The Plant Journal (2013) 76, 675–686
doi: 10.1111/tpj.12331
Loss-of-function of OsSTN8 suppresses the photosystem II core protein phosphorylation and interferes with the photosystem II repair mechanism in rice (Oryza sativa) Krishna Nath1,2,†, Roshan Sharma Poudyal1,†, Joon-Seob Eom3,†, Yu Shin Park4, Ismayil S. Zulfugarov1,5, Sujata R. Mishra1, Altanzaya Tovuu1,6, Nayeoon Ryoo3, Ho-Sung Yoon7, Hong Gil Nam2, Gynheung An3, Jong-Seong Jeon3,* and Choon-Hwan Lee1,* 1 Department of Molecular Biology, Pusan National University, Busan 609–735, Korea, 2 Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology, Daegu 711–873, Korea, 3 Crop Biotech Institute and Graduate School of Biotechnology, Kyung Hee University, Yongin 446–701, Korea, 4 Center for Core Research Facilities, Daegu Gyeongbuk Institute of Science and Technology, Daegu 711–873, Korea, 5 Department of Biology, North-Eastern Federal University, 58 Belinsky Street, Yakutsk 677–027, Russia, 6 Department of Biology, Mongolian State University of Agriculture, Zaisan 17024, Ulaanbaatar, Mongolia, and 7 Department of Biology, Kyungpook National University, Daegu 702–701, Korea Received 3 May 2013; revised 7 August 2013; accepted 2 September 2013; published online 16 September 2013. *For correspondence (e-mails
[email protected];
[email protected]). † These authors contributed equally to this work.
SUMMARY STN8 kinase is involved in photosystem II (PSII) core protein phosphorylation (PCPP). To examine the role of PCPP in PSII repair during high light (HL) illumination, we characterized a T–DNA insertional knockout mutant of the rice (Oryza sativa) STN8 gene. In this osstn8 mutant, PCPP was significantly suppressed, and the grana were thin and elongated. Upon HL illumination, PSII was strongly inactivated in the mutants, but the D1 protein was degraded more slowly than in wild-type, and mobilization of the PSII supercomplexes from the grana to the stromal lamellae for repair was also suppressed. In addition, higher accumulation of reactive oxygen species and preferential oxidation of PSII reaction center core proteins in thylakoid membranes were observed in the mutants during HL illumination. Taken together, our current data show that the absence of STN8 is sufficient to abolish PCPP in osstn8 mutants and to produce all of the phenotypes observed in the double mutant of Arabidopsis, indicating the essential role of STN8-mediated PCPP in PSII repair. Keywords: STN8 kinase, high light illumination, photosystem II core protein phosphorylation, D1 protein degradation, photosystem II repair, rice.
INTRODUCTION During photosynthesis, plants convert light into chemical energy, but excessive light is harmful to plants. Even in low light, photosystem II (PSII) may be damaged in proportion to the light intensity (Tyystja€rvi and Aro, 1996; Jansen et al., 1999; Oguchi et al., 2009). A damaged PSII must be repaired at all light intensities, and the overall level of photosynthetic activity is reduced when the rate of repair does not match the rate of photodamage (Prasil et al., 1992). The repair of photodamaged PSII, particularly turnover of the D1 protein, is thought to be a key regulatory step of the PSII repair cycle (Yokthongwattana and Melis, 2006). In this cycle, the damaged and phosphorylated PSII © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd
is first relocated from the grana to the stromal lamellae, where the damaged D1 protein is degraded after dephosphorylation by specific proteases (Bailey et al., 2002; KapriPardes et al., 2007; Sun et al., 2010; Kley et al., 2011; Schuhmann and Adamska, 2012; Wagner et al., 2012; Kirchhoff, 2013a), and newly synthesized D1 is inserted into PSII (Ohad et al., 1985; Aro et al., 1993; Rintama€ki et al., 1996; Tikkanen et al., 2008; Tikkanen and Aro, 2012). The phosphorylation/dephosphorylation cycle of PSII core proteins has been identified as one of the regulatory mechanisms that is responsible for protecting PSII from photoinhibition (Tikkanen et al., 2008; Fristedt et al., 2009; 675
676 Krishna Nath et al. Goral et al., 2010; Tikkanen and Aro, 2012). A serine/threonine protein kinase, STN8, has recently been identified in Arabidopsis thaliana as specific for the phosphorylation of the PSII core proteins D1, D2, CP43, PsbH and TSP9 (Bonardi et al., 2005; Vainonen et al., 2005). STN7 kinase in Arabidopsis was found to be involved not only in phosphorylation of light-harvesting complex II (LHCII) protein (Bellafiore et al., 2005; Bonardi et al., 2005), but also in PSII core protein phosphorylation (PCPP) (Bonardi et al., 2005; Tikkanen et al., 2008). Complete inhibition of PCPP was therefore only observed in the Arabidopsis stn7/stn8 double mutant (Bonardi et al., 2005; Tikkanen et al., 2008). Recently, the role of PCPP in the PSII repair cycle was questioned because complete inhibition of PCPP did not have any significant effects on PSII activity and D1 turnover rate in Arabidopsis stn8 and stn7/stn8 double mutants under high light (HL) illumination (Bonardi et al. (2005). However, under prolonged HL illumination, the PSII of the stn7/stn8 double mutants became more sensitive, and degradation of photodamaged D1 protein was slower compared with wild-type (WT) plants (Tikkanen et al., 2008; Fristedt et al., 2009). In addition, in the double mutants, the lack of PCPP prevents the disassembly of PSII–LHCII supercomplexes during HL illumination, which blocks the migration of damaged PSII units from the grana to the stroma (Tikkanen et al., 2008; Tikkanen and Aro, 2012). In the thylakoid structure, PCPP facilitates the lateral mobility of membrane proteins, and the restriction in lateral migration of D1 in stn8 and stn7/stn8 mutants of Arabidopsis led to formation of extended appressed grana regions of thylakoids (Fristedt et al., 2009). More recently, several reports proposed a central role for PCPP in dynamic restructuring of thylakoid membranes in the light, which involves expansion of granal thylakoid lumen (Kirchhoff et al., 2011) and lateral shrinkage and vertical de-stacking of grana (Herbstova et al., 2012; Kirchhoff, 2013b). Although evidence for the important role of PCPP in PSII repair is accumulating, details of its regulatory mechanisms and the regulatory networks are unclear as yet, and there are several inconsistencies in the results obtained from algae, dicots and monocots (Chen et al., 2013; Nath et al., 2013). In view of the above reports, we here examined the characteristics of a T–DNA insertional STN8 knockout mutant, osstn8, of the monocot rice (Oryza sativa). In comparison with earlier findings in the stn8 mutant of dicot Arabidopsis, we observed clear phenotypes in the osstn8 mutants. Our results indicate that the functional redundancy between STN8 and STN7 during PCPP is much lower in rice than that in Arabidopsis, and that the absence of STN8 is sufficient to abolish PCPP in osstn8 mutants to produce all of the phenotypes observed in the double mutant of Arabidopsis, supporting the essential role of PCPP by STN8 kinase in PSII repair in rice.
