 50% of WT levels in amiR-TerC, and to approximately 40% of WT levels in terc–1TerC–GFP. The nuclear-encoded Lhcb2 subunit behaved similarly, with levels of 45% in amiR-TerC and 54% in terc–1TerC–GFP relative to the WT level (Figure 5a,b). WTTerC–GFP retained WT levels of the inspected proteins. In contrast to the reduced amount of PSII subunits in amiR-TerC and terc–1TerC–GFP, the levels of PSII assembly factors were heterogeneous. Whereas the amount of ALB3 and LPA2 remained at WT levels in all genotypes, the amount of LPA1 and PAM68 was reduced in amiR-TerC and terc–1TerC–GFP (Figure 5a,b). Regardless of the amount of PSII proteins, the ability to form PSII complexes was maintained in amiR-TerC and terc–1TerC–GFP, as deduced from blue native gel electrophoresis experiments (Figure S4 and Methods S3). Synthesis of thylakoid membrane proteins in terc–1TerC–GFP and amiR-TerC

Figure 4. Electron micrographs of WT, WTTerC–GFP, terc–1TerC–GFP and amiRTerC chloroplasts. (a–c) WT (a) and WTTerC–GFP chloroplasts (b), and a close-up view of a WTTerC–GFP chloroplast (c). L, lipid; S, starch. (d–f) A moderately (d) and a strongly affected chloroplast (e) from terc– 1TerC–GFP, and a close-up of the strongly affected chloroplast (f). T, thylakoid; S, starch. (g–i) Analogous examples from amiR-TerC., that is a moderately (g) and a strongly affected chloroplast (h, i). T, thylakoid.

To investigate rates of synthesis of thylakoid membrane protein in our four genotypes, in vivo pulse-labeling experiments were performed After pulse labeling, thylakoid membrane proteins from the four genotypes were separated into PsaA/B, the ATPase a/b subunits, CP47, CP43, D2 and D1 (Figure 5c). In both terc–1TerC–GFP and amiR-TerC samples, incorporation of radioactivity was less efficient, and in WTTerC–GFP samples, incorporation was slightly higher than in WT samples. The level of incorporation of [35S]Met into the ATPase a/b subunits and the D1 and D2 proteins was approximately 50% in terc–1TerC–GFP and amiR-TerC compared to WT. However, radiolabeled CP47


TerC function in thylakoid membrane protein synthesis 349 Figure 5. Accumulation of thylakoid membrane proteins in WT, WTTerC–GFP, terc–1TerC–GFP and amiR-TerC. (a) Total leaf proteins were subjected to immunoblot analysis. Protein samples (3.5 mg fresh weight) were loaded in the lanes marked WT, WTTerC–GFP, terc–1TerC–GFP and amiR-TerC. Half the amount of wild-type proteins was loaded in the lane marked WT 1/2. Immunoblots were probed with antibodies raised against individual subunits of PSII core proteins, Lhcb2 and assembly factors. Actin was used as a loading control. (b) Quantification of thylakoid membrane proteins from (a) and two further replications. Protein levels relative to those in wild-type were calculated. Values are means SD. (c) Thylakoid membrane proteins of leaves were pulse-labeled for 20 min. Samples containing 106 cpm (counts per minute) were loaded in the lanes marked WT, WTTerC–GFP, terc–1TerC–GFP and amiR-TerC. A sample containing half the amount of radioactivity was loaded in the lane marked WT 1/2. (d) After pulse labeling, leaves were infiltrated with cold Met and incubated for 2 h. Samples containing 106 cpm (counts per minute) were loaded in the lanes marked WT, WTTerC–GFP, terc–1TerC–GFP and amiR-TerC. A sample containing half the amount of radioactivity was loaded in the lane marked WT 1/2.

