Functional dissection of two Arabidopsis PsbO proteins PsbO1 and PsbO2 Reiko Murakami 1 , Kentaro Ifuku 1 , Atsushi Takabayashi 1 , Toshiharu Shikanai 2 , Tsuyoshi Endo 1 and Fumihiko Sato 1 1 Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto, Japan 2 Graduate School of Agriculture, Kyushu University, Higashiku, Fukuoka, Japan In oxygenic photosynthesis, the multisubunit protein complex photosystem II (PSII) uses light energy to oxidize water and to form molecular oxygen [1–3]. Water oxidation occurs at a catalytic site of PSII that contains four manganese atoms, and PSII contains sev- eral extrinsic subunits that play important roles in sta- bilizing the active manganese site. One of these, the nuclear-encoded PsbO protein in PSII, has a molecular mass of approximately 26 kDa, although it has been called extrinsic 33-kDa protein traditionally, and is syn- thesized with a transit sequence that targets the protein to the thylakoid lumen [4,5]. PsbO is present in all oxygen-evolving organisms. It appears to play a central role in stabilization of the manganese cluster and is essential for efficient and stable oxygen evolution. To better understand the role of PsbO, mutants that lack the psbO gene have been established. PsbO has been deleted using genetic methods in cyanobacteria [6,7] and in green algae [8], and such mutants have been maintained under heterotrophic conditions. In contrast, no PsbO mutant has been obtained in higher plants due to the essential role of this protein in pho- tosynthesis. Previously, however, we isolated an Ara- bidopsis mutant [9,10] with a defect in the psbO1 gene Keywords Arabidopsis thaliana; isoform; oxygen evolution; psbO1; psbO2 Correspondence T. Endo Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto 606–8502, Japan Fax: +81 75 7536398 Tel: +81 75 7536381 E-mail: tuendo@kais.kyoto-u.ac.jp (Received 24 December 2004, revised 17 February 2005, accepted 1 March 2005) doi:10.1111/j.1742-4658.2005.04636.x PsbO protein is an extrinsic subunit of photosystem II (PSII) and has been proposed to play a central role in stabilization of the catalytic manganese cluster. Arabidopsis thaliana has two psbO genes that express two PsbO proteins; PsbO1 and PsbO2. We reported previously that a mutant plant that lacked PsbO1 (psbo1) showed considerable growth retardation despite the presence of PsbO2 [Murakami, R., Ifuku, K., Takabayashi, A., Shika- nai, T., Endo, T., and Sato, F. (2002) FEBS Lett 523, 138–142]. In the pre- sent study, we characterized the functional differences between PsbO1 and PsbO2. We found that PsbO1 is the major isoform in the wild-type, and the amount of PsbO2 in psbo1 was significantly less than the total amount of PsbO in the wild-type. The amount of PsbO as well as the efficiency of PSII in psbo1 increased as the plants grew; however, it never reached the total PsbO level observed in the wild-type, suggesting that the poor activity of PSII in psbo1 was caused by a shortage of PsbO. In addition, an in vitro reconstitution experiment using recombinant PsbOs and urea-washed PSII particles showed that oxygen evolution was better recovered by PsbO1 than by PsbO2. Further analysis using chimeric and mutated PsbOs suggested that the amino acid changes Val186 fi Ser, Leu246 fi Ile, and Val204 fi Ile could explain the functional difference between the two PsbOs. Therefore we concluded that both the lower expression level and the inferior functionality of PsbO2 are responsible for the phenotype observed in psbo1. Abbreviations F m , maximum fluorescence yield at closed PSII centers; F 0 , minimum fluorescence yield at open PSII centers; F v , F m ) F 0 ; PSII, photosystem II. FEBS Journal 272 (2005) 2165–2175 ª 2005 FEBS 2165 (psbo1), as PsbO proteins in Arabidopsis are encoded by two genes, psbO1 and psbO2 [11]. On the other hand, many other plant species have only one psbO gene. The second gene in Arabidopsis would be a unique result of a duplication of the genome and chro- mosomes some 28–48 million years ago, before the divergence of Arabidopsis from Brassica about 14–24 million years ago [12,13]. Our previous analysis showed that psbo1 exhibited weak photosynthetic activity and considerable growth retardation compared with the wild-type [10]. This observation raised the question of why the psbO2 gene could not complement the defect in the psbO1 gene, since PsbO1 and PsbO2 are highly homologous with regard to the primary structure. In this study, we characterized the differences in the accumulation and biochemical activity of