RESULTS Identification of a T–DNA insertional mutant of OsSTN8 kinase From the TIGR rice database (http://rice.plantbiology.msu.edu/analyses_search_locus.shtml), we identified a serine/threonine protein kinase gene (LOC_Os05 g40180) on chromosome 5 that is highly homologous to the Arabidopsis AtSTN8 gene, and designated this OsSTN8. OsSTN8 encodes a protein of 591 amino acids that shows more than 70% sequence similarity with AtSTN8 (Figure S1). OsSTN8 also shares a relatively low sequence similarity (less than 45%) with the homologs AtSTN7 and OsSTN7 (Figure S1). In their kinase domains, OsSTN8 and AtSTN8 share a conserved catalytic kinase domain (Figure S1), suggesting that they belong to a serine/threonine protein kinase family. To elucidate the function of OsSTN8, its knockout mutant was obtained from a pool of T–DNA insertional rice lines generated using a T–DNA vector (Jeong et al., 2002) (http://www.postech.ac.kr/life/pfg/risd/index.html). OsSTN8 has three exons and two introns plus 5′ and 3′ UTR regions. Determination of the flanking sequences of the T–DNA by inverse PCR as described by An et al. (2003) revealed that the T–DNA was inserted in the second exon (Figure 1a). The homozygous mutant plants were identified from segregating progeny plants by PCR using gene- and T–DNA-specific primers (Figure 1b). Of a total of eight plants, three plants homozygous for the T–DNA insertion were selected (Figure S2) and analyzed in subsequent experiments as osstn8 mutants. RT–PCR analysis indicated that the endogenous OsSTN8 transcript is absent from these homozygous osstn8 mutants (Figure 1c). To ensure that the mutation in OsSTN8 is responsible for the phenotypes observed in the mutants in this study, we transformed this mutant with a WT copy of OsSTN8 cDNA under the control of the maize ubiquitin (Ubi) promoter. More than ten independent transgenic rice plants were obtained, among which a line with high expression of OsSTN8 was selected (Figure 1c) and analyzed in subsequent experiments as osstn8 complemented line 1 (Comp–1). Leaf-preferential expression of OsSTN8 in rice The expression pattern of OsSTN8 was determined by real-time PCR analysis (Figure 1d). OsSTN8 was found to be highly expressed in leaves (photosynthetic organ). Its expression was much lower in roots, flowers and immature seeds. In addition, OsSTN7 was also found to be preferentially expressed in the leaves (Figure 1e). These results are consistent with previous findings for Arabidopsis homologs (Bellafiore et al., 2005; Bonardi et al., 2005), suggesting that both STN7 and STN8 are thylakoid membrane-associated protein kinases.
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 76, 675–686
Role of STN8 for PSII repair in rice 677 Figure 1. Characterization of rice STN8 kinase mutant plants. (a) Schematic representation of the OsSTN8 gene in rice. The insertion site of the T–DNA in exon 2 of OsSTN8 is indicated. The untranslated region (UTR), exons (E1, E2 and E3) and introns (I1 and I2) are represented as white boxes, black boxes and lines between exons, respectively. The primers used for genotyping (P1, P2 and P3) are indicated by black arrows. (b) Genotyping and PCR analysis of genomic DNA isolated from WT and osstn8 mutant plants. The WT gene-specific primers (P1 and P2) amplify a 927 bp product, whereas amplification of the osstn8 mutant gene using P1 and a primer for the T–DNA left border (P3) yields a 542 bp product. (c) RT–PCR analysis of WT, osstn8 mutant and osstn8 complemented (Comp–1) plants using gene-specific primers. OsUBQ5 was amplified as an internal control. (d, e) Real-time PCR organ-specific gene expression analysis of OsSTN7 and OsSTN8 in WT plants. L, leaf tissues; R, root tissues; F, flower tissues; S, seed tissues.
