Distribution of the extrinsic proteins as a potential marker for the evolution of photosynthetic oxygen-evolving photosystem II Isao Enami 1 , Takehiro Suzuki 1 , Osamu Tada 1 , Yoshiko Nakada 1 , Kumi Nakamura 1 , Akihiko Tohri 1 , Hisataka Ohta 1 , Isao Inoue 2 and Jian-Ren Shen 3 1 Department of Biology, Faculty of Science, Tokyo University of Science, Tokyo, Japan 2 Institute of Biological Science, University of Tsukuba, Japan 3 Department of Biology, Faculty of Science, Okayama University, and PRESTO, JST, Japan The appearance of oxygenic photosynthetic organisms was a key event in the evolution of our green bio- sphere. The organisms developed the machinery using solar energy to oxidize water into oxygen and to reduce CO 2 with an endless supply of reducing equiva- lents. The release of oxygen as the byproduct of the water-splitting reaction has not only created an oxygen atmosphere but also the ozone layer needed to shield terrestrial plants and animals from ultraviolet radi- ation. The water-splitting reaction takes place in a thyla- koid membrane-located multiprotein-pigment complex known as photosystem II (PSII). The PSII complex contains a number of intrinsic proteins and 3–4 extrin- sic proteins associated with the luminal side. So far the PSII membrane fragments and core complexes that are highly active in oxygen evolution and retain all of the extrinsic proteins have been isolated from cyanobac- teria [1–3], red alga [4,5], Euglena [6], green alga [7] and higher plants [8,9]. Among these PSII complexes Keywords evolution; immunological assay; oxygen evolution; photosystem II; PSII extrinsic proteins Correspondence I. Enami, Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka 1–3, Shinjuku-ku, Tokyo 162–8601, Japan Tel: +81 471241501 (ext. 5022) Fax: +81 332600322 E-mail: enami@rs.noda.tus.ac.jp (Received 14 June 2005, revised 8 August 2005, accepted 11 August 2005) doi:10.1111/j.1742-4658.2005.04912.x Distribution of photosystem II (PSII) extrinsic proteins was examined using antibodies raised against various extrinsic proteins from different sources. The results showed that a glaucophyte (Cyanophora paradoxa) having the most primitive plastids contained the cyanobacterial-type extrinsic proteins (PsbO, PsbV, PsbU), and the primitive red algae (Cyanidium caldarium) contained the red algal-type extrinsic proteins (PsO, PsbQ¢, PsbV, PsbU), whereas a prasinophyte (Pyraminonas parkeae), which is one of the most primitive green algae, contained the green algal-type ones (PsbO, PsbP, PsbQ). These suggest that the extrinsic proteins had been diverged into cyanobacterial-, red algal- and green algal-types during early phases of evo- lution after a primary endosymbiosis. This study also showed that a hapto- phyte, diatoms and brown algae, which resulted from red algal secondary endosymbiosis, contained the red algal-type, whereas Euglena gracilis resul- ted from green algal secondary endosymbiosis contained the green algal- type extrinsic proteins, suggesting that the red algal- and green algal-type extrinsic proteins have been retained unchanged in the different lines of organisms following the secondary endosymbiosis. Based on these immuno- logical analyses, together with the current genome data, the evolution of photosynthetic oxygen-evolving PSII was discussed from a view of distribu- tion of the extrinsic proteins, and a new model for the evolution of the PSII extrinsic proteins was proposed. Abbreviations C-PsbV and C-PsbU, cyanobacterial PsbV and PsbU proteins; G-PsbQ, green algal PsbQ protein; H-PsbP and H-PsbQ, higher plant PsbP and PsbQ proteins; R-PsbQ¢, R-PsbV and R-PsbU, red algal PsbQ¢, PsbV and PsbU proteins; PSII, photosystem II. 5020 FEBS Journal 272 (2005) 5020–5030 ª 2005 FEBS from a wide variety of organisms, the major intrinsic core proteins are largely conserved, whereas the extrin- sic proteins which form the oxygen-evolving center of PSII are significantly different among different plant species. Among the extrinsic proteins, the 33 kDa pro- tein (PsbO) which plays an important role in maintain- ing the stability and activity of the manganese cluster is present in all of the oxygenic photosynthetic organ- isms. In contrast, the other extrinsic proteins that function to optimize the availability of Ca 2+ and Cl – cofactors for water oxidation are different among dif- ferent plant species. Cyanobacterial and red algal PSII complexes contain cytochrome c 550 (PsbV) and the 12 kDa protein (PsbU) [1–5,10]. In red algal PSII, a fourth extrinsic protein, the unique 20 kDa protein is present in addition to these three extrinsic proteins [5]. The 20 kDa protein that is required for the effective binding of PsbV and PsbU in red algal PSII [5] has some similarities to PsbQ of green algae in their amino acid sequences; this 20 kDa protein was named PsbQ¢ [11]. In contrast, Euglena, green algal and higher plant PSII complexes contain the 23 kDa (PsbP) and 17 kDa (PsbQ) proteins instead of PsbV and PsbU [6–9]. Recently, however, PsbP- and PsbQ-like proteins were also found in cyanobacterial PSII [3], and they have been suggested to regulate the PSII function in the prokaryotic cyanobacteria [12,13]. The PsbV and PsbU proteins in cyanobacterial and red algal PSII showed some similar functions to those of the PsbP and PsbQ proteins in green algal and higher plant PSII [1,5,14]. These facts imply that PsbV and PsbU were replaced by PsbP and PsbQ during evolution from prokaryotic cyanobacteria and the primitive eukaryotic red algae to the green lineage Euglena, green algae and higher plants, and PsbQ¢ may be