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and CP43 accumulated to less than 50% in both genotypes (Figure 5c). Radiolabeled thylakoid proteins were stable for at least 2 h in all four genotypes, as determined by a chase experiment with unlabeled Met (Figure 5d). Based on this observation and on results obtained with terc–1 (Figure 2b), we conclude that TerC primarily effects de novo synthesis of thylakoid membrane proteins, with particular effect on CP47 and CP43. In addition, kinetic studies of PSII assembly in amiR-TerC and WT showed no major difference in PSII assembly, confirming that TerC mostly functions during earlier steps (Figure S3 and Methods S3). TerC–GFP is detected in the low-molecular-weight range The two genotypes WTTerC–GFP and terc–1TerC–GFP provided the opportunity to study the distribution of TerC–GFP. Sucrose gradient centrifugation experiments were performed. We found no difference between WT and the genotypes WTTerC–GFP, terc–1TerC–GFP and amiR-TerC with respect to the distribution of LHCII and PSI complexes. Probing of gradient fractions using anti-CP47, anti-CP43 and anti-D2 detected the expected PSII complexes from approximately 200 kDa to > 450 kDa in all four genotypes (Figure 6a). In addition, CP43 was identified in lowermolecular-weight fractions (Ma et al., 2007), i.e. at approximately 67–160 kDa, in WT and WTTerC–GFP, respectively (Figure 6a). Particularly noteworthy was the strong reduction in the amount of CP43 in these fractions (fractions 6– 8) in amiR-TerC and terc–1TerC–GFP (Figure 6a,b). Moreover,

these low-molecular-weight fractions (fractions 6–9) also contained the TerC–GFP fusion protein (Figure 6a). When blue-native gel electrophoresis experiments were performed, the GFP signal in terc–1TerC–GFP presumably represented the TerC–GFP fusion protein itself, which has a predicted molecular weight of 64 kDa. A GFP signal with the same mobility was detected in WTTerC–GFP; however, a small amount of TerC–GFP co-migrated with PSII complexes in WTTerC–GFP (Figure S4). TerC interacts in particular with ALB3 If TerC is involved in insertion of thylakoid membrane proteins, it may well interact with subunits of the PSII complex. To test this notion, we used a modified split-ubiquitin system (Pasch et al., 2005). The results indicated that TerC does not interact with the PSI subunits PsaA and PsaB, nor with ferredoxin or the ATPase IV subunit (AtpI). Furthermore, it did not interact with components of the signal recognition pathway (FtsY) (Kogata et al., 1999) or the secretory thylakoid targeting pathway (SecY and SecE) (Roy and Barkan, 1998; Schu€ nemann et al., 1999) (Figure 7a and Figure S5). However, with regard to PSII subunits, TerC interacted with D1, D2 and CP43, but not with CP47, PsbE or PsbO (Figure 7a). Furthermore, TerC also interacted with LPA2, and with other known PSII assembly factors such as LPA1, PAM68 and ALB3, but not with LPA3 or HCF136 (Figure 7a and Figure S5). The interactions of TerC with ALB3, LPA2 and CP43 suggest a role for TerC during insertion of CP43 into the thylakoid membrane.


350 Anja Schneider et al. Figure 6. Sucrose-gradient analysis of thylakoid membrane complexes. (a) Thylakoids (1 mg of chlorophyll ml1) from WT, WTTerC–GFP, terc–1TerC–GFP and amiR-TerC were solubilized and fractionated. The positions of LHCII, PSII and PSI complexes, and the molecular mass markers equine myoglobin (17.8 kDa), bovine serum albumin (67 kDa), rabbit aldolase (160 kDa) and horse ferritin (450 kDa) are indicated. Nineteen fractions were collected, and proteins were identified by immunoblotting. No signal was obtained with anti-GFP in the WT and amiR-TerC lanes. (b) The distribution of CP43 over the 19 fractions from the sucrose gradients was determined from (a). The relative amounts of CP43 (total of 1 for each genotype) were calculated from three replications. Values are means SD. Asterisks indicate values that are statistically significantly different compared with WT (Student’s t test, *P < 0.05; **P < 0.02).

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To study further whether interaction also occurs in vivo, chloroplasts from WT and terc-1TerC–GFP plants were isolated. Proteins were cross-linked, and solubilized thylakoid membranes were incubated with magnetic beads coupled to anti-GFP antibody. Co-immunoprecipitated samples were subjected to immunoblot analysis. Our results showed that TerC–GFP and ALB3 were immunoprecipitated by the anti-GFP antibody (Figure 7b). This result provides additional evidence for an interaction between TerC and ALB3. However, we did not detect interactions of TerC–GFP with D1, D2 and CP43, presumably because TerC is only loosely and/or transiently associated with these components in vivo and thus these interactions are difficult to trap.