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Phenotypic analysis of osstn8 mutant plants The growth of osstn8 mutants was slower than that of WT (Figure S3a,c), and the growth retardation resulted in a flowering delay of approximately 7–10 days (Figure S3b). The contents of photosynthetic pigments were not significantly altered in osstn8 mutants compared with WT, but the chlorophyll a/b ratio was 3.2 in WT and 3.0 in osstn8 mutant leaves (Tables S1 and S2). PSII core protein phosphorylation is significantly repressed in osstn8 mutant leaves Because Arabidopsis STN8 is required for PCPP (Bonardi et al., 2005; Vainonen et al., 2005), we analyzed the endogenous phosphorylation of thylakoid membrane proteins in both WT and osstn8 mutant leaves by immunoblotting using a phosphothreonine-specific antibody (Figure 2a). In dark-adapted WT leaves, the phosphorylation of D1 and D2 was negligible, but phosphorylation of CP43 was detectable. By illumination with either growth light (GL) at 500 lmol m 2 sec 1 or HL at 2000 lmol m 2 sec 1 for 3 h, phosphorylation of the reaction center proteins D1 and D2 was strongly induced, but that of CP43 was not significantly altered. In osstn8 plants, phosphorylation of all PSII core proteins was significantly repressed. Phosphorylation of LHCII was also induced under GL and was negligible under HL, but was not significantly repressed by mutation of OsSTN8 (Figure 2a). In addition, the state transition was not altered in osstn8 mutants compared with WT (Figure S4). The extent of phosphorylation of PSII core proteins
and LHCII in the transgenic Comp–1 plants carrying a wildtype copy of OsSTN8 gene was comparable to that of WT (Figure 2a), suggesting that the specific inhibition of PCPP in osstn8 mutants is due to the mutation of OsSTN8. Taken together, these data indicate that OsSTN8 kinase is responsible for PCPP, but not for the phosphorylation of LHCII proteins or the state transition, in monocot rice plants. osstn8 mutant leaves are more susceptible to HL illumination Because osstn8 mutants with a significant repression of PCPP (Figure 2a) showed growth retardation (Figure S3), we compared the photochemical efficiency of PSII, the Fv/ Fm, between WT and osstn8 mutant leaves. Under GL, the Fv/Fm values for osstn8 mutant leaves were very similar to those for WT leaves (approximately 0.8; Figure 2b), indicating that the mutant leaves remained healthy. However, osstn8 mutant leaves were more susceptible to HL illumination at 2000 lmol m 2 sec 1 than WT leaves (Figure 2b). When WT and mutant leaves were placed under dim light at 20 lmol m 2 sec 1 intensity for PSII recovery after HL illumination for 3 h, the Fv/Fm values from both WT and mutant leaves increased at similar rates, although the value reached in the mutants after 12 h was considerably less than that in the WT (Figure 2c). When we treated both WT and mutant leaves with light intensities varying from 1500 to 3300 lmol m 2 sec 1 for 3 h, dose-dependent photoinactivation of PSII was observed, and the mutant leaves were found to be more susceptible to HL illumination than WT leaves (Figure S5).
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 76, 675–686
678 Krishna Nath et al. lincomycin, the decrease in the Fv/Fm in the mutants was more rapid than that in WT (Figure 2b), suggesting that the PSII repair system is significantly impaired in osstn8 leaves. Upon treatment with lincomycin, the decreased Fv/Fm did not recover under dim light in either plant type (Figure 2c), indicating that lincomycin infiltration was sufficient to block the de novo synthesis of D1 protein almost completely, which was also found to be the case in the samples illuminated with HL at various intensities (Figure S5).
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D1 protein degradation is suppressed in osstn8 mutant leaves
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Figure 2. Phosphorylation of thylakoid proteins and PSII photoinactivation during HL illumination. (a) Immunoblotting analysis of phosphoproteins in WT and osstn8 thylakoid membranes using a phosphothreonine antibody. Thylakoid membrane extracts containing 3 lg of chlorophyll were loaded in each lane. The SDS– PAGE results after Coomassie blue staining (CBS) are shown in the bottom panel. D, dark-adapted for 15 h; GL, growth light intensity (500 lmol m 2 sec 1); HL, high light intensity (2000 lmol m 2 sec 1) for 3 h. (b, c) Decrease in the photochemical efficiency of PSII or Fv/Fm during photoinhibition under 2000 lmol m 2 sec 1 light intensity in the presence/absence of the plastid protein synthesis inhibitor lincomycin (Lin) for up to 6 h (b), and subsequent recovery of 3 h photoinhibited samples under dim light intensity of 20 lmol m 2 sec 1 for up to 12 h (c). Values are means SD of five replicates.