an intermediate between the PsbQ-like proteins in cyanobacteria and the mature PsbQ protein in higher plants. The distribution of these extrinsic pro- teins among various organisms therefore provides a clue to elucidate the evolutionary process of the oxygen- evolving complexes. In addition to these biochemical studies, genome- wide analysis of the extrinsic proteins has been largely advanced, owing to the sequencing of whole plastids and genomes of a number of photosynthetic organ- isms. Recently, De Las Rivas et al. [15] summarized the nature and composition of the extrinsic proteins of different organisms using knowledge from complete genome sequences and current databases. Their bio- informatics analysis to explore the known sequences of the extrinsic proteins revealed that: (a) PsbO is present in all of the oxygenic photosynthetic organisms; (b) PsbV and PsbU are present in all cyanobacteria ana- lyzed, including Gloeobacter violaceus, which is consid- ered to be the most primitive cyanobacterium and a red alga (Cyanidium caldarium), but not in green algae and higher plants. In the three green oxyphotobacteria analyzed, PsbV and PsbU are present only in Prochlo- rococcus marinus MIT9313 but not in the strains MED4 and SS120. (c) PsbP is present in green algae and higher plants, and psbP-like genes were also found in all cyanobacteria and green oxyphotobacteria ana- lyzed. (d) PsbQ is present in green algae and higher plants, and psbQ-like genes were found in most of cyanobacteria and a red alga (C. caldarium; PsbQ¢), but not in G. violaceus and green oxyphotobacteria. These genome sequences provide valuable information for the distribution of the extrinsic proteins among dif- ferent plant species, although their information is lim- ited by the plant species of which the complete genome sequences had been determined. In spite of these advanced biochemical and genome- wide analyses, there is little information on the ext- rinsic proteins of non-green algae including the Glaucophyceae, Haptophyceae, Prasinophyceae, Bacil- larriophyceae (diatom) and Phaeophyceae (brown algae), which are considered to hold important posi- tions in the evolution of oxygenic photosynthetic organisms. In this study, we examined the distribution of the extrinsic proteins in these organisms using anti- bodies raised against PsbV, PsbU, PsbQ¢, PsbP and PsbQ from cyanobacterial, red algal, green algal and higher plant PSII complexes. Based on the immuno- logical analyses and the current genome data, we proposed a new model for the evolution of the PSII extrinsic proteins in which the model proposed by Thornton et al. [12] was modified. Results Specificities of antibodies used in this study For the wide-detection of the extrinsic proteins in various plant species, seven antibodies [anti-(H-PsbP), anti-(H-PsbQ), anti-(G-PsbQ), anti-(R-PsbQ¢), anti-(R- PsbV), anti-(R-PsbU) and anti-(C-PsbU)] were used in this study. Figure 1 shows the reactivities of cyanobacte- rial, red algal, green algal and higher plant PSII with these antibodies. Cyanobacterial PSII complex isolated from Thermosynechococcus vulcanus (Fig. 1A) reacted with the antibodies against red algal PsbV [lane 5; anti- (R-PsbV)] and cyanobacterial PsbU [lane 7; anti-(C- PsbU)], but not with the antibody against red algal PsbU [lane 6; anti-(R-PsbU)]. Immunoblot analysis using thylakoid membranes of T. vulcanus yielded the same results. In contrast, red algal PSII complex from I. Enami et al. Evolution of PSII extrinsic proteins FEBS Journal 272 (2005) 5020–5030 ª 2005 FEBS 5021 C. caldarium (Fig. 1B) reacted with anti-(R-PsbV) (lane 5) and anti-(R-PsbU) (lane 6), but not with anti- (C-PsbU) (lane 7). These facts suggest that anti- (R-PsbV) can be used as a common antibody for PsbV among different species but anti-(R-PsbU) and anti- (C-PsbU) have a high species-specificity and cannot be used as a common antibody to detect the presence of this protein among different species. These may be due to the low sequence homology of PsbU between the cyanobacterium and red alga. This is also consistent with our previous report that while the structure and function of PsbV have been largely conserved between cyanobacteria and red algae, those of PsbU have been changed in the two organisms [16]. The antibody against red algal PsbQ¢ [anti-(R-PsbQ¢)] reacted with red algal PSII complex (lane 4, Fig. 1B) but not with the cyanobacterial PSII complex (lane 4, Fig. 1A), consis- tent with the fact that the purified cyanobacterial PSII does not contain the PsbQ ¢ -like protein. Both of the cyanobacterial and red algal PSII complexes did not react with any antibodies against the extrinsic proteins of green algal and higher plant PSII (lanes 1–3, Fig. 1A,B). These are consistent with the results from recent crystallographic analysis of Thermosynecococcus PSII in which PsbV and PsbU were clearly detected but PsbQ¢ as well as PsbP and PsbQ were not [17–19]. Green algal PSII complex (Fig. 1C) from Chlamydo- monas reinhardtii reacted with antibodies against higher plant PsbP [lane 1; anti-(H-PsbP)] and green algal PsbQ [lane 3; anti-(G-PsbQ)], but not with the antibody against higher plant PsbQ [lane 2; anti-(H-PsbQ)]. Simi- larly, higher plant PSII membrane fragments (Fig. 1D) from spinach reacted with anti-(H-PsbP) (lane 1) and anti-(H-PsbQ) (lane 2) but not with anti-(G-PsbQ) (lane 3). These results suggest that anti-(H-PsbP) can be used as a common antibody for PsbP among different spe- cies, but anti-(H-PsbQ) and anti-(G-PsbQ) cannot due to their high species-specificity. These may reflect the low homology of