these iso- forms to clarify their functional differences. A detailed immunoblot analysis using the mutant, psbo1 , and the wild-type showed that a lower level of PsbO2 would limit the photosynthetic activity. Additional in vitro experiments with urea-washed PSII particles reconsti- tuted with PsbOs revealed that oxygen-evolution was better recovered by PsbO1 than by PsbO2, though the two isoforms had a similar binding affinity for the PSII particles. These results showed that the differ- ences in both biochemical activity and the level of accumulation were responsible for the poor photosyn- thetic activity in psbo1. We further dissected the functional differences between PsbO1 and PsbO2, as these isoforms have quite a similar primary structure, and differ at only 11 amino acids. A previous investigation [5] pointed out the importance of Cys at positions 29 and 52 of mature PsbO1 for forming a disulfide bridge [14,15], and Val at position 148 for producing a b-sheet [16], as well as carboxylic residues that have been reported to be involved in the interaction between PsbO and PSII [17,18]. Moreover, these amino acid residues are conserved in PsbO2. In addition, the predictions regarding the structures of Arabidopsis PsbO1 and PsbO2 based on PSII of Thermosynechococcus elonga- tus [19] (protein data bank accession number 1S5L) were similar. Thus, to identify the amino acids respon- sible for the poor oxygen-evolving activity of PsbO2, we prepared chimeric proteins derived from PsbO1 and PsbO2. Further, site-directed mutagenesis showed that the replacement of Val186 of mature PsbO1 with Ser and of Leu246 with Ile decreased the oxygen- evolving activity, whereas substitution of Val-204 with Ile increased the oxygen-evolving activity. Based on these results, we discuss the physiological role of the duplicated PsbO in Arabidopsis. Results PsbO isoform level and the efficiency of PSII To understand the reason for the loss of photosyn- thetic activity in psbo1, we measured the protein levels of PsbOs and the photosynthetic activity in mutant and wild-type plants. Whereas the wild-type plants maintained strong photosynthetic activity throughout their growth, psbo1 plants showed a gradual increase in electron transport activity, represented by the chlo- rophyll fluorescence parameter, F v ⁄ F m (the maximum photochemical efficiency in PSII) [20,21], as they grew (Fig. 1). PsbO1 and PsbO2 can be distinguished on SDS ⁄ PAGE, since PsbO1 migrated more slowly than PsbO2 [10]. Immunoblot analysis showed that the major isoform in the wild-type was PsbO1, which com- prised about 90% of the total amount of PsbO based on densitometry of the immunoblot membranes (Fig. 2A). The amount of PsbO2 was much greater in psbo1 than in the wild-type (Fig. 2A,B), indicating that the expression of psbO2 was activated in a compensa- tory manner. At an early growth stage, the PsbO2 level in psbo1 accounted for 40% of the total in the wild- type. The amount of PsbO2 increased considerably in mature psbo1, and reached about 70% of the total in the wild-type. This change in the level of PsbO2 coincided with the change in F v ⁄ F m in immature and mature psbo1 plants; i.e. 0.54 ± 0.05 and 0.68 ± 0.03, respectively. The accumulation of PsbO2 in mature plants clearly led to more efficient PSII. Similarly, immunoblot analysis of other PSII pro- teins, PsbA and PsbP, showed that the amounts of 0.3 0.4 0.5 0.6 0.7 0.8 0.9 020406080100 da y s after g ermination wild-type psbo1 Fv/Fm Fig. 1. Potential quantum yield of PSII (F v ⁄ F m ) in leaves of the wild- type (r) and psbo1 (h) during the course of plant growth. Values represent averages and standard deviations for 10–15 plants. Functional dissection of PsbO1 and PsbO2 R. Murakami et al. 2166 FEBS Journal 272 (2005) 2165–2175 ª 2005 FEBS PsbA and PsbP were also smaller in psbo1 than in the wild-type (Fig. 2A,B), whereas the levels of these pro- teins also increased as psbo1 grew. As PsbO stabilizes the manganese cluster and PSII core, and it has often been reported that PsbO deficiency affects the amount of PsbA and PsbP [22–24], it is very likely that the amount of PsbOs limits the amounts of PsbA and PsbP. These results suggest that the amount of PsbOs in psbo1 is the critical limiting factor for photosyn- thesis and growth. Biochemical activities of PsbO1 and PsbO2 in oxygen evolution As it was clear that the shortage of PsbOs in psbo1 is the critical limiting factor for photosynthesis, we car- ried out in vitro experiments using recombinant PsbOs and urea-washed