To test whether the higher susceptibility to photoinhibition in the mutant leaves is due to accelerated damage to PSII or impaired repair of photodamaged PSII, the plants were illuminated in the presence of lincomycin, which blocks PSII repair by inhibiting chloroplast protein synthesis. The addition of lincomycin induced a more pronounced degree of PSII inactivation, and the Fv/Fm decreased at similar rates between WT and osstn8 mutant leaves (Figures 2b and S5). The results obtained in the presence of lincomycin suggest that the rate of PSII inactivation in the mutant leaves under HL illumination does not differ from that of WT leaves. However, in the absence of
To examine whether the susceptibility of osstn8 mutants to HL illumination is related to the preferred degradation pathway for D1 protein, immunoblotting experiments for D1 protein and its degradation products were performed. This analysis revealed that the level of D1 protein decreased in both WT and Comp–1 leaves after HL illumination in the absence of lincomycin, but the extent of the decrease was less in osstn8 mutants compared with WT or Comp–1 lines (Figure 3a,b). In the presence of lincomycin, where de novo synthesis of D1 protein is blocked, the difference in the level of D1 protein between the mutant and WT (or Comp–1) leaves after HL illumination was greater (Figure 3a), suggesting that the degradation of photodamaged D1 protein is repressed in the mutant leaves. This result was verified by comparing the band intensities of degradation products of D1 protein. The level of D1 fragment accumulation was less in osstn8 mutant leaves compared with WT (or Comp–1) leaves, particularly in the absence of lincomycin (Figure 3b). These results were verified using an antibody raised against the DE loop of D1 protein (Figure 3c), suggesting that the degradation process for D1 is repressed as a consequence of blockage of PCPP by the mutation in the OsSTN8 gene. Production of reactive oxygen species in WT and osstn8 mutants under HL illumination Due to the higher susceptibility of osstn8 mutants to HL illumination, we expected higher reactive oxygen species (ROS) accumulation in the mutants compared with WT. Therefore, we measured the level of the two major ROS species, superoxide and hydrogen peroxide (H2O2), produced under HL illumination by histochemical staining of the leaves using nitroblue tetrazolium (NBT) and 3,3′–diaminobenzidine (DAB), respectively. Interestingly, both superoxide and H2O2 were more highly produced in the mutants after HL illumination than in WT (Figure 4a,b). To confirm these results in the thylakoids, we first monitored the increase of dihydroethidium (DHE) fluorescence as a measure of superoxide production by addition of 25 lM DHE to a solution containing 10 lg thylakoids/ ml. Upon HL illumination at 800 lmol m 2 sec 1, DHE fluorescence started to increase, and the rate of the initial increase was much higher in osstn8 mutants compared
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 76, 675–686
Role of STN8 for PSII repair in rice 679
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Figure 3. D1 protein degradation during HL illumination. (a–c) Immunoblotting analysis of D1 protein and its degradation fragments before and after HL illumination (2000 lmol m 2 sec 1) for 3 h [( ) and (+)] in the presence and absence of lincomycin (+Lin and Lin). Lhcb1 was used as a loading control. (a) Immunoblotting of the full-length D1 protein using an antibody raised against D1C, the C–terminal end. Thylakoids containing 1 lg chlorophyll were loaded into each lane. (b, c) Immunoblotting analysis of D1 protein degradation using an antibody raised against D1C and against the DE loop of D1 protein, respectively. Thylakoid extracts containing 5 lg of chlorophyll were loaded into each lane.
with WT (Figure 4c). The difference in the amount of DHE produced between the plants after 5 min did not increase further during illumination for 15 min. When we monitored the production of H2O2 in thylakoids by measuring changes in 2′,7′–dichlorofluorescein diacetate (DCFDA) fluorescence under HL illumination, the accumulation of H2O2 was almost linear after the start of illumination, but a difference between the mutant and WT was observed after illumination for only 2.5 min (Figure 4d), suggesting that superoxide is probably produced earlier and that H2O2 is newly generated or converted from superoxide by the action of enzymes such as superoxide dismutase. The photoinactivation of photosystem I is similar between WT and osstn8 mutants Because superoxide is believed to be produced by photosystem I (PSI) in plants under stress conditions
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Figure 4. Measurement of ROS and oxidation of thylakoid membrane proteins. (a, b) Histochemical assays for superoxide anion radicals and H2O2 by NBT and DAB staining, respectively. Leaf fragments were cut under water using a sharp scissors to avoid mechanical damage. The leaf fragments were then incubated in 6 mM NBT solution containing 50 mM HEPES buffer (pH 7.5) for 3 h in darkness, or in 5 mM DAB solution containing 10 mM MES (pH 3.8) for 8 h in darkness. (c, d) Quantitative analysis of superoxide anion radicals and H2O2 in vitro by measuring the relative fluorescence of DHE and DCFDA, respectively, in the thylakoids. (e) Immunoblotting analysis of oxidation of thylakoid membrane proteins in WT and osstn8 mutant plants before and after HL illumination at a 1500 lmol m 2 sec 1 light intensity (upper panel), and SDS–PAGE after Coomassie blue staining (lower panel). Thylakoid membrane extracts containing 5 lg chlorophyll were loaded into each lane.
(Asada, 1999; Sonoike, 2006), we measured PSI activity as the relative amount of far-red light-induced P700+ (DA810/A810) in the leaves of WT and osstn8 mutant plants. The PSI activity in the mutant leaf segments was found to be similar to that of WT (Table 1). After HL illumination at 2000 lmol m 2 sec 1 for 1 h, the PSI activity decreased by 41.6% and 39.1% in the WT and the osstn8 mutant, respectively, compared with their controls, indicating that the photoinactivation of PSI in osstn8 mutants was not more severe than that in WT.
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 76, 675–686
680 Krishna Nath et al. Table 1 Photoinactivation of PSI in WT and osstn8 mutant rice plants under HL illumination at room temperature
DA810/A810
Before HL illumination
After HL illumination
WT
osstn8
WT
osstn8
0.67 0.07
0.69 0.05
0.39 0.06
0.42 0.13
PSI activity (DA810/A810) was measured before and after HL illumination at 2000 lmol m 2 sec 1 for 1 h. Each data point represents the mean standard deviation of at least four separate experiments.
Figure 5. Separation of PSII–LHCII supercomplexes from lincomycin-infiltrated leaf fragments by BN-PAGE. Thylakoids from WT and osstn8 mutants were solubilized in n–dodecyl-b–D– maltoside, and separated by 5–13.5% BN-PAGE. An equal volume of 10 lg chlorophyll was loaded into each lane. Before isolation of thylakoids, leaves were either dark-adapted for 15 h (D) or high light-illuminated at 2000 lmol m 2 sec 1 for 3 h (HL).