the PsbQ protein between green algae and higher plants, as shown by De Las Rivas et al. [15] that the sequence homologies (number of identical resi- dues out of the total residues) of PsbP and PsbQ are 61 and 29% between spinach and C. reinhardtii, respect- ively. In addition, the green algal and higher plant PSII did not react with any antibodies against the cyanobac- terial and red algal extrinsic proteins (lanes 4–7, Fig. 1C,D), suggesting the absence of these proteins in the green algal and higher plant PSII. Plant species having cyanobacterial-type extrinsic proteins Glaucophyta as represented by Cyanophora paradoxa, are a group of unique photosynthetic eukaryotes that possess a special type of plastid called cyanelle. The cya- nelle is surrounded by a peptidoglycan wall [20] and possesses a central body that resembles a cyanobacterial carboxysome [21] which is not present in the plastids of the primitive eukaryotes red algae. This has been taken as evidence implying that the cyanelle is originated from endosymbiotic cyanobacteria [22] and that C. paradoxa first branched during the evolutionary process of chloro- plasts [23]. Shibata et al. [21] isolated the thylakoid membranes and PSII particles from C. paradoxa and reported that PsbV could be detected by heme-staining, but PsbU could not be detected by anti-(C-PsbU) in the thylakoid membranes and PSII particles of C. paradoxa. Here we used the seven antibodies against the extrinsic proteins to detect the presence of homologous proteins A B C D Fig. 1. Reactivities of the PSII complexes isolated from Thermosyn- echococcus vulcanus (A), Cyanidium caldarium (B), Chlamydo- monas reinhardtii (C), and the PSII membrane fragments from Spinacia oleracea (D) with antibodies raised against their extrinsic proteins. Lane 1, anti-(H-PsbP); lane 2, anti-(H-PsbQ); lane 3, anti- (G-PsbQ); lane 4, anti-(R-PsbQ¢); lane 5, anti-(R-PsbV); lane 6, anti- (R-PsbU); lane 7, anti-(C-PsbU). Evolution of PSII extrinsic proteins I. Enami et al. 5022 FEBS Journal 272 (2005) 5020–5030 ª 2005 FEBS in the thylakoid membranes of C. paradoxa (Fig. 2). The C. paradoxa thylakoid membranes reacted with anti-(R-PsbV) (lane 5) and anti-(R-PsbU) (lane 6) but not with anti-(C-PsbU) (lane 7); the latter is consistent with the result of Shibata et al. [21]. The presence of PsbV in C. paradoxa is consistent with the presence of the psbV gene in the complete sequences of the cyanelle genome [24], in which the psbU gene was not found. The fact, however, that C. paradoxa thylakoid membranes reacted with anti-(R-PsbU) apparently indicates the presence of this protein in this alga, and the absence of this gene in the cyanelle genome suggested that this gene has been transferred to the nuclear genome in this organism, as in the case of red algae. On the other hand, the failure of cross-reaction with anti-(C-PsbU) suggests that C. paradoxa PsbU has a higher homology with the red algal protein than with the cyanobacterial one. The C. paradoxa thylakoid membranes also contained a band cross-reacted with anti-(R-PsbQ¢), the apparent molecular mass of which was remarkably higher than that of PsbQ¢ (lane 4) in the red algal PSII. In addition, this polypeptide band did not disappear by 1 m alkaline Tris-treatment (data not shown), which is known to remove all of the extrinsic proteins from higher plant [25], cyanobacterial [10], and red algal PSII [4]. This suggests that the band cross-reacted with anti-R-PsbQ¢ in the thylakoid membranes of C. paradoxa is not an extrinsic protein homologous to the red algal PsbQ¢ pro- tein. The C. paradoxa thylakoid membranes did not react with any antibodies against the green algal and higher plant extrinsic proteins (lanes 1–3). Thus, we con- clude that C. paradoxa has the cyanobacterial-type extrinsic proteins (PsbV and PsbU). Plant species having red algal-type extrinsic proteins Molecular, morphological and phylogenetic data sug- gest that taxonomically diverse groups of chlorophyll c-containing protists comprising cryptophytes, hapto- phytes and photosynthetic stramenopiles (diatoms and brown algae, etc.) share a common plastid that arose from ancient secondary endosymbiosis involving red algae [26–28]. Therefore, it is very interesting to see whether the red algal-type extrinsic proteins (PsbQ¢, PsbV and PsbU) have been retained in these algae or if they have been replaced by the green algal-type ones (PsbP and PsbQ). Figure 3 shows the reactivities of the thylakoid membranes isolated from a diatom (Fig. 3A, Cheaeo- toceros gracilis), a haptophyte (Fig. 3B, Pavlova gyrans), and two brown algae (Fig. 3C, Laminria Fig. 2. Reactivities of the thylakoid membranes isolated from Cyanophora paradoxa with antibodies raised against various extrin- sic proteins. Lane 1, anti-(H-PsbP); lane 2, anti-(H-PsbQ); lane 3, anti-(G-PsbQ); lane 4, anti-(R-PsbQ¢); lane 5, anti-(R-PsbV); lane 6, anti-(R-PsbU); lane 7, anti-(C-PsbU). A B DC Fig. 3. Reactivities of the thylakoid membranes isolated from Che- aeotoceros gracilis (A), Pavlova gyrans (B), Laminria japonica (C) and Undaria pinnatifida (D) with antibodies raised against various extrinsic proteins. Lane 1, anti-(H-PsbP); lane 2, anti-(H-PsbQ); lane 3, anti-(G-PsbQ); lane 4, anti-(R-PsbQ¢); lane 5, anti-(R-PsbV); lane 6, anti-(R-PsbU); lane 7, anti-(C-PsbU). I. Enami et al. Evolution of PSII extrinsic proteins FEBS Journal 272 (2005) 5020–5030 ª 2005 FEBS 5023 japonica; and Fig. 3D, Undaria pinnatifida) with the seven antibodies against the extrinsic proteins. All of these thylakoid membranes reacted with anti-(R- PsbQ¢) (lane 4) and