PSII particles from spinach to clarify the qualitative differences between PsbO1 and PsbO2. Arabidopsis PsbO has been reported to be able to bind urea-washed PSII particles of spinach and to restore the oxygen-evolving activity as effectively as spinach PsbO [16,25]. We carried out reconstitution procedures according to their methods. First, we examined the oxygen-evolving activity as a function of the PsbOs ⁄ PSII ratio in the assay medium (Fig. 3A). The maxi- mum activity with PsbO2 was about 80% of that with PsbO1, whereas the maximum oxygen-evolving activity reconstituted with both PsbO1 and PsbO2 was achieved at about 2 PsbOs ⁄ PSII. The restoration curve obtained with our recombinant PsbOs closely resem- bled that for spinach PsbO and Arabidopsis PsbO1 reported by Betts et al . [25]. PsbO1 and PsbO2 also showed a similar affinity for urea-washed PSII parti- cles (Fig. 3B,C); the binding of both PsbOs with urea- washed PSII particles was saturated at about 2 PsbOs ⁄ PSII, like the oxygen-evolving activity. Competition analysis of PsbO1 and PsbO2 To confirm that the two PsbOs have a similar binding affinity, the competition between them for binding sites on PSII particles was analyzed. Increasingly larger amounts of an equimolar mixture of PsbO1 and PsbO2 were incubated with urea-washed PSII particles (Fig. 4A,B), and the relative amounts of bound-PsbO1 wild-type y oun g wild-type mature psbo1 y oun g psbo1 mature PsbO PsbA PsbP Relative protein accumulation 140 120 100 80 60 40 20 0 PsbA PsbP wild-type MY psbo1 MY wild-type MY psbo1 MY CBB-staining PsbO1 PsbO2 A B Fig. 2. Protein analysis by SDS ⁄ PAGE (15%). (A) Coomassie brilliant blue-staining and immunoblot analysis with polyclonal antibodies against spinach PsbO, PsbP and PsbA. Antibodies against PsbO detect two signals with slightly different migrations, upper band; PsbO1, lower band; PsbO2. Thylakoid membranes were loaded on a chlorophyll basis; equivalent to 5 lg of chlo- rophyll for Coomassie brilliant blue-staining and 1 lg for immunoblot analysis. The arrow shows PsbOs in Coomassie brilliant blue-staining. Y, young plants (leaf size, about 0.5 cm); M, mature plants (leaf size, about 2.5 cm). (B) The relationship between the accumulation of PsbO (black), PsbP (mid-grey) and PsbA (light grey). The protein level was quantified by densitometry. Values are relative to the protein level in the young wild-type (100%). Standard deviations were calculated from three measurements. R. Murakami et al. Functional dissection of PsbO1 and PsbO2 FEBS Journal 272 (2005) 2165–2175 ª 2005 FEBS 2167 and bound-PsbO2 with urea-washed PSII particles were quantified by the densitometry of Coomassie bril- liant blue-stained proteins on a SDS ⁄ PAGE plate. As PsbO1 migrated more slowly than PsbO2, the two could be distinguished in the binding analysis [10,26–28]. Urea-washed PSII particles incubated with two moles of PsbOs per mole of PSII particles (one mole of each protein added, 1 : 1) had similar amounts of PsbO1 and PsbO2 (approximately PsbO1 ⁄ PsbO2 ¼ 51 : 49). The oxygen-evolving activity with a mixture of one mole each of PsbO1 and PsbO2 was higher than that with four moles of PsbO2, but lower than that with four moles of PsbO1. Although the differ- ence was small, the same results were obtained consis- tently. As the levels of both proteins were saturated at about 2 PsbOs ⁄ PSII, we added an excessive amount of PsbOs per PSII (two moles of each protein, 2 : 2). The two proteins bound equally with urea-washed PSII (approximately 50 : 50) and the oxygen-evolving activ- ity was as high as the sample; 1 : 1. These results sug- gested that the two proteins had a similar affinity for urea-washed PSII. Next, we examined the effect of increasing the propor- tion of PsbO1 on the reconstitution of PSII. The ratio of PsbO1 to total PsbOs bound with urea-washed PSII particles and the oxygen-evolving activity demonstrated equal competition between PsbO1 and PsbO2 for PSII particles (Fig. 4C). These competition analyses were consistent with the analysis of the reconstitution with PsbO1 and PsbO2, in terms of similar binding affinity. Chimeric PsbOs and surface charge To examine which amino acid of PsbO2 is responsible for the poor oxygen-evolving activity, we prepared chi- meric proteins with PsbO1 and PsbO2 in Escherichia coli (Fig. 5A). For example, the chimeric protein PsbO1-1-2 has the N-terminal and middle parts of PsbO1 combined to the C-terminus of PsbO2. Each of the parts contains about 80 amino acids, and, respect- ively, has