Oxidation of thylakoid proteins is elevated in osstn8 plants The higher production of ROS in the mutant leaves was confirmed by comparing the oxidation of thylakoid proteins between WT and osstn8 mutant leaves during HL illumination at 1500 lmol m 2 sec 1 for up to 3 h. In osstn8 plants, PSII core proteins were found to be carbonylated more rapidly than in WT (Figure 4e). Mobilization of PSII–LHCII supercomplexes is suppressed in osstn8 mutant plants In Arabidopsis stn7/stn8 double mutants, the PSII–LHCII supercomplex is unlikely to be properly disassembled to enable mobilization of damaged proteins for degradation during HL illumination (Tikkanen et al., 2008). In the BN-PAGE experiment (Figure 5), mobilization of PSII supercomplexes was observed in WT plants during HL illumination, and this process was significantly blocked in osstn8 mutants. This result suggests that defects in PCPP in osstn8 mutant leaves block the disassembly of PSII–LHCII supercomplexes. The thylakoid membrane structure of the mutants differs from that of WT Several striking features were observed in the thylakoid membrane structure in osstn8 chloroplasts (Figure 6).
Transmission electron microscopy analysis revealed that stacking of the grana is significantly reduced and the length of grana is dramatically increased in the chloroplasts of osstn8 mutants under natural growth conditions (Figure 6b,d,f). Under HL illumination at 2000 lmol m 2 sec 1, a significant increase in grana stacking and a slight increase in the width of the grana was observed in WT (Figure 6g,i,k), but not in the osstn8 plants (Figure 6h,j,l). DISCUSSION The results obtained in this study of a monocot model rice plant strongly support the classical view of the PSII repair cycle (Aro et al., 1993), which was challenged by Bonardi et al. (2005) on the basis of studies performed in stn8 single and stn7/stn8 double mutants of Arabidopsis that suggested that protein phosphorylation by STN8 kinase was not essential for PSII repair. In rice, mutation of OsSTN8 alone was found to be sufficient to significantly suppress PCPP (Figure 2a), whereas this is only partially blocked in the Arabidopsis stn8 single mutants (Bonardi et al., 2005; Tikkanen et al., 2008; Fristedt et al., 2009). Defects in PCPP in osstn8 mutants were also sufficient to suppress the mobilization of PSII supercomplexes under HL illumination as shown by BN-PAGE (Figure 5). Because the damaged PSII was mostly unable to be mobilized for degradation in osstn8 mutants, the degradation of D1 protein was largely suppressed in these plants (Figure 3a–c). Lincomycin infiltration experiments (Figures 2b, 3a–c and S5) further suggested that the rate of photodamage in PSII in osstn8 mutants was similar to that in WT, but that the repair process was blocked in the mutants. Hence, PSII of the osstn8 mutants is highly susceptible to HL illumination (Figure 2b), although PSII inactivation under HL illumination was not found previously to be significant in Arabidopsis stn7/stn8 mutants (Bonardi et al., 2005) or was only noticeable following prolonged illumination (Tikkanen et al., 2008). Compared with Arabidopsis, the dose of HL supplied to rice leaves was not very high. Tikkanen et al. (2008) observed a significant difference between WT and the double mutants only after 6 h under 1000 lmol photons m 2 sec 1, which corresponds to a dose of 6000 lmol photons m 2 and is much stronger than the treatment we applied to rice (the dose given for 1 h illumination in Figure 2b is only 2000 lmol photons m 2), and the dose given for Figure S5 is in the range of 4500– 9900 lmol photons m 2. In addition the growth light intensity for Arabidopsis was 100 lmol photons m 2 sec 1 and that for rice was 600 lmol photons m 2 sec 1. Although the STN8 kinase is specific to PCPP in Arabidopsis, STN7 also appears to be involved in this process, because significant suppression of both PCPP and mobilization of PSII supercomplexes was only found to occur in the stn7/stn8 double mutant of Arabidopsis (Bellafiore et al., 2005; Bonardi et al., 2005; Vainonen et al., 2005;
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 76, 675–686
Role of STN8 for PSII repair in rice 681
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Figure 6. Transmission electron microscope analysis of chloroplasts from WT and osstn8 mutant plants. (a–f) Sections from WT and osstn8 leaves before HL illumination. (g–l) Sections from WT and osstn8 leaves after HL illumination. (a, b, g, h) Low resolution; scale bar = 500 nm. (c, d, i, j) Intermediate resolution; scale bar = 200 nm. (e, f, k, l) High resolution; scale bar = 100 nm.