anti-(R-PsbV) (lane 5) but not with any other antibodies, except the diatom thylakoid membranes which reacted with anti-(H-PsbP) (lane 1, Fig. 3A). In order to confirm the presence of PsbP in the diatom thylakoid membranes, we treated the mem- branes with 1 m alkaline Tris and performed western blot analysis on the membranes and Tris extracts, respectively. The results showed that the bands cross- reacted with anti-(R-PsbQ¢) and anti-(R-PsbV) were released by 1 m Tris-treatment, whereas the band cross-reacted with anti-(H-PsbP) was not extracted and remained in the membranes (data not shown). Similar results were obtained with another diatom, Phaeod- actylum tricornutum (not shown). The behavior of this band in the diatom is thus similar to that of the PsbP- like protein in cyanobacteria [3,12]. The fact that C. gracilis, P. gyrans, L. japonica and U. pinnatifida contained bands cross-reacted with anti-(R-PsbQ¢) and anti-(R-PsbV), but not the anti- bodies against the green algal and higher plant extrinsic proteins (except diatom) implies that these chlorophyll c-containing algae have the red algal-type extrinsic proteins but not green algal-type ones. We could not, however, detect the presence of PsbU in these algae which plays a role in optimizing the availability of Cl – cofactors for water oxidation [5,29]. This may be due to the high species-specificity of the antibody against PsbU as described above. PsbU must be present in PSII containing PsbV, because PsbU is known to have a strong interaction with PsbV and is required, in cooperation with PsbV, for maintaining the high activity of oxygen evolution in the absence of Cl – and Ca 2+ [5,29]. In fact, the psbU gene has been found in the genome of two diatoms, P. tricornutum and Thalassiosira pseudonana, whose complete genome sequences are available in the current databases [30,31], which sup- ports the presence of PsbU in diatoms. In addition, we recently purified a PSII complex from a diatom C. gracilis, and found that this PSII complex con- tained PsbO, PsbQ¢, PsbV, PsbU as the extrinsic proteins by means of immunological analysis and N-terminal sequencing (data not shown). Complete plastid genome sequences also showed that PsbV is present in the red algae Porphyra purpurea [32], Cya- nidioschzon merolae [33] and C. caldarium [34], and in a diatom, Odontella sinensis [35]. Based on these results, we conclude that diatoms, haptophyte and brown algae contain the red algal-type extrinsic pro- teins (PsbQ¢, PsbV and PsbU). Plant species having green algal-type extrinsic proteins Prasinophytes are considered to be the most primitive green algae [36]. Thylakoid membranes of a prasino- phyte, Pyraminonas parkeae, cross-reacted with anti- (H-PsbP) but not with other antibodies (Fig. 4A). Thylakoid membranes of an euglenophyte Euglena gracilis, which is considered to have originated from a green algal secondary endosymbiosis, also cross-reac- ted with anti-(H-PsbP) but not with other antibodies (Fig. 4B). Although these algal thylakoid membranes did not cross-react with antibodies against green algal and higher plant PsbQ, the presence of PsbQ in isola- ted PSII of E. gracilis has been confirmed recently [6]. The failure of cross-reaction of the thylakoid mem- branes from prasinophyte and euglenophyte with anti- bodies against green algal and higher plant PsbQ may be due to the high species-specificity of the antibody against PsbQ as mentioned above. In fact, PsbQ is required, in cooperation with PsbP, for the high oxy- gen-evolving activity in the absence of Cl – and Ca 2+ , and has been found to be present in all of the PSIIs retaining PsbP that have been purified from higher plants [8,9], green alga [7] and Euglena [6]. Thus, it is most likely that Prasinophytes also contain PsbQ. The thylakoid membranes of P. parkeae and E. gracilis did not react with any antibodies against the red algal and cyanobacterial extrinsic proteins (lanes 4–7). Thus, the present results indicate that Prasinophyceae and Euglenophyceae contain the green algal-type extrinsic proteins (PsbP and PsbQ) but not the red algal-type ones. AB Fig. 4. Reactivities of the thylakoid membranes isolated from Pyraminonas parkeae (A) and Euglena garcilis (B) with antibodies raised against various extrinsic proteins. Lane 1, anti-(H-PsbP); lane 2, anti-(H-PsbQ); lane 3, anti-(G-PsbQ); lane 4, anti-(R-PsbQ¢); lane 5, anti-(R-PsbV); lane 6, anti-(R-PsbU); lane 7, anti-(C-PsbU). Evolution of PSII extrinsic proteins I. Enami et al. 5024 FEBS Journal 272 (2005) 5020–5030 ª 2005 FEBS Discussion In this study, we examined the distribution of the extrinsic proteins among various plant species by immunological assay with antibodies raised against seven extrinsic proteins. The results were summarized in Table 1. As shown in Table 1, a glaucophyte con- tained the cyanobacterial-type extrinsic proteins (PsbU and PsbV), and chlorophyll a ⁄ c-containing algae dia- toms, haptophyte and brown algae such as retained the red algal-type extrinsic proteins (PsbQ¢, PsbV and PsbU), whereas chlorophyll a ⁄ b-containing algae pra- sinophyte, Euglena, green alga and higher plant, had the green algal-type extrinsic proteins (PsbP and PsbQ). The distribution of the extrinsic proteins obtained in this study was also incorporated into the current phylogenetic tree as shown in Figure 5. Table 1. Distribution of the PSII extrinsic proteins among various plant species revealed by immunological assays. ‘ +’ and ‘–’ desig- nate the presence and absence of each extrinsic protein confirmed by the immunological assays in this study, and (+) shows the pres- ence