three, five and three amino acid replacements between PsbO1 and PsbO2. SDS ⁄ PAGE analysis of chimeric PsbOs showed that PsbO1-1-2 and PsbO2-1-1 migrated as slowly as PsbO1, and only PsbO1-2-1 migrated as PsbO2 did (Fig. 5B). Anion-exchange chromatography was also performed, as PsbO1 was eluted at a higher NaCl 012345 PsbO1 PsbO2 100 80 60 40 20 0 untreated PSII urea-washed PSII 0.5 1 2 4 0.5 1 2 4 PsbO1 PsbO2 012345 140 120 100 80 60 40 20 0 PsbO1 PsbO2 A B C Fig. 3. The oxygen-evolving activity of urea-washed PSII reconstitu- ted with PsbO1 and PsbO2 and the binding affinity for urea-washed PSII of PsbO1 and PsbO2. (A) The relative oxygen-evolving activity of urea-washed PSII reconstituted with PsbO1 (r) and PsbO2 (h). The maximum rate of oxygen evolution (100%) equals the rate measured for untreated PSII minus that for urea-washed PSII. (B) Coomassie brilliant blue-staining. Urea-washed PSII reconstituted with PsbO1 and PsbO2 were separated on SDS ⁄ PAGE (15%). An amount equivalent to 2 lg of chlorophyll was loaded in each lane. The arrow shows PsbOs in Coomassie brilliant blue-staining. (C) Quantification of PsbO1 (r) and PsbO2 (h) bound to urea-washed PSII particles. Values quantified by densitometry were plotted against PsbOs added per PSII. Values are relative to the untreated PSII (100%). Standard deviations were calculated from five meas- urements. Functional dissection of PsbO1 and PsbO2 R. Murakami et al. 2168 FEBS Journal 272 (2005) 2165–2175 ª 2005 FEBS concentration than was PsbO2. PsbO1-2-1 again showed a chromatographic elution profile similar to PsbO2, whereas the profiles of other chimeric proteins were similar to that of PsbO1 (data not shown). In fact, these results were consistent with the theoretical pI values; the theoretical pI values of PsbO1, PsbO1-1-2 and PsbO2-1-1 were calculated to be 4.96 (calculated molecular mass, 26565 Da), 4.95 (26567 Da) and 4.96 (26579 Da), respectively, and those of PsbO2 and PsbO1-2-1 were calculated to be 5.05 (26571 Da) and 5.06 (26555 Da), respectively (to compute pI ⁄ Mw, http://kr.expasy.org). These results suggest that, in terms of surface charge, PsbO1-2-1 was similar to PsbO2 and the other chimeric PsbOs were similar to PsbO1. Ppu MI Bam HI PsbO1 PsbO2 PsbO1-1-2 PsbO2-1-1 PsbO1-2-1 A B C Fig. 5. Oxygen-evolving activity of chimeric proteins derived from PsbO1 and PsbO2. (A) A model of the chimeric PsbOs. In chimeric PsbOs, a part of PsbO1 (C-terminus, middle and N-terminus) was replaced by a corresponding part of PsbO2. Each of the parts con- tains about 80 amino acids, and, respectively, has three, five and three amino acid replacements between PsbO1 and PsbO2. (B) Coomassie brilliant blue-staining on SDS ⁄ PAGE (15%) of PsbO1, PsbO2 and chimeric PsbOs. They were expressed using a pET-sys- tem in Escherichia coli and purified by anion-exchange chromato- graphy. Protein (0.5 lg) was loaded in each lane. (C) Oxygen-evol- ving activities of urea-washed PSII reconstituted with PsbO1, PsbO2 and chimeric PsbOs. The maximum rate of oxygen evolu- tion (100%) equals the rate measured for untreated PSII minus that for urea-washed PSII. untreated PSII urea-washed PSII PsbO PsbO2 (1:1) (2:2) 10020 40 60 800 120 0 0.2 0.4 0.6 0.8 1 100 80 60 40 20 0 120 A B C Fig. 4. Competition of PsbO1 and PsbO2 for the reconstitution of PSII. (A) Reconstitution of PSII with an equimolar mixture of PsbO1 and PsbO2. Reconstituted PSII particles were separated on a 15% SDS ⁄ PAGE and stained by Coomassie brilliant blue. An amount equivalent to 2 lg of chlorophyll was loaded in each lane. PsbO1, PsbO2; 4 mol PsbO1 or PsbO2 per PSII, 1 : 1; 1 mole PsbO1 and 1mole PsbO2 per PSII, 2 : 2; 2 mole PsbO1 and 2 mole PsbO2 per PSII. (B) Relative oxygen-evolving activity of PSII reconstituted with an equimolar mixture of PsbO1 and PsbO2. The maximum rate of oxygen evolution (100%) equals the rate measured for untreated PSII minus that for urea-washed PSII. (C) Reconstitution of PSII with PsbOs in which the ratio of PsbO1 to total PsbO was varied. The relative oxygen-evolving activity (r) and the ratio of bound PsbO1 : PsbOs (h). The maximum rate of oxygen evolution (100%) equals the rate measured for untreated PSII minus that for urea- washed PSII. The ratio of bound PsbO1 : PsbOs was estimated from the Coomassie brilliant blue-staining on SDS ⁄ PAGE. Standard deviations were calculated from five measurements. R. Murakami et al. Functional dissection of PsbO1 and PsbO2 FEBS Journal 272 (2005) 2165–2175 ª 2005 FEBS 2169 However, the oxygen-evolving activity