Down-regulation of TerC expression in the lpa2 mutant causes a seedling-lethal phenotype To study the physiological effects of diminished TerC function in combination with less efficient PSII assembly, we introduced the amiR-TerC construct into the lpa2 and pam68–2 mutants and the PSI mutant psad1–1 (Ihnatowicz et al., 2004), and analyzed the T1 generation (Table 2). Twenty-one kanamycin-resistant lpa2 seedlings were recovered, which is equivalent to a transformation efficiency of 0.2%. The transformation efficiencies were higher in WT, psad1–1 and pam68–2 (Table 2). All primary transformed lines in the WT, psad1–1 and pam68–2 backgrounds developed pale-green and variegated leaves


TerC function in thylakoid membrane protein synthesis 351

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Table 2 Overview of generated amiR-TerC transgenic lines

WT psad1–1 pam68–2 lpa2

T1 transformants/ total seeds

Transformation frequency, %

T2 seed production

11/2.750 18/3.600 15/3.750 21/10.500

0.4 0.5 0.4 0.2

5 6 9 0

The indicated genotypes were subjected to floral dipping using an Agrobacterium strain harboring plasmid p35S:amiR-TerC.

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Figure 7. Interaction studies of TerC with thylakoid membrane proteins. (a) The full-length TerC protein was N–terminally fused to Cub (C terminal half of ubiquitin) (TerCCub) and selected proteins were C–terminally fused to NubG (N terminal half of ubiquitin). Alg5 (an endoplasmic reticulum membrane protein) fused to NubG served as a negative control, and Alg5 fused to NubI (the wild-type Nub) served as a positive control. Yeast colonies were grown on permissive medium lacking Leu and Trp (–LT), and interaction of the Cub (C) and NubG (N) halves of ubiquitin is indicated by the ability to grow on selective medium lacking Leu, Trp and His (–LTH). (b) Co-immunoprecipitation analysis using terc–1TerC–GFP. Solubilized thylakoid membranes were incubated with anti-GFP antibody, and the precipitated proteins separated, followed by immunoblot analysis. Thylakoid membranes isolated from wild-type served as a negative control. Input, solubilized thylakoid membranes; Co–IP, co-immunoprecipitated proteins; S.n., supernatant.

(Figure 8a). Five plant lines (amiR-TerC and amiR-TerC#2 to amiRTerC#5) in the WT background, six in the psad1–1 background (psad1–1 amiR-TerC#1 to psad1–1 amiR-

TerC#6) and nine in the pam68–2 background (pam68–2 amiR-TerC#1 to pam68–2 amiR-TerC#9) were established for seed production (Table 2). None of the 21 transformed lpa2 lines completed the life cycle. These lpa amiR-TerC lines were grouped into three categories. One group developed albinotic cotyledons, the second developed yellowish leaves and the third developed pale-green leaves (Figure 8a,b). For further analysis, two individuals (lpa2 amiR-TerC#5 and lpa2 amiR-TerC#6) belonging to the third group were transferred to greenhouse conditions. A representative line of each genotype is shown in Figure 8(c). As expected, amiR-TerC#4 showed a clear increase in F0 and a decrease in FV/FM (Figure 8c and Table S1) compared to WT. The psad1–1 amiR-TerC#3 line behaved in a similar manner, considering that the FV/FM value in the parental line psad1–1 is already decreased due to a pleiotropic effect (Ihnatowicz et al., 2004). An interesting difference was found between pam68–2 amiR-TerC#3 and lpa2 amiR-TerC#5. In both parental lines, pam68–2 and lpa2, a similar decrease in FV/FM resulting from impairment in PSII was observed (Figure 8c and Table S1). In pam68–2 amiR-TerC#3, a further decrease in FV/FM was evident. The lpa2 amiR-TerC#5 line died early (see above), but young leaves showed a strong reduction in FV/FM (Figure 8c and Table S1). Thus, the combination of diminished TerC function and complete loss of LPA2 resulted in lethality at the seedling stage. DISCUSSION Here we describe a role for TerC in assisting synthesis of thylakoid membrane proteins. Because TerC is an integral membrane protein (Kwon and Cho, 2008; Figure S1) with eight predicted a-helices (http://aramemnon.botanik.unikoeln.de/), it is tempting to speculate that TerC assists this process at the membrane insertion step. Complete loss of TerC results in early lethality at the seedling stage (Kwon and Cho, 2008). In particular, synthesis of thylakoid membrane proteins is abrogated in the terc–1 mutant (Figure 2), and partitioning of thylakoid membranes into grana stacks and stroma lamellae does not occur (Figure 1). In order to be able to dissect the underlying mechanisms, and to avoid problems associated with pleiotropic effects, a