of each extrinsic protein deduced from genomic sequence data or functional requirements (see text for details), although it was not detected by the immunological assays. Psb P Psb Q Psb Q¢ Psb V Psb U Cyanobacteria – – – + + Glaucophyceae – – – + + Red algae – – + + + Diatoms – – + + (+) Haptophyceae – – + + (+) Brown algae – – + + (+) Prasinophyceae + (+) – – – Euglenophyceae + (+) – – – Green algae + + – – – Higher plants + + – – – Fig. 5. Phylogenetic tree of the PSII extrin- sic proteins. See text for details. I. Enami et al. Evolution of PSII extrinsic proteins FEBS Journal 272 (2005) 5020–5030 ª 2005 FEBS 5025 Current knowledge indicates that a single primary endosymbiosis, in which a photosynthetic cyanobac- teria-like prokaryote was engulfed and retained by a eukaryotic phagotroph, resulted in the primordial alga. The primordial alga then gave rise through vertical evolution to the Glaucophyta, Rhodophyta (red algae) and Chlorophyta (green algae) [26] (Fig. 5). These pri- mary plastids are surrounded by two envelope mem- branes. Our immunological studies showed that a glaucophyte, C. paradoxa that has the most primitive plastids [23], contained the PsbV and PsbU proteins as the extrinsic proteins (Figs 1 and 2). A primitive red alga, C. caldarium that has the most ancient chloro- plast-genome [34], contained the PsbQ¢ protein in addi- tion to the cyanobacterial extrinsic proteins (Fig. 1) [4,5]. A prasinophyte, P. parkeae which is one of the most primitive green algae [36], contained the PsbP and probably PsbQ as the extrinsic proteins. These results suggest that the extrinsic proteins had been diverged into three types, cyanobacterial-, red algal- and green algal-types during early phases of evolution after the primary endosymbiosis. A variety of plant species were formed by subse- quent one or several secondary endosymbiosis event(s), in which an unicellular algal species was engulfed by another amoeboid eukaryote [37], and the plant king- dom can be divided into two evolutionary lineages: the red lineage containing chlorophyll a ⁄ c and the green lineage characterized by chlorophyll a ⁄ b [38] (Fig. 5). These plastids are surrounded by 3–4 envelope mem- branes. In this study, it was found that plant species belong to the red lineage (C. caldarium, C. gracilis, P. gyrans, L. japonica and U. pinnatifida) contained the red algal-type extrinsic proteins (Figs 1 and 3). In con- trast, species belong to the green lineage (P. parkeae, E. garcilis, C. reinhardtii, spinach) contained the green algal-type extrinsic proteins (Figs 1 and 4). These indi- cate that organisms derived from the red algal or green algal secondary endosymbiosis have unchangeably retained their red algal-type or green algal-type ex- trinsic proteins, respectively. Thus, we propose that organisms containing cryptomonads, heterokonts, dinoflagellates and apicomplexa that belong to the red lineage, contain the red algal-type extrinsic proteins, although the extrinsic proteins in these organisms were not examined in this study. Cyanobacteria are known to contain psbP- and psbQ-like genes in addition to the psbO, psbV and psbU genes [15], suggesting that all of the genes enco- ding cyanobacterial-, red algal- and higher plant-type extrinsic proteins are already present in cyanobacteria. Among these gene products, the PsbO, PsbV and PsbU proteins function as the extrinsic proteins in cyanobacteria and most likely also in Glaucophyta. In fact, Shen et al. [1,2,10] purified PSII complex retain- ing PsbO, PsbV and PsbU but not PsbP- and PsbQ- like proteins from the cyanobacterium T. vulcanus. The PSII complex is highly active in oxygen evolution in the absence of Cl – and Ca 2+ and its crystallographic analysis showed the existence of PsbO, PsbV and PsbU but not PsbP- and PsbQ-like proteins [17–19]. On the other hand, Thornton et al. [12] and Summerfield et al. [13] reported recently that the PsbP- and PsbQ-like proteins in Synechocystis 6803 are regulatory proteins necessary for the maintenance of optimally active PSII in nutrient-limiting media depleted of Cl – ,Ca 2+ or iron. The psbP- and psbQ-deletion mutants of Synecho- cystis 6803, however, showed photoautotrophic growth rates similar to those of wild-type under normal growth conditions. Therefore, Thornton et al. [12] mentioned that the PsbP- and PsbQ-like proteins do not share the critical roles that PsbO and PsbV play in cyanobacterial PSII-dependent growth. In addition, the cyanobacterial PsbP- and PsbQ-like proteins are a kind of lipoproteins but not characterized as the ext- rinsic PSII proteins [12]. Thus, the PsbO, PsbV and PsbU proteins are the typical extrinsic proteins in cyanobacterial PSII, and the cyanobacterial PsbP- and PsbQ-like proteins are regulatory lipoproteins that are necessary in nutrient-limiting media. On the other hand, the PsbO, PsbQ¢, PsbV and PsbU proteins func- tion as the extrinsic proteins in a primitive red alga, C. caldarium [4,5] and probably in the red lineage, whereas the PsbO, PsbP and PsbQ proteins function as the extrinsic proteins in Prasinophyceae, Euglena [6], green algae [7] and higher plants [8,9], and probably in the green lineage. These results are consistent with the existence of three types of extrinsic proteins mentioned above, namely, cyanobacterial- (PsbO, PsbV, PsbU), red algal- (PsbO, PsbQ¢, PsbV, PsbU) and green algal- types (PsbO, PsbP, PsbQ) (Fig. 5). Several complete sequences of nuclear and chloro- plast genomes have been accumulated since the report of De Las Rivas et al. [15] which summarized the com- position of the extrinsic proteins