of urea- washed PSII particles reconstituted with PsbO1-2-1 was similar to that of the particles reconstituted with PsbO1 (Fig. 5C). A lower level of activity was observed with PsbO1-1-2, a chimera with the C-ter- minal sequence of PsbO2. Although the difference was small, the same results were obtained repeatedly. These results suggest that the difference in surface charge between PsbO1 and PsbO2 did not affect the difference in oxygen-evolving activity. C-Terminal amino acid substitution and oxygen-evolving activity PsbO1 and PsbO1-1-2 had three amino acid changes; Val186 (PsbO1) to Ser (PsbO2), Val204 to Ile, and Leu246 to Ile (Fig. 6A). To examine which replace- ment was responsible for the difference in the oxygen- evolving activity, we prepared mutated PsbO1 in which an amino acid was substituted for the corresponding one in PsbO2 (V186S, V204I and L246I). Measure- ment of the oxygen-evolving activity upon reconstitu- tion with the spinach PSII core showed that the activity levels with V186S and L246I were apparently lower than that with PsbO1 (Fig. 6B), suggesting that these two amino acids were responsible for the lower level of activity. Unexpectedly, V204I showed stronger oxygen-evolving activity than PsbO1. We next prepared double-mutated proteins with two amino acid substitutions; i.e. V186S ⁄ V204I, V186S ⁄ L246I and V204I ⁄ L246I. The oxygen-evolving activity reconstituted with V186S ⁄ V204I and V204I ⁄ L246I was similar to that with PsbO1; levels were higher than for V186S and L246I and lower than for V204I (Fig. 6B). The activity reconstituted with V186S ⁄ L246I was much weaker than that with V186S, L246I or PsbO2. These results confirmed that the replacement of Val186 with Ser and Leu146 with Ile led to a reduction in the level of oxygen-evolving activity, while the replacement of Val204 with Ile led to an increase. Location of amino acids that differ between PsbO1 and PsbO2 The locations of three amino acids, Val186 Val204 and Leu246, were predicted using the Thermosynechococcus elongatus PsbO [19] (protein data bank accession num- ber; 1S5L) as a template. The prediction suggested that Val186, Val204 and Leu246 were dispersed in different secondary structures; i.e. in the a-helix, in the b-sheet and near the b-sheet, respectively (Fig. 7). The predic- tion also supported the notion that these replacements independently affected the oxygen-evolving activity. As expected, the predicted structure of Arabidopsis PsbO2 was very similar to the structure of PsbO1 (data not shown), and the substitution of amino acids between PsbO1 and PsbO2 would not modify the overall struc- ture of PsbO. Discussion The existence of two psbO genes enabled the isolation of psbo1 which lacked psbO1 expression and showed weak photosynthetic activity. In this study, we exam- ined the functional role of two PsbOs in Arabidopsis. Careful characterization of the PsbOs in psbo1 and the wild type (Fig. 2A) showed that psbo1 showed a much greater accumulation of PsbO2 than in the wild type. This finding suggests a compensational mechanism that stimulates the expression of PsbO2 when a functional psbO1 gene is absent. The shortage of PsbOs caused a photosynthetic defect in psbo1, especially in young psbo1. However, mature psbo1 exhibited increased lev- els of PsbO and improved photosynthetic activity, as estimated from the increased F v ⁄ F m , but the molecular mechanism of this adaptation to a genetic defect is not clear. Our immunoblot analysis also showed that the accumulation of PsbOs limited the levels of other PSII proteins as well as the efficiency of PSII. This result was consistent with findings in early studies using higher-plant PSII that the extraction of PsbO from PSII affected the stability of PsbA and the assembly of other extrinsic proteins [22–24]. In this respect, the quite different phenotypes observed in psbo1 and psbO-deletion mutants of green algae and cyanobacteria might lead to a clearer understanding of the structure of the oxygen-evolving complex [6,7]. First, although a psbO-deletion mutant of the Chlamydomonas reinhardtii [8] also had an enhanced turnover of core PSII polypeptide, PsbA, a psbO- deletion mutant of cyanobacteria accumulated PSII reaction centers at nearly normals levels. The differ- ence in the behavior of the deletion mutants between higher plants, green algae and cyanobacteria suggests a difference in the relationship between the extrinsic proteins. Green algae and higher plants contain PsbP and PsbQ, whereas cyanobacteria contain PsbV and