352 Anja Schneider et al.

(a)

(b)

(c)

Figure 8. Generation of amiR-TerC lines in various genotypes. (a) Kanamycin selection of amiR-TerC plant lines. WT, psad1–1, pam68–2 and lpa2 lines were grown on MS medium, and the respective transformed lines were grown on MS medium containing kanamycin. Two to three independent transformants are shown. lpa2 amiR-TerC plants displayed albinotic (#1, circled), yellowish (#2) and pale-green (#3) phenotypes. Note that kanamycin-sensitive seedlings do not produce roots. (b) In total, 21 transformed lpa2 amiR-TerC lines were isolated and grouped into three categories. (c) Kanamycin-resistant plant lines were transferred to soil, cultivated at a photon flux density of 100 lmol photons m2 sec1 for 1 week, and the photosynthetic parameters F0 (middle panel) and FV/FM (bottom panel) were recorded. The color scale at the bottom indicates the signal intensities. WT, psad1–1, pam68–2 and lpa2 plants at similar developmental stages as the respective amiR-TerC plants were used as controls.

TerC is required for efficient insertion of thylakoid membrane proteins

Figure 9. Model for the role of TerC in CP43 de novo synthesis. In this model, the integral membrane protein TerC works in combination with ALB3, presumably at the co-translational membrane insertion step of CP43 (black). Putative additional factors are indicated by a question mark. LPA2 and LPA3, two other thylakoid membrane proteins, assist with CP43 assembly into PSII, and both interact with ALB3 (Chi et al., 2012). In this model, ALB3 works as a mediator connecting these two processes. For clarity, the low-molecular-mass subunits are omitted.

knockdown allele of TerC (amiR-TerC) was generated. The amiR-TerC plants showed an increase in F0, while FV/FM and the effective quantum yield of PSII were reduced (Figure 3 and Table 1). Similar data were obtained with the partially complemented line terc–1TerC–GFP (Figure 3 and Table 1). Given the effect of TerC on accumulation of Lhcb2 (Figure 5) and grana formation (Figure 4), the increased F0 level may be caused by fewer assembled antenna proteins and/or a reduced state of plastoquinone acceptors in the dark. Thus, in both of these lines, the severe terc–1 phenotype is attenuated, allowing us to study the function of TerC.

The terc–1 mutant, when grown in dim light, lacked the core proteins of PSII, namely D1, CP47, CP43 and D2 (Figure 2a), and in vivo labeling of chloroplast proteins directly demonstrated that the rates of synthesis of PSII core proteins in the terc–1 mutants were more than tenfold lower than in WT (Figure 2b). Polysome association of the corresponding transcripts is not affected in the mutant (Kwon and Cho, 2008), so the rapid loss of newly synthesized PSII core proteins may only be attributed to inhibition of their co-translational integration into thylakoids. To date, only D1 has been shown experimentally to be integrated into PSII co-translationally, being inserted into the thylakoid membrane as the nascent D1 precursor (Zhang et al., 1999). The translational and post-translational regulatory mechanisms that prevent accumulation of PSII subunits in the absence of assembly partners (Zhang et al., 1999; Baena-Gonzalez and Aro, 2002; Zerges, 2002; Minai et al., 2006) may explain the complete absence of PSII core proteins in the terc–1 mutant, but the profound defects in thylakoid membrane organization seen in the terc–1 mutant imply that TerC has a broader role. Only a few of the tightly appressed lamellae that are characteristic of the PSII assembly factor mutants hcf136 (Meurer et al., 1998) and alb3 (Sundberg et al., 1997) were found in the terc–1 mutant (Figure 1). In contrast, a tobacco psbA deletion mutant, which lacks the D1 subunit of PSII, retained the