in different organ- isms. Based on these complete genome data, we summarized the occurrence and comparison of the extrinsic proteins in various plant species in Table 2. The gene encoding the extrinsic PsbO was excluded in Table 2, because this gene is present in all of the oxy- genic photosynthetic organisms. As described by De Las Rivas et al. [15], all of the genes encoding the PsbP-like, PsbQ-like, PsbV and PsbU proteins were found in Synechocystis 6803 and in all of cyanobac- teria analyzed (data not shown). In a primitive red alga, C. merolae, the genes encoding the PsbP-like, Evolution of PSII extrinsic proteins I. Enami et al. 5026 FEBS Journal 272 (2005) 5020–5030 ª 2005 FEBS PsbQ-like and PsbU proteins were detected in its nuc- lear genome [39] and the gene encoding the PsbV pro- tein was found in its chloroplast genome [33]. The psbV gene was also found in the chloroplast genome of other red algae, C. caldarium [34] and P. purpurea [32]. The transit peptide analysis of the cloned gene from C. caldarium showed that the psbV gene was remained in the plastid [16], while the genes of psbO, psbQ¢ and psbU were transferred to the nucleus [40], consistent with the results of nuclear and chloroplast genome analyses in red algae. These indicate that all of the genes encoding the PsbP-like, PsbQ-like, PsbV and PsbU proteins in cyanobacteria have been retained in red algae after primary endosymbiosis. Recently, com- plete nuclear and chloroplast genome sequences of a diatom, Thalassiosira pseudonana, were determined [31], in which the genes encoding PsbU (nuclear gen- ome) and PsbV (chloroplast genome) were detected but the genes encoding PsbP and PsbQ could not be found. However, when using psbP and psbQ genes from the red alga C. merolae as references, homolog- ous psbP and psbQ genes were found to be present in another diatom Phaeodactylum tricomutan. Using the sequences from the diatom P. tricomutan as references, the homologous psbQ gene was found in the diatom T. pseudonana, but the psbP gene was not found. Com- plete plastid genome analysis also showed that the gene encoding PsbV is present in the chloroplast of a diatom, Odontella sinensis [35]. In the green lineage, ancestral chloroplast genome sequences of a pra- sinophte, Mesostigma viride, was completely deter- mined [41] in which the gene encoding PsbV was not detected. The gene encoding PsbV was also not detec- ted in the complete sequences of E. gracilis chloroplast DNA [42]. In a green alga (C. reihardtii) and higher plant (Oryza sativa), the genes encoding PsbP and PsbQ are present but the genes encoding PsbV and PsbU are not detected. Based on the current genome data and the immuno- logical results in this study, we propose a new model for the evolution of the PSII extrinsic proteins (Fig. 5). The prokaryotic cyanobacteria contain five genes for Table 2. Homology search of the PSII extrinsic proteins (PsbP, PsbQ, PsbV and PsbU) in the complete sequences of nuclear and chloroplast genomes of various species. The search was conducted using spinach sequences for PsbP and PsbQ, and using Synechocystis sp. PCC6803 sequences for PsbV and PsbU. Percentage of identity to these reference sequences is indicated. The E-value from BLAST [45] is also indicated as a decimal number or as an exponential. PsbP family PsbQ family PsbV PsbU Cyanobacteria Synechocystis sp. PCC6803 Presence 27% (4e)7) Presence 24% (10e)7) Presence 100% Presence 100% Rhodophyceae (red algae) Cyanidioschyzon merolae Nuclear DNA Presence 25% (0.039) Presence 25% (7e)5) Absence Presence 33% (5e)11) Chloroplast DNA Absence Absence Presence 40% (6e)27) Absence Cyanidium caldarium Chloroplast DNA Absence Absence Presence 45% (2e)30) Absence Bacillariophyceae (diatoms) Thalassiosira pseudonana Nuclear DNA ? ? Absence Presence 35% (4e)10) Chloroplast DNA Absence Absence Presence 48% (1e)34) Absence Odontella sinensis Chloroplast DNA Absence Absence Presence 49% (3e)33) Absence Prasinophyceae Mesostigma viride Chloroplast DNA Absence Absence Absence Absence Euglenophyceae Euglena gracilis Chloroplast DNA Absence Absence Absence Absence Chlorophyceae (green algae) Chlamydomonas reinhardtii Nuclear DNA Presence 61% (2e)59) Presence 29% (5e)7) Absence Absence Chloroplast DNA Absence Absence Absence Absence Higher plant Oryza sativa Nuclear DNA Presence 82% (1e)88) Presence 69% (2e)44) Absence Absence Chloroplast DNA Absence Absence Absence Absence I. Enami et al. Evolution of PSII extrinsic proteins FEBS Journal 272 (2005) 5020–5030 ª 2005 FEBS 5027 the PSII extrinsic protein PsbO, PsbP, PsbQ, PsbV and PsbU. All of these five genes were retained in the primitive red algae (C. merolae and C. caldarium), and at least four out of the five genes (psbO, psbQ¢, psbV, psbU) are present and their gene products function as the extrinsic proteins in the algae of red lineage which contain Haptophyta, diatoms and brown algae and are characterized by chlorophyll a ⁄ c. The psbP gene is pre- sent in some of the algae in the red lineage but may be lost in other part of the red lineage. In the green lin- eage containing Prasinophyceae, Euglenophyta, green algae and higher plants which are characterized by chlorophyll a ⁄ b, the genes for psbV and psbU have been lost and PsbO, PsbP and PsbQ are present and function in their PSII exclusively. Thornton et al. [12] mentioned in their model of evolution of the PSII extrinsic proteins that PsbV was lost in a red alga, C. merolae. Based on this they poin- ted out that the evolutionary history of the water oxi- dation domain in the red algae may be more complex as biochemical