Tikkanen et al., 2008). In contrast, our analyses in rice reveal that the mutation in OsSTN8 alone is sufficient to produce all of the phenotypes observed in the double mutants of Arabidopsis, which suggests that the functional redundancy of STN7 and STN8 for PCPP is much less in the monocot model plant rice, compared with the model dicot plant Arabidopsis. In addition, the growth of the osstn8 mutant was slightly retarded (Figure S3), and flowering and seed harvest time were delayed compared with WT (Figure S3). In contrast to PCPP, phosphorylation of LHCII protein is specific to STN7 in Arabidopsis (Bellafiore et al., 2005; Tikkanen et al., 2008), and the process is not influenced significantly by the mutation of OsSTN8 in rice (Figure 2a). The phosphorylation of CP43 was found not to be lightinduced, unlike other PSII core proteins including D1 and D2 (Figure 2a), suggesting that the phosphorylation of
CP43 is differently regulated from that of D1 and D2, although all of these PSII core proteins are phosphorylated by STN8 in rice. Differential regulation of CP43 phosphorylation was also reported previously in Arabidopsis (Bonardi et al., 2005; Vainonen et al., 2005; Tikkanen et al., 2008). We obtained the same result in rice in our current study (Figure 5). CP43 phosphorylation was reported to be important for mobilization of PSII supercomplexes in Arabidopsis (Dietzel et al., 2011). In addition to CP43, CP29 is physically close to CP47, a PSII core protein, and CP24, a monomer of LHCII (Yakushevska et al., 2003), is involved in HL-induced disassembly of PSII–LHCII supercomplexes in Arabidopsis (Fristedt and Vener, 2011), and this disassembly is an essential step in the PSII repair cycle (Tikkanen et al., 2008; Tikkanen and Aro, 2012). In the PSII repair cycle, photodamaged and phosphorylated PSII moves from the granal to stromal areas for
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 76, 675–686
682 Krishna Nath et al. subsequent degradation of the D1 protein, which is replaced by newly synthesized D1 in the newly assembled PSII complex (Aro et al., 1993; Tikkanen et al., 2008; Tikkanen and Aro, 2012). In this linear process, the PSII repair cycle may be blocked either at the phosphorylation step or the degradation step. When the degradation step was blocked in a previous study by mutation of the responsible proteases, the repair cycle appeared to stop working in both FtsH knockout mutants (Bailey et al., 2002) and deg1 mutants (Kapri-Pardes et al., 2007). In these mutants, the degradation of D1 was significantly suppressed, indicating that the degradation steps involving these two proteases are unique to the PSII repair cycle. However, an earlier study indicated that inhibition of DEG5 or DEG8 protease does not seem to block the repair cycle completely, but a double mutation of these two proteases may do so (Sun et al., 2007). Similarly, partial inactivation of PSII was observed in the deg5 and deg7 mutants, and the degree of PSII photoinactivation was more severe in the corresponding double mutants (Sun et al., 2010). In this study, blockade of the degradation step in the PSII repair cycle in osstn8 mutants in the absence of lincomycin was found to be partial based on the results of our PSII photoinactivation experiments (Figures 2b and S5). However, the phosphorylation step for the photodamaged D1 protein was significantly blocked in osstn8 mutants (Figure 2a). PSII supercomplexes were not mobilized efficiently in these mutants (Figure 5), and we thus speculate that the non-mobilized and damaged but unphosphorylated D1 protein may be degraded slowly by unknown housekeeping proteases, although the degradation of D1 protein after mobilization is mainly governed by FtsH and Deg proteases. The present analysis of the oxidation of thylakoid proteins suggest that PSII core proteins are oxidized to a greater extent in osstn8 mutants than in WT (Figure 4e). In Arabidopsis, a preferential oxidation of photosynthetic machineries was observed only in the stn7/stn8 double mutants, which lack complete PCPP (Tikkanen et al., 2008). Under stress, superoxide is believed to be produced mostly by PSI (Asada, 1999; Sonoike, 2006). However, suppression of the far-red light-induced P700+ formation in the leaves was not more severe in the mutants compared with WT either before or after HL illumination (Table 1). Although singlet oxygen is known to be mainly formed at PSII under HL conditions (Telfer et al., 1994), we observed more production of superoxide in the osstn8 mutants with greater carbonylation of PSII core proteins. Although several researchers have also demonstrated the lightinduced generation of superoxide in PSII (Ananyev et al., 1994; Cleland and Grace, 1999; Pospısil, 2009), the possible site of superoxide production in osstn8 mutants and the underlying mechanism of this superoxide production remain to be further investigated.
Under GL, osstn8 mutants showed growth retardation and also delayed flowering and seed harvesting times compared with WT (Figure S3). In contrast, no such phenotype was reported in the Arabidopsis single stn8 mutant (Bonardi et al., 2005; Tikkanen et al., 2008). Indeed, growth retardation was observed only in the stn7 and stn7/stn8 double mutants in Arabidopsis (Bonardi et al., 2005; Frenkel et al., 2007; Tikkanen et al., 2008). These results suggest that suppression of PCPP significantly influences the structure and function of plants under normal growth conditions. Grana stacking was reduced, and the grana became longer in osstn8 mutants (Figure 6), as occurs in the stn7/stn8 mutant of Arabidopsis (Fristedt et al., 2009). This structural rearrangement of the entire network of thylakoid membranes with elongated grana is probably caused by the hindrance of lateral migration of photodamaged PSII complexes due to a deficiency of PCPP, in agreement with Fristedt et al. (2009). Recently, several reports proposed a central role for PCPP in the dynamic restructuring of thylakoid membranes in the light that involves a swollen lumen, lateral shrinkage of grana and vertical destacking of grana, allowing the physical contact between proteases and damaged PSII required for the initiation of PSII repair during photoinhibition (Kirchhoff et al., 2011; Herbstova et al., 2012; Kirchhoff, 2013b). Under HL illumination, the PSII repair cycle involving PCPP, the migration of the PSII supercomplex and dynamic structural rearrangement of thylakoid membrane are blocked in the osstn8 mutants, which thus showed susceptibility to HL illumination (Figure 2b), although the Arabidopsis stn7/stn8 mutants became susceptible only after prolonged illumination (Tikkanen et al., 2008). However, the migration of PSII core proteins was not observed in monocots, including barley and maize under abiotic stress conditions (Chen et al., 2009; Liu et al., 2009), and this inconsistency with the classical model for the PSII repair cycle remains unresolved. In summary, we show here that the functional redundancy during PCPP between STN7 and STN8 kinases appears to be much less in rice than in Arabidopsis. By comparing the phenotypes caused by mutation of STN7 and/or STN8 kinases between these two plant species, we speculate that all characteristic features of the osstn8 mutant described in the present study are the direct consequences of suppression of PCPP. The higher accumulation of ROS and the oxidation of PSII reaction center core proteins in thylakoid membranes during HL illumination in osstn8 plants is probably the result of unmobilized PSII supercomplexes with damaged but unphosphorylated PSII core proteins. Taken together, our current results clearly support the idea that phosphorylation of PSII core proteins, in particular D1 protein, by STN8 is involved in the PSII repair mechanism during photoinhibition.