TerC function in thylakoid membrane protein synthesis 353 ability to form grana stacks, albeit of drastically reduced size and number (Baena-Gonzalez et al., 2003). In amiRTerC and terc–1TerC–GFP, the results are less clear-cut. Thylakoids with nearly WT morphology were observed alongside structures in which the normal distinction between grana and stroma lamellae was blurred (Figure 4). This indicates that a minimum level of functional TerC protein is required for formation of grana. The thylakoid membrane phenotype is accompanied by a strong reduction in all core proteins of PSII in amiR-TerC and terc–1TerC–GFP (Figure 5a), and may be attributed to a reduction in their de novo synthesis, particularly CP47 and CP43 (Figure 5c). The reduction in the steady-state level of CP43 primarily affects the unassembled protein in amiR-TerC and terc– 1TerC–GFP (Figure 6). Further downstream events, such as PSII monomer or PSII dimer formation, appeared to be less affected, if at all, in amiR-TerC and terc–1TerC–GFP (Figures S3 and S4). In the terc–1 mutant, the complete elimination of de novo synthesis of PSII core subunits and the ATPase a and b subunits eventually leads to seedling lethality. TerC interacts with ALB3 So far, knowledge of the precise mechanism of co-translational insertion of PSII core proteins has proved elusive. We showed that de novo synthesis of CP47 decreased in amiR-TerC and terc–1TerC–GFP; however, TerC does not interact with CP47 in split-ubiquitin assays (Figure 7a). Hence, the mechanism for CP47 membrane insertion remains unclear at present. More progress has been achieved in the understanding of CP43 and D1 biogenesis in Arabidopsis. The current model for CP43 assembly involves the participation of LPA2, LPA3 and ALB3, while the two PSII assembly factors PAM68 and LPA1 are involved during D1 biogenesis (Chi et al., 2012). The possibility that TerC plays a role in inserting ALB3 and other assembly factors into the thylakoid membrane may be excluded, because they accumulated in the terc–1 mutant (Figure 2a). Nevertheless, TerC interacts with ALB3, LPA2, LPA1 and PAM68 in yeast two-hybrid split-ubiquitin assays (Figure 7a). The interactions in vivo may be transient and sequential, and indeed only the interaction of TerC–GFP and ALB3 was confirmed (Figure 7b). Formation of a complex of TerC and ALB3 therefore seems possible. As shown by sucrose-gradient fractionation analysis, TerC–GFP fractionates between 67 and 160 kDa (Figure 6), and ALB3 was found throughout the sucrose gradient, particularly in two main complexes of approximately 60–140 kDa and > 600 kDa, respectively (Ma et al., 2007). It is tempting to speculate that TerC, in conjunction with the general assembly factor ALB3, facilitates insertion of thylakoid membrane proteins. The effect on D1 biogenesis is not as strong as one may have expected; however, a number of amiR-TerC transgenic lines generated in the WT, pam68–2 and psad1–1 backgrounds did not survive