data suggests that the red alga C. calda- rium has PsbV but not PsbP [11]. As mentioned above, however, the gene encoding PsbV was found in the plastid genome of the red algae C. merolae [33] and C. caldarium [34], and the gene product (PsbV) was detected in the PSII complex of C. caldarium [4,5]. Thus, all red algae examined so far contained the psbV gene. The psbQ gene encoding the PsbQ-like lipoprotein in cyanobacteria seems to have been changed to the gene encoding the PsbQ¢ extrinsic protein, which is required for effective binding of the PsbV and PsbU proteins in the red lineage, and to the gene encoding the PsbQ extrinsic protein, which functions in optimizing the availability of Ca 2+ and Cl – cofactors for water oxida- tion in the green lineage. In fact, all of the thylakoid membranes from diatoms (C. gracilis and P. tricor- nutum), a haptophyte (P. gyrans) and brown algae (L. japonica and U. pinnatifida) in the red lineage reac- ted with antibody against red algal PsbQ¢ but not with antibody against green algal and higher plant PsbQ (Fig. 3). This indicates that PsbQ¢ is present in the red lineage. On the other hand, the present immunological assays showed that no PsbP was detected in diatoms, haptophyte and brown algae. The psbP gene was found in P. tricomutan but not in T. pseudonana, sug- gesting that the psbP gene was lost at least in some algae of the red lineage after the red algal secondary endosymbiosis. The psbP gene encoding the PsbP-like lipoprotein in cyanobacteria seems to have been chan- ged to the gene encoding the PsbP extrinsic protein which functions in optimizing the availability of Ca 2+ and Cl – cofactors for water oxidation in the green lin- eage. The distribution of PsbP- and PsbQ-like proteins in various plant species, however, has to be investi- gated further by immunological assays with antibodies raised against these proteins. In the green lineage, the genes encoding PsbV and PsbU may have been lost during early phases after the primary endosymbiosis (see Fig. 5), because the psbV gene was not detected in ancestral chloroplast genome sequence of a prasinophyte, M. viride (Table 2) and no PsbV and PsbU proteins were found in a primitive green alga, P. parkeae as well as E. garcilis, C. rein- hardtii and spinach in the present immunological assays. Experimental procedures Preparation of antibodies against various extrinsic proteins The genes encoding PsbQ¢, PsbV and PsbU from a red alga, C. caldarium, and PsbU from a cyanobacterium, T. vulcanus and PsbQ from a green alga, C. reinhardtii, were cloned and sequenced by means of PCR and a rapid amplification of cDNA ends (RACE) procedure [40]. The cloned genes were expressed in Escherichia coli as fusion- proteins with His-tag and calmodulin, and the resulted proteins were purified with His-bind resin and calmodulin- affinity column [29]. The recombinant protein of PsbV (cytochome c 550 ) was an apoprotein with no heme c attached. These recombinant proteins were used for pre- paration of the antibodies against red algal PsbQ¢, PsbV and PsbU, cyanobacterial PsbU and green algal PsbQ. The antibodies against spinach PsbP and PsbQ were generously provided by T. Horio and T. Kakuno. Preparation of thylakoid membranes and PSII complexes from various species Cyanobacterial and red algal PSII complexes were puri- fied from T. vulcanus and C. caldarium , according to Shen et al. [10] and Enami et al. [4], respectively. Spinach PSII membrane fragments (BBY-type PSII) were prepared according to Berthold et al. [8] with slight modifications [43]. Green algal PSII complex and Euglena thylakoid membranes were prepared from C. reinhardtii having His- tagged CP47 and E. garcilis according to Suzuki et al. [7,6, respectively]. Thylakoid membranes from a glauco- phyte (C. paradoxa), a haptophyte (P. gyrans), diatoms (C. gracilis and P. tricornutum) and a prasinophyte (P. parkeae NIES no.) were prepared by centrifugation after disruption of their cells with glass beads according to Suzuki et al. [7]. Thylakoid membranes from brown algae (L. japonica and U. pinnatifida) were prepared by Evolution of PSII extrinsic proteins I. Enami et al. 5028 FEBS Journal 272 (2005) 5020–5030 ª 2005 FEBS centrifugation after homogenization of their sporophyte with blender. Immunological assays PSII complexes and thylakoid membranes from various organisms were solubilized by 2% lithium lauryl sulfate and 75 mm dithiothreitol. The solubilized samples (10 lg chlorophyll in each lane) were applied to an SDS ⁄ poly- acrylamide gel containing a gradient of 16–22% polyacryl- amide and 7.5 m urea [44]. For western blotting, proteins on the gel were transferred onto a poly(vinylidene difluo- ride) membrane, reacted with respective antibodies and visualized with biotinylated anti-rabbit IgG. Acknowledgements We thank Drs H. Koike and Y. Kashino, University of Hyogo, for the generous supply of cells of C. parad- oxa, C. gracilis and P. tricornutum. The present work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to I.E. (10640638 and 13640658). References 1 Shen J-R & Inoue Y (1993) Binding and functional properties of two new extrinsic components, cytochrome c-550 and a 12-kDa protein, in cyanobacterial photosys- tem II. Biochemistry 32, 1825–1832. 2 Shen J-R, Burnap RL & Inoue Y (1995) An indepen- dent role of cytochrome c-550 in cyanobacterial photo- system II as revealed by double-deletion mutagenesis of the psbO and psbV genes in Synechocystis sp. PCC 6803. Biochemistry 34, 12661–12668. 