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 76, 675–686
Role of STN8 for PSII repair in rice 683 EXPERIMENTAL PROCEDURES Plant materials and growth conditions Wild-type rice (O. sativa cultivar ‘Hwayoung’), the OsSTN8 knockout mutant osstn8, and complemented transgenic rice lines were used in the experiments. osstn8 lines were selected from a T–DNA inserted mutant pool that was generated by transformation with a T–DNA vector, pGA2707, containing the promoterless GUS gene next to the right border of the T–DNA (Jeon et al., 2000; Jeong et al., 2002). T–DNA flanking sequences were then determined as previously described (An et al., 2003). The T2 progeny of homozygous mutant lines of osstn8, transgenic lines of complemented osstn8, and WT plants were grown in a greenhouse at 30/26°C with a 16 h/8 h day/night photoperiod.
Genetic complementation The full-length OsSTN8 cDNA was amplified by PCR using the primers 5′-CGGGATCCCGAGAGCCCCCTCCTCTCCAT-3′ (forward) and 5′-GACTAGTCCTTCCTCCTCCTTTTCCCTC TCTC-3′ (reverse) (the underlined sequences correspond to BamHI and SpeI sites, respectively). A cDNA clone (AK287774) from the Knowledgebased Oryza Molecular Biological Encyclopedia (KOME, http:// cdna01.dna.affrc.go.jp/cDNA/) was used as the template. The PCR product was cloned into the pGEM–T Easy vector (Promega http:// www.promega.com/) and confirmed by sequencing. As described by Eom et al. (2011), the insert digested with BamHI and SpeI was placed between the maize (Zea mays) Ubiquitin1 (Ubi1) promoter and the nopaline synthase (Nos) terminator of the Ubi/NC4300 binary vector carrying the phosphomannose isomerase gene as a selectable marker. The resulting construct, Ubi1::OsSTN8, was used to transform the osstn8 mutant as described previously (Lucca et al., 2001).
RNA extraction and RT–PCR analysis Total RNA was extracted from leaf, root, flower and immature seed tissues using TRI Reagentâ (Life Technologies, http:// www.lifetechnologies.com/) according to the manufacturer’s instructions. The first-strand cDNA was synthesized from 1 lg total RNA using M–MLV reverse transcriptase (Promega), and used as the template for subsequent PCR amplification. All PCR reactions were performed using OsSTN8 gene-specific primers and OsUBQ5 housekeeping gene primers as an internal control (Jain et al., 2006).
photochemical efficiency of PSII (Fv/Fm), was calculated using the equation Fv/Fm = (Fm – Fo)/Fm.
Photoinhibitory treatments Healthy leaves were chosen from WT and osstn8 mutant rice plants, and approximately 2–3 cm leaf segments were prepared in water to protect against severe wounding, and then floated on water or immersed in 3 mM lincomycin. For photoinhibitory treatments, leaf segments were placed under 2000 lmol m 2 sec 1 light intensity for 6 h using a metal halide lamp equipped with a water bath to reduce heat. Approximately 2–3 cm leaf segments were infiltrated with or without 3 mM lincomycin by submergence overnight in darkness. During HL illumination, the 3 mM lincomycin solution was replaced with 1 mM lincomycin to reduce the toxic effects of this agent. To observe the recovery of damaged PSII after HL illumination, leaf segments were maintained in dim light at 20 lmol m 2 sec 1 for up to 12 h.
Immunoblotting and OxyBlotTM analysis For immunoblotting, leaf segments were dark-adapted overnight with or without 3 mM lincomycin, and then exposed to either GL at 500 lmol m 2 sec 1 or HL at 2000 lmol m 2 sec 1 for 3 h. Thylakoid membranes were isolated as described by Oh et al. (2009), and chlorophyll concentration in isolated thylakoids was measured as described by Porra et al. (1989). SDS–PAGE and immunoblotting were performed as described by Towbin et al. (1979). PCPP was detected using a phosphothreonine antibody (Cell Signaling, http://www.cellsignal.com/). D1 protein and its degradation fragments were detected using anti-D1 antibodies as described by Miyao (1994). To analyze the oxidation of thylakoid membrane proteins, whole plants were dark-adapted overnight, and leaf segments were illuminated at 1500 lmol m 2 sec 1 for up to 3 h. The oxidation of proteins in thylakoid membranes was detected using an OxyBlotTM protein oxidation detection kit (Millipore, http:// www.millipore.com/) according to the manufacturer’s instructions.
Qualitative and quantitative measurement of ROS
For quantitative real-time PCR, gene-specific PCR primers and fluorogenic probes for the TaqMan assay were designed by the Assays-by-Design service (Applied Biosystems, http://www. appliedBiosystems.com/), and their sequences are listed in Table S3. All reactions were performed in triplicate using TaqMan Universal PCR Master Mix and an ABI PRISM 7500 sequence detector (Applied Biosystems) according to the manufacturer’s instructions. Changes in gene expression were analyzed by the comparative cycle threshold (DDCt) method using Sequence Detection Systems software version 1.2 (Applied Biosystems).
Qualitative and quantitative measurements of ROS were performed as recommended by Zulfugarov et al. (2011). A qualitative or histochemical assay for ROS detection was performed in detached leaf segments as previously described by Fryer et al. (2002) and Li et al. (2010). Briefly, for determination of superoxide anion radicals, leaf samples were immersed in 6 mM NBT solution containing 50 mM HEPES buffer (pH 7.5) for 2 h in the dark. For H2O2 detection, detached leaves were immersed in 5 mM DAB solution containing 10 mM MES (pH 3.8) for 8 h in the dark. Pigments were extracted from leaf segments using absolute ethanol at 65°C by shaking in a water bath. ROS assays for thylakoids were performed during photoinhibitory treatment of thylakoids under an 800 lmol m 2 sec 1 intensity of light for 5–15 min. For detection of superoxide, the DHE fluorescence was measured as described by Georgiou et al. (2005). Similarly, measurement of the DCFDA fluorescence was used for H2O2 detection (Hempel et al., 1999). The fluorescence emission spectrum of the individual ROS sensors was measured using a F–4500 fluorescence spectrophotometer (Hitachi, http://www.hitachi-hitec.com/).