beyond the T1 generation (Table 2). We suggest a model in which ALB3 and TerC act at the co-translational membrane insertion step (Figure 9). In the case of CP43, further processing of CP43 may be expedited by LPA2 and LPA3. This model is in accordance with the proposal that LPA2 and LPA3 act downstream of CP43 membrane insertion to incorporate CP43 into CP43-free PSII to form PSII monomers (Cai et al., 2010; Chi et al., 2012). This view is also supported by in planta studies using amiR-TerC and lpa2 (Figure 8). Inefficient CP43 synthesis and thylakoid membrane insertion, combined with inefficient assembly of CP43 into the PSII monomer, are incompatible with photoautotrophic growth and development. CONCLUSION In the present study, we analyzed the role of TerC during PSII biogenesis. The phenotypes of terc–1 and alb3 mutant plants are highly similar, and the proteins TerC and ALB3 interact. The YidC/Oxa1/ALB3 family of proteins is ubiquitously distributed (Funes et al., 2011), and these proteins function in bacteria, mitochondria and chloroplasts as membrane protein insertases. Predicted homologs of TerC are found not only in plants and green algae, but also in many bacterial taxa (Kwon and Cho, 2008). A TerC homolog is found in some but not all cyanobacteria (Figure S6 and Methods S4), indicating that TerC is dispensable for PSII assembly within this taxon, or that its function evolved during endosymbiosis. EXPERIMENTAL PROCEDURES Plant material and vector construction The terc–1 mutant (SALK_014739; Alonso et al., 2003) contains a T–DNA insertion in the second intron of the gene (Kwon and Cho, 2008). The terc–1, psad1–1 (Ihnatowicz et al., 2004), lpa2 (SALK_067468, Ma et al., 2007) and pam68–2 (SALK_044323, Armbruster et al., 2010) mutations possess the Col–0 genetic background. To generate a construct expressing an artificial miRNA targeting TerC under the control of the CaMV 35S promoter, a suitable target site was identified as described at http://wmd3.weigel word.org. Cloning of p35S:amiR-TerC was performed as described by Schwab et al. (2006) and Kunz et al. (2009). Using the miR319a precursor-containing plasmid pRS300 as a template, together with suitable primers (Table S2), a PCR product that included the artificial miRNA coding sequence (50 -TAAGAGTACAGCAGGAA GCGG-30 ) was generated and recombined into vector pGWB2 (Nakagawa et al., 2007), yielding plasmid p35S:amiR-TerC. Cloning of p35S:TerC-GFP was performed using PCR primers bearing attB adaptors (Table S2) and Arabidopsis first-strand cDNA. The amplified full-length TerC cDNA was recombined into pDonor 201 (Invitrogen, http://www.lifetechnologies.com/), and fused upstream of the GFP coding sequence and downstream of the 35S promoter by subsequent recombination into pB7FWG2 (Karimi et al., 2002), yielding plasmid p35S:TerC-GFP. Both plasmids were introduced into Arabidopsis by floral-dip transformation (Clough and Bent, 1998). The 35S:amiR-TerC


354 Anja Schneider et al. construct was stably introduced into Col–0, psad1–1, pam68–2 and lpa2 strains, and individual transgenic plants were selected on the basis of their resistance to kanamycin (50 mg l1). Successful integration of the transgene was confirmed by PCRbased genotyping using genomic DNA (Liu et al., 1995). The 35S:TerC-GFP construct was stably introduced into Col–0 and terc–1, and individual transgenic plants (WTTerC–GFP and terc– 1TerC–GFP, respectively) were selected on the basis of their resistance to Basta by spraying with 0.5 ml l1 Basta (Bayer, http:// www.bayer.de which contains 200 g l1 glufosinat- ammonium). Genotyping of complemented homozygous terc–1 plants was performed by PCR, and successful integration of the transgene was confirmed (Table S2).

Growth conditions Segregating terc–1 plants were grown on 1 x Murashige and Skoog (MS) medium (Duchefa, http://www.duchefa.com) supplemented with 1% sucrose, at a photon flux density of approximately 4 lmol photons m2 sec1 white light (provided by lightemitting diodes) under a 12 h light/12 h dark cycle. Unless stated otherwise, WT and transformed Arabidopsis lines were grown for 4 weeks in a growth chamber (Percival, http://www.percival-scientific.com) equipped with 17 W cool white fluorescent lamps under a 12 h light/12 h dark cycle at a photon flux density of approximately 100 lmol photons m2 sec1. Arabidopsis plants used for microscopy and kanamycin selection were surface-sterilized and grown on MS medium. Arabidopsis plants used for transformation and Basta selection were grown in a temperature-controlled greenhouse under a 16 h light/8 h dark cycle.

RNA preparation and real-time PCR Total RNA was isolated from ground Arabidopsis leaves using TRIzol reagent (Invitrogen), followed by DNase I (New England Biolabs, https://www.neb.com) treatment, and 1 lg aliquots of RNA were used as template for first-strand cDNA synthesis using SuperScript III (Invitrogen). For quantitative real-time PCR, SYBR Green Supermix (Bio–Rad, http://www.bio–rad.com/) was used, and PCR was performed on an iQ5 multicolor real-time PCR detection system (Bio–Rad) using TerC- and ubiquitin-specific (Czechowski et al., 2005) primer pairs (Table S2). Quantification of relative expression levels was performed using the comparative Ct method (Livak and Schmittgen, 2001).