PsbU. There is an apparent difference between PsbP and PsbV, as PsbP cannot bind to the PSII core or function in the absence of PsbO, but PsbV alone functions effectively in the absence of PsbO. Second, PsbP was accumulated at normal levels in the Chlamydomonas reinhardtii psbO-deletion mutant, sug- gesting a difference between higher plants and green algae in the regulation of levels of extrinsic proteins. Functional dissection of PsbO1 and PsbO2 R. Murakami et al. 2170 FEBS Journal 272 (2005) 2165–2175 ª 2005 FEBS Arabidopsis thaliana O1 Arabidopsis thaliana O2 Nicotiana tabacum Solanum tuberosum Lycopersicon esculentum Pisum sativum Oryza sativa Spinacia oleracea Volvox carteri Chlamydomonas reinhardtii Euglena gracilis Bigelowiella natans Synechocystis sp. PCC 6803 Anabaena sp. PCC 7120 Thermosynechococcus elongatus BP-1 Prochlorococcus marinus SS120 Prochlorococcus marinus MIT9313 Synechococcus sp.WH8102 Triticum aestivum Fritillaria agrestis * Synechocystis sp. PCC 6803 Prochlorococcus marinus SS120 Prochlorococcus marinus MIT9313 Synechococcus sp.WH8102 Arabidopsis thaliana O1 Arabidopsis thaliana O2 Nicotiana tabacum Solanum tuberosum Lycopersicon esculentum Pisum sativum Oryza sativa Spinacia oleracea Volvox carteri Chlamydomonas reinhardtii Euglena gracilis Bigelowiella natans Anabaena sp. PCC 7120 Thermosynechococcus elongatus BP-1 Triticum aestivum Fritillaria agrestis * Arabidopsis thaliana O1 Arabidopsis thaliana O2 Nicotiana tabacum Solanum tuberosum Lycopersicon esculentum Pisum sativum Oryza sativa Spinacia oleracea Volvox carteri Chlamydomonas reinhardtii Euglena gracilis Bigelowiella natans Synechocystis sp. PCC 6803 Anabaena sp. PCC 7120 Thermosynechococcus elongatus BP-1 Prochlorococcus marinus SS120 Prochlorococcus marinus MIT9313 Synechococcus sp.WH8102 Triticum aestivum Fritillaria agrestis * Relative oxygen-evolving activity 0 20 40 60 80 100 120 PsbO1 PsbO2 V186S V204I L246I 186&204 186&246 204&246 AB Fig. 6. Determination of the amino acid changes responsible for the alteration of protein function. (A) Alignments around the replacements between PsbO1 and PsbO1-1-2. The alignments were made with CLUSTAL W. Asterisks indicate substituted residues with Arabidopsis PsbO1 and PsbO2. Arabidopsis PsbO1 and PsbO2 are underlined. (B) Oxygen-evolving activities reconstituted with PsbO1, PsbO2, and mutated- PsbOs. The maximum rate of oxygen evolution (100%) equals the rate measured for untreated PSII minus that for urea-washed PSII. Stand- ard deviations were calculated from five measurements. R. Murakami et al. Functional dissection of PsbO1 and PsbO2 FEBS Journal 272 (2005) 2165–2175 ª 2005 FEBS 2171 Interestingly, PsbO1 and PsbO2 showed different biochemical activity at reconstituting oxygen-evolving activity with urea-washed PSII isolated from spinach; the restoration of oxygen-evolving activity with PsbO2 was about 80% of that with PsbO1, whereas the bind- ing affinities of PsbO1 and PsbO2 were similar. A recent study on the structure of PSII [19] suggested that PSII has one copy of PsbO, but early works [16,25] and our study (Fig. 3) showed that the maximum oxygen-evolving activity reconstituted with both PsbO was achieved at about two PsbOs per PSII. It is not clear why about two PsbOs per PSII are nee- ded for the maximum activity. The chimeric PsbO derived from PsbO1 and PsbO2 clearly indicated that the surface charge was not crit- ical for the different activities of PsbO1 and PsbO2, whereas negative and positive charges of amino acids have been reported to be important for the interaction between PsbO and PSII [5,17,18]. The analysis using the chimeric PsbOs showed that three amino acid replacements at the C-terminus affected the restoration of oxygen-evolving activity. As predicted in Fig. 7, the three amino acids were dispersed in PsbO. Interestingly, although Val186 was not conserved in higher plants, green algae and cyano- bacteria, its substitution with Ser decreased oxygen- evolving activities. On the other hand, when Val204 of PsbO1, which is conserved in higher plants except for Arabidopsis PsbO2, was substituted with the Ile of PsbO2, this mutated-protein restored the oxygen-evol- ving activity better than PsbO1. It should be noted that the amino acid at this position is substituted with Ile in both green algae and cyanobacteria. By contrast, Leu246 is conserved in all higher plants and green algae, except in PsbO2. PsbO2 has Ile246, which is substituted with Ile or Val in cyanobacteria, except for Prochlorococcus marinus which has Lys. The