Measurement of chlorophyll fluorescence
BN-PAGE
Chlorophyll fluorescence at room temperature was determined by measuring the minimum fluorescence (Fo) and maximum fluorescence (Fm) values from detached leaf segments using a PAM 2000 portable fluorometer (Walz) after dark incubation for 30 min. The
BN-PAGE was performed as described by Reisinger and Eichacker (2006) with some modifications. Briefly, thylakoid membranes were isolated in grinding buffer containing 50 mM HEPES (pH 7.6), 0.3 M sorbitol, 10 mM sodium chloride, 5 mM magnesium chloride,
Real-time PCR analysis of organ-specific gene expression
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684 Krishna Nath et al. 2 mM EDTA and 5 mM sodium ascorbate. The homogenate was filtered through three layers of Miracloth (Calbiochem, http:// www.emdmillipore.com/) and centrifuged at 16 000 g for 10 min at 4°C. The pellets were then washed three times with washing buffer containing 50 mM HEPES (pH 7.6), 0.3 M sorbitol, 10 mM sodium chloride and 5 mM magnesium chloride. Approximately 20 lg thylakoids were resuspended in 20% glycerol, 25 mM Bis/ Tris, 10 mM magnesium chloride with 2% n dodecyl-b–D–maltoside for 30 min at 4°C. The solubilized fraction was loaded onto a 5– 13.5% BN gel.
Measurement of PSI activity The redox state of P700 was determined using a pulse amplitudemodulated fluorometer (PAM101/102/103, Walz) as described by Kim et al. (2005) in reflectance mode. The device was equipped with a dual-wavelength (810/870 nm) emitter/detector unit (ED–P700DW) consisting of an LED driver unit and an emitter/ detector unit (Walz). The PSI activity was expressed as the relative amount of far-red light-induced P700+ in the leaf segments (DA810/ A810) after 5 min of pre-illumination with 120 lmol m 2 sec 1 actinic white light (Klughammer and Schreiber, 1994; Ivanov et al., 1998; Kim et al., 2005). A810 is the absorbance signal after application of saturating far red light, and DA810 is the saturating lightinduced change in the absorbance signal during illumination with actinic light.
Figure S1. Deduced amino acid sequence alignment for Oryza sativa STN8 kinase and homologs from rice and Arabidopsis. Figure S2. Genotyping and PCR analysis of genomic DNA isolated from eight individual plants. Figure S3. Phenotypes of WT and the osstn8 mutant. Figure S4. Measurement of state transitions in WT and osstn8 mutant plants. Figure S5. Dose-dependent photoinactivation of PSII in WT and osstn8 mutant plants. Table S1. Photosynthetic pigment contents and chlorophyll a/b ratio in WT and osstn8 mutant plants before and after HL illumination. Table S2. Total chlorophyll content per leaf area in WT and osstn8 mutant plants before HL illumination. Table S3. Gene-specific primers and probes used for real-time PCR analysis. Method S1. Please refer to each supplementary method in the text, and renumber as required so consecutive in the text. Database search and sequence analysis. Method S2. Genotyping and PCR analysis of the T–DNA-inserted mutant line. Method S3. Measurement of state transition. Method S4. Measurement of light intensity-dependent photoinhibition. Method S5. Measurement of photosynthetic pigments by HPLC.
Transmission electron microscopy analysis Transmission electron microscopy analysis was performed using leaves before and after HL illumination at 2000 lmol m 2 sec 1 for 3 h. Detached leaves were cut into small pieces (1 mm 9 3 mm) and vacuum-infiltrated in 2.5% glutaraldehyde containing 0.1 M sodium cacodylate buffer (pH 7.2) for 20 min, prior to fixing at 4°C for 20 h in the same buffer containing 1% osmium tetroxide (OsO4). The samples were then briefly washed in 0.1 M sodium cacodylate buffer (pH 7.2) and dehydrated with a graded series of ethanol from 30% to absolute ethanol to complete extraction of pigments from the samples. Dehydrated samples were twice vacuum-infiltrated with propylene oxide for 30 min, embedded in Epon 812 embedding medium (Electron Microscopy Sciences, http://www.emsdiasum.com/), and then polymerized at 60°C for 30 h. Leaf sections of 70 nm thickness were prepared using an ultra-microtome, and collected on an EM grid. Ultra-thin sections were stained using 2% uranium acetate followed by 2% lead citrate, and viewed using a transmission electron microscope (JEM–1011, JEOL, http://www.jeol.co.jp/).
ACKNOWLEDGEMENTS This work was supported by a grant from the National Research Foundation of Korea, funded by the Korean Government Ministry of Education Science and Technology (numbers 2010-0011395, 2011-0017947 and 2012-0004968). J.–S.J. was supported by a grant from the Next-Generation BioGreen 21 Program (PJ00948406), Rural Development Administration of the Korean Ministry of Food, Agriculture, Forestry and Fisheries. H.G.N. was supported by a grant from the National Research Foundation of Korea through the National Research Support Program (number 2010-0020417). The authors thank Suleyman I. Allakhverdiev (Russian Academy of Sciences, Russia) for critically reading the manuscript.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article.
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