Chlorophyll fluorescence measurements The photosynthetic performance of PSII was assessed by chlorophyll a fluorescence measurements using Imaging PAM or Dual PAM fluorometers (Walz, http://www.walz.com/). Plants were darkadapted for 30 min and exposed to a blue measuring beam to determine the minimal fluorescence (F0). Then a saturating 0.8 sec light flash (2800 lmol photons m2 sec1 for the Imaging PAM fluorometer or 5000 lmol photons m2 sec1 for the Dual PAM fluorometer) was applied to measure the maximum fluorescence (FM), and the maximum quantum yield of PSII (FV/FM) was calculated (Maxwell and Johnson, 2000). A 10 min exposure to actinic light (635 nm, 62 lmol photons m2 sec1) was used to measure steady-state fluorescence. Further saturating light flashes were used to calculate the effective quantum yield of PSII (ΦII) (Maxwell and Johnson, 2000).

Protein analysis Total proteins were isolated from Arabidopsis leaves in 50 mM Tris, pH 8.0, 10 mM EDTA, pH 8.0, 2 mM EGTA, 10 mM dithiothreitol

and 0.8% w/v SDS, incubated at 95°C for 10 min and separated on Tris-glycine SDS gels (12% acrylamide). Thylakoid isolation was performed as described by Bassi et al. (1985). Thylakoid samples for sucrose-gradient fractionation were diluted to a chlorophyll concentration of 1 mg ml1, and solubilized using a final concentration of 1.0% w/v b–dodecyl maltoside. Sucrose-gradient centrifugation was performed as described previously (Armbruster et al., 2010), and the gradient was divided into 19 fractions. Equal aliquots of proteins from all fractions were separated on Tris-glycin SDS gels (12% acrylamide). For in vivo labeling, seedlings or 4–5 leaves obtained from Arabidopsis lines were incubated in 1 mCi of [35S]Met in a total volume of 300 ll for 20 min in the presence of 20 lg ml1 cycloheximide at light levels of 4 lmol photons m2 sec1 (for terc–1) or 60 lmol photons m2 sec1 (for transformed lines). In pulsechase experiments, the chase was initiated by adding cold Met to a final concentration of 10 mM. Subsequently thylakoid proteins were prepared and fractionated on denaturing gradient Tris-glycin SDS gels (8-12% acrylamide). Proteins were transferred to polyvinylidene fluoride membranes and used for immunoblot detection with specific antibodies (Table S3) and the enhanced chemiluminescence system (Pierce, http:// www.piercenet.com/), or radioactive signals were detected using a Typhoon phosphor imager (GE Healthcare, http://www.gelifesciences.com). For signal quantification, Bio–1D advanced software (Vilber Lourmat, http://www.labindia.com) was used.

Split-ubiquitin assay The TerC coding sequence, with and without transit peptide, was cloned into the multiple cloning site in pAMBV4, and the coding sequences for mature thylakoid proteins (Pasch et al., 2005; Armbruster et al., 2010) were cloned into the multiple cloning site of pADSL (Pasch et al., 2005). Interaction studies were performed using the DUALmembrane kit (Dualsystems Biotech, http:// www.dualsystems.com/) as described by Pasch et al. (2005).

Co-immunoprecipitation assay Chloroplasts from WT and terc–1TerC–GFP plants were isolated as described previously (Stoppel et al., 2012). Proteins were crosslinked using DSP (Dithiobis [succinimidyl propionate]) at 50 mM. Chloroplasts were lysed in 30 mM HEPES/KOH, pH 7.7, 10 mM magnesium acetate and 60 mM potassium acetate in the presence of a protease inhibitor cocktail (Roche, http://www.roche-appliedscience.com/) by incubation on ice for 30 min. After centrifugation, 30 min at 4°C, at 38 000 g, the pelleted thylakoid fraction was solubilized on ice in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA and 1% v/v Nonidet P–40 (Sigma-Aldrich, http://sigma-aldrich.com) in the presence of protease inhibitor cocktail. Thylakoid proteins (2 mg) were incubated with 30 ll of GFP-Trapâ M beads (Chromotek, http://www.chromotek.com/) for 1 h at 4°C with rotation. Beads were washed thoroughly and finally resuspended in NuPAGEâ-LDS sample buffer (Life Technologies, http://www.life technologies.com/) supplemented with 50 mM dithiothreitol. The precipitated proteins were subjected to immunoblot analysis.