replace- ment of Leu with Ile was shown to result in a reduc- tion of the restoration of oxygen-evolving activity. It has been reported that Leu at position 246 was critical for PsbO to bind PSII and the restoration of oxygen- evolving activity in spinach [29]. Although our recon- stitution experiment with PsbO2 showed a similar affinity for PSII particles as PsbO1, the importance of this amino acid residue for oxygen-evolving activity was consistent with the results of previous works [29,30]. The similar chemical properties of Leu and Ile might explain their similar binding affinities. It has also been reported that the C-terminal peptide of PsbO (15 amino acids) competed with mature PsbO to bind the PSII core, suggesting that the C-terminus of PsbO plays an important role in the interaction with an integral part of the PSII core [31]. Identification of the importance of the three amino acid residues in PsbO, especially V204I, could be useful for understanding the efficiency of PSII. In vivo and in vitro experiments have shown that both a shortage of the PsbOs and a lower level of oxy- gen-evolving activity resulted in the poor photosyn- thetic activity and retarded growth in psbo1. In most higher plants, such as spinach, pea and rice, PsbO is encoded by only a single gene [26,32]. The existence of a duplicated psbO gene in Arabidopsis should help us to understand the molecular selection of duplicated genes. L246I V186S V186S V204I L246I A B Fig. 7. The location of three amino acid substitutions between PsbO1 and PsbO2. (A) Stereoview of Thermosynechococcus elong- atus PsbO. The corresponding positions of Val186, Val204 and Leu246 in Thermosynechococcus elongatus PsbO were predicted by SwissModel (http://swissmodel.expasy.org//SWISS-MODEL. html) and are shown in red and indicated by an arrow. (B) Stereo- view of PsbO, PsbA and Mn clusters in Thermosynechococcus elongatus. PsbO: green, PsbA: blue, Mn clusters: pink, The posi- tions corresponding to Val-186, Val-204 and Leu-246 are shown in red and indicated by an arrow. These figures were generated with P YMOL (http://pymol.sourceforge.net/). Functional dissection of PsbO1 and PsbO2 R. Murakami et al. 2172 FEBS Journal 272 (2005) 2165–2175 ª 2005 FEBS In Arabidopsis, psbQ was also encoded by two nuc- lear genes (At4g21280 and At4g05180) with about 82% similarity at the gene level. On the other hand, psbP (At1g06680) has a similar but nonfunctional gene (At2g30790) (about 84% similarity at the gene level); it encodes a much smaller protein than PsbP, suggesting that the isogene of psbP recently lost its function. Simi- larly, other subunits in PSII encoded by nuclear genes, such as psbR and psbS, are encoded by a single gene. The investigation of PsbQ isogenes should provide another clue as to the physiological role of duplicated genes for extrinsic proteins in the oxidation of water. Although the duplicated genes would initially have had the same functions, the accumulation of mutations over time has resulted in either the functional loss of one copy or functional divergence between them (our data and [33]). It is not clear how much functional differenti- ation exists between PsbO1 and PsbO2 and whether the inferior psbO2 will lose its activity. At present, the data- base (Brassica Genome Gateway, http://brassica.bbsrc. ac.uk) suggests that Brassica rapa and Brassica napus have a psbO1-like gene (accession number; Brassica rapa, BQ791144 and Brassica napus, AF139818) and a psbO2-like gene (accession number; Brassica rapa, BG543314 and Brassica napus, CD821869), indicating that any potential evolutionary pressure to remove the psbO2 gene is not strong. Indeed, PsbO2 might have some physiological significance; for example, the poor oxygen-evolving activity with PsbO2 might be advanta- geous under excessive light conditions. Conclusions The psbO2 gene could not complement the defect in the psbO1 gene for both quantitative and qualitative reasons: a shortage of PsbOs and the poor oxygen- evolving activity of PsbO2. The functional difference between PsbO1 and PsbO2 in the restoration of oxygen-evolving activity was ascribed to three amino acid replacements at the C-terminus; Val186 in PsbO1 for Ser in PsbO2, Val204 for Ile, and Leu246 for Ile. Experimental procedures Plant growth and measurements of chlorophyll fluorescence Seeds of the wild-type and psbo1 were sown in soil after cold treatment for 24 h at 4 °C. Seeds were obtained from Lehle Seeds (Round Rock, TX, USA). They were grown at 23 ± 0.5 °C under white light (30 lmolÆphotons ⁄ m )2 Æs) with a 9-h light (from 09:00–18:00 h): 15-h dark cycle for 2 months. The chlorophyll fluorescence parameter (F m ) F 0 ) ⁄ F m (¼ F v ⁄ F m ), was measured using a PAM2000 chlorophyll fluorometer (Waltz, Effeltrich, Germany). The minimum fluorescence at the open PSII centers, F o , was determined by measuring light. F m (the maximum chlorophyll fluores- cence at closed PSII centers in the dark) was measured by applying a 1-s pulse of saturating white light. Immunoblot analysis Thylakoid membranes were isolated from wild-type and psbo1 mutant leaves according to the method of Endo et al. [34]. Protein samples were prepared in sample buffer [50 mm Tris ⁄ HCl pH 6.8, 5% sodium dodecylsulfate (SDS), 5% glycerol, and 5% 2-mercaptoethanol], and analyzed by 15% SDS ⁄ PAGE to evaluate visually the level of protein expression with Coomassie brilliant blue staining [35]. Thyl- akoid membranes were loaded on a chlorophyll basis. The protein analysis with respect to the protein content showed the results similar to that on a chlorophyll basis. The thy- lakoid proteins were also transferred to a polyvinylidene difluoride membrane with a semidry blotting system [36], and detected with rabbit antiserum against spinach PsbO, kindly provided by the late A. Watanabe of the University of Tokyo, or rabbit antiserum against spinach D1 protein (PsbA), provided by Y. Yamamoto (Division of Integrated Life Science, Kyoto University, Japan). The chlorophyll concentration was measured by spectrophotometry in 80% acetone [37]. The amounts of PsbO, PsbA and PsbP in plants were quantified from immunoblots and bound PsbOs to urea- washed PSII were quantified from Coomassie brilliant blue- stained plates. Image analysis was performed using the public-domain software nih-image (version 1.62). Preparation of recombinant PsbOs The recombinant PsbO1 and PsbO2 were expressed and purified as described previously [38]. The expression vectors for the chimeric proteins were produced by digestion of psbO1- and psbO2-pET 21d + with BamHI, NotIor PpuMI and by the ligation of resultant fragments. Site- directed mutagenesis was performed according to the proto- col for the Quick-change Site-directed Mutagenesis Kit (Stratagene) and a Technical Review for Long polymerase- chain reaction using KOD plus (TOYOBO, Osaka, Japan). All mutant constructs were confirmed by DNA sequencing. These mutant proteins were purified similar to the original PsbO1 and PsbO2. Reconstitution of PSII with recombinant PsbOs Photosystem II membranes were isolated from spinach leaves purchased at a local market according to the method R. Murakami et al. Functional dissection of PsbO1 and PsbO2 FEBS Journal 272 (2005) 2165–2175 ª 2005 FEBS 2173 of Ghanotakis et al. [39]. Native extrinsic proteins in PSII were extracted by incubation in urea-washed buffer (50 mm Mes–NaOH (pH 8.3) and 3 m urea), to give urea-washed PSII particles. Recombinant PsbOs were mixed with urea- washed PSII particles and incubated at room temperature (25 °C) for 1 h in the dark to reconstitute PSII [23]. The concentration of PSII was estimated on the basis of chl concentrations and a stoichiometry of 250 chlorophylls per PSII complex. Photosystem II activity was measured as oxygen evolution at 25 °C with a Clark-type Oxygen electrode (Hansatech, UK) in the presence of 2 mm DCBQ (dichloro- p-quinone) as the electron acceptor for PSII. Red satura- tion actinic light, at an intensity of 2000 lmolÆphotons ⁄ m )2 Æs, was provided by an incandescent lamp that was used in conjunction with an HA50 heat-absorbing filter and an R-60 red optical filter (Kenko, Tokyo, Japan). Alignment of PsbOs Similarity was evaluated using the clustal w program [40–42]. The alignment parameters used were: protein mass matrix: BLOSUM series, gap opening penalty (GOP): 10.0, gap extension penalty (GEP): 0.05 and Delay divergent sequences: 40%. Acknowledgements This study was supported in part by a COE Scientific Research Grant from the Ministry of Education, Cul- ture, Sports, Science and Technology of Japan. 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(A) A model of the chimeric PsbOs replacements between PsbO1 and PsbO2 . SDS ⁄ PAGE analysis of chimeric PsbOs showed that PsbO1 -1-2 and PsbO2 -1-1 migrated as slowly as PsbO1 , and only PsbO1 -2-1 migrated as PsbO2 did (Fig. 5B) amount equivalent to 2 lg of chlorophyll was loaded in each lane. PsbO1 , PsbO2 ; 4 mol PsbO1 or PsbO2 per PSII, 1 : 1; 1 mole PsbO1 and 1mole PsbO2 per PSII, 2 : 2; 2 mole PsbO1 and 2 mole PsbO2 per PSII.