Electron microscopy Leaves of agar-grown Arabidopsis lines used for transmission electron microscopy were processed as described by Breuers et al. (2012) with slight modifications. As fixative, 2% glutaraldehyde and 4% formaldehyde in 100 mM sodium phosphate buffer, pH 7.4, was used, osmication was performed in 2% osmium tetroxide for 120 min at 4°C, and the samples were stained by


TerC function in thylakoid membrane protein synthesis 355 incubation in 2% uranyl acetate at 4°C overnight. Sections were viewed using a JEM-2100 transmission electron microscope (JEOL, http://www.jeol.de/).

ACKNOWLEDGMENTS This work was supported by funds from the Deutsche Forschungsgemeinschaft (LE 1265/20–1) to D.L. We thank Roberto Barbato (Universita del Piemonte Orientale, Alessandria, Department of Environmental and Life Sciences) and Lixin Zhang (Chinese Academy of Sciences, Beijing, Institute of Botany) for providing antibodies, Theresa Rottmann (Universita€t Bayreuth, Zellbiologie/ Elektronenmikroskopie) and Silke Funke (Ruhr-Universita€t Bochum, AG Molekularbiologie pflanzlicher Organellen) for technical assistance, and Paul Hardy for critical reading of the manuscript.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Chloroplast localization and topology of the TerC–GFP fusion protein. Figure S2. Target for artificial microRNA. Figure S3. Two-dimensional blue native/SDS–PAGE of in vivo labeled thylakoid membrane complexes from WT and amiR-TerC. Figure S4. Two-dimensional blue native/SDS–PAGE of steadystate levels of thylakoid membrane complexes. Figure S5. Split-ubiquitin analysis of TerC full-length and mature proteins. Figure S6. Phylogenetic analysis. Table S1. Photosynthetic performance of amiR-TerC lines. Table S2. Primers used in this study. Table S3. Antibodies used in this study. Methods S1. Fluorescence microscopy. Methods S2. Topology studies. Methods S3. Two-dimensional blue native/SDS–PAGE. Methods S4. Phylogenetic analysis.

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The extreme Albino3 (Alb3) C terminus is required for Alb3 stability and function in Arabidopsis thaliana.

MPH1 is a thylakoid membrane protein involved in protecting photosystem II from photodamage in land plants.

Water-soluble chlorophyll protein is involved in herbivore resistance activation during greening of Arabidopsis thaliana.

Photosystem II Subunit PsbS Is Involved in the Induction of LHCSR Protein-dependent Energy Dissipation in Chlamydomonas reinhardtii.

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

Identification of the basic amino acid residues on the PsbP protein involved in the electrostatic interaction with photosystem II.

The AMP-Activated Protein Kinase KIN10 Is Involved in the Regulation of Autophagy in Arabidopsis.

The SERRATE protein is involved in alternative splicing in Arabidopsis thaliana.

Discovery of a chlorophyll binding protein complex involved in the early steps of photosystem II assembly in Synechocystis.

Restoration of light induced photosystem II inhibition without de novo protein synthesis.

BEACH-domain proteins act together in a cascade to mediate vacuolar protein trafficking and disease resistance in Arabidopsis.

Photosystem II Repair and Plant Immunity: Lessons Learned from Arabidopsis Mutant Lacking the THYLAKOID LUMEN PROTEIN 18.3.

A VAMP-associated protein, PVA31 is involved in leaf senescence in Arabidopsis.

COLD SHOCK DOMAIN PROTEIN 3 is involved in salt and drought stress tolerance in Arabidopsis.

Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B.

Quality control of Photosystem II: reversible and irreversible protein aggregation decides the fate of Photosystem II under excessive illumination.

An Organelle RNA Recognition Motif Protein Is Required for Photosystem II Subunit psbF Transcript Editing.

Frataxin Is Localized to Both the Chloroplast and Mitochondrion and Is Involved in Chloroplast Fe-S Protein Function in Arabidopsis.

Luminidependens (LD) is an Arabidopsis protein with prion behavior.

Protein Machineries Involved in the Attachment of Heme to Cytochrome c: Protein Structures and Molecular Mechanisms.

The light-harvesting chlorpohyll-protein complex of photosystem II. Its location in the photosynthetic membrane.

Studies on the light-induced loss of the D1 protein in photosystem-II membrane fragments.

MS-based cross-linking analysis reveals the location of the PsbQ protein in cyanobacterial photosystem II.

The status of cysteine residues in the extrinsic 33 kDa protein of spinach photosystem II complexes.