3 Kashino Y, Lauber WM, Carroll JA, Wang Q, Whitmarsh J, Satoh K & Pakrasi HB (2002) Proteomic analysis of a highly active photosystem II: preparation from the cyano- bacterium Synechocystis sp. PCC 6803 reveals the presence of novel polypeptides. Biochemistry 41, 8004–8012. 4 Enami I, Murayama H, Ohta H, Kamo M, Nakazato K & Shen J-R (1995) Isolation and characterization of a photosystem II complex from the red alga Cyanidium caldarium: association of cytochrome c-550 and a 12 kDa protein with the complex. Biochim Biophys Acta 1232, 208–216. 5 Enami I, Kikuchi S, Fukuda T, Ohta H & Shen J-R (1998) Binding and functional properties of four extrin- sic proteins of photosystem II from a red alga, Cyani- dium caldarium, as studied by release-reconstitution experiments. Biochemistry 37, 2787–2793. 6 Suzuki T, Tada O, Makimura M, Tohri A, Ohta H, Yamamoto Y & Enami I (2003) Isolation and character- ization of oxygen-evolving photosystem II complexes retaining the PsbO, P and Q proteins from Euglena gracilis. Plant Cell Physiol 45, 1168–1175. 7 Suzuki T, Minagawa J, Tomo T, Sonoike K, Ohta H & Enami I (2003) Binding and functional properties of the extrinsic proteins in oxygen-evolving photosystem II particle from a green alga, Chlamydomonas reinhardtii having his-tagged CP47. Plant Cell Physiol 44, 76–84. 8 Berthold DA, Babcock GT & Yocum CF (1981) A highly resolved oxygen-evolving photosystem II pre- paration from spinach thylakoid membranes. FEBS Lett 134, 231–234. 9 Kuwabara T & Murata N (1982) Inactivation of photosynthetic oxygen evolution and concomitant release of three polypeptides in the photosystem II particles of spinach chloroplasts. Plant Cell Physiol 23, 533–539. 10 Shen J-R, Ikeuchi M & Inoue Y (1992) Stoichiometric association of extrinsic cytochrome c550 and 12 kDa protein with a highly purified oxygen-evolving photo- system II core complex from Synechococcus vulcanus. FEBS Lett 301, 145–149. 11 Ohta H, Suzuki T, Ueno M, Okumura A, Yoshihara S, Shen J-R & Enami I (2003) Extrinsic proteins of photo- system II. An intermediate member of PsbQ protein family in red algal PSII. Eur J Biochem 270, 4156–4163. 12 Thornton LE, Ohkawa H, Roose JL, Kashino Y, Keren N & Pakrasi HB (2005) Homologs of plant PsbP, PsbQ, proteins are necessary for regulation of photosystem II and activity in the cyanobacterium Synechocystis 6803. Plant Cell 16, 2164–2175. 13 Summerfield TC, Shand JA, Bentley FK & Eaton-Rye JJ (2005) PsbQ (Sll1638) in Synechocystis sp. PCC 6803 is required for photosystem II activity in specific mutants and in nutrient-limiting conditions. Biochemistry 44, 805– 815. 14 Enami I, Yoshihara S, Tohri A, Okumura A, Ohta H & Shen J-R (2000) Cross-reconstitution of various extrinsic proteins and photosystem II complexes from cyanobac- teria, red algae and higher plants. Plant Cell Physiol 41, 1354–1364. 15 De Las Rivas J, Balsera M & Barber J (2004) Evolution of oxygenic photosynthesis: genome-wide analysis of the OEC extrinsic proteins. Trends Plant Sci 9, 18–25. 16 Enami I, Iwai M, Akiyama A, Suzuki T, Okumura A, Katoh T, Tada. O, Ohta H & Shen J-R (2003) Compar- ison of binding and functional properties of two extrin- sic components, Cyt c550 and a 12 kDa protein, in cyanobacterial PSII with those in red algal PSII. Plant Cell Physiol 44, 820–827. 17 Kamiya N & Shen J-R (2003) Crystal structure of oxygen- evolving photosystem II from Thermosynechococcus vulcanus at 3.7-A ˚ resolution. Proc Natl Acad Sci USA 100, 98–103. I. Enami et al. Evolution of PSII extrinsic proteins FEBS Journal 272 (2005) 5020–5030 ª 2005 FEBS 5029 [...]... TH Jr, Wsamann C & Staehlin LA (1983) Structure of the thylakoid and envelope membranes of the cyanelles of Cyanophora paradoxa Plant Physiol 71, 409–419 21 Shibata M, Kashino Y, Satoh K & Koike H (2001) Isolation and characterization of oxygen-evolving thylakoid membranes and photosystem II particles from a glaucocystophyte, Cyanophora paradoxa Plant Cell Physiol 42, 733–741 22 Hall WT & Claus G (1963)... Ann NY Acad Sci 361, 154–165 39 Matsuzaki M, Misumi O, Shin IT, Maruyama S, Takahara M, Miyagishima SY, Mori T, Nishida K, Yagisawa F, Nishida K, Yoshida Y et al (2004) Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D Nature 428, 653–657 40 Ohta H, Okumura A, Okuyama S, Akiyama A, Iwai M, Yoshihara S, Shen J-R, Kamo M & Enami I (1999) Cloning, expression of the psbU... (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism Science 306, 79–86 5030 I Enami et al 32 Reith M & Munholland J (1993) A high-resolution gene map of the chloroplast genome of the red alga Porphyra purpurea Plant Cell 5, 465–475 33 Ohta N, Matsuzaki M, Misumi O, Miyagishima SY, Nozaki H, Tanaka K, Shin IT, Kohara Y & Kuroiwa T (2003) Complete sequence and analysis... analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae DNA Res 10, 67–77 34 Glockner G, Rosenthal A & Valentin K (2000) The structure and gene repertoire of an ancient red algal plastid genome J Mol Evol 51, 382–390 35 Kowallik KV, Stoebe B, Schaffran I, Kroth-Pancic P & Freier U (1995) The chloroplast genome of a chlorophyll a + c-containing alga, Odontella sinensis Plant Mol... (1963) Ultrastructural studies on the blue-green algal symbiont in Cyanophora paradoxa Korschikoff J Cell Biol 19, 551–563 23 Martin W, Stoebe B, Goremykin V, Hapsmann S, Hasegawa M & Kowallik KV (1998) Gene transfer to the nucleus and the evolution of chloroplasts Nature 393, 162–165 24 Stirewalt VL, Michalowski CB, Lofelhardt W, Bohnert HJ & Bryant DA (1981) Nucleotide sequence of the cyanelle genome... psbU gene, and functional studies of the recombinant 12-kDa protein of photosystem II from a red alga Cyanidium caldarium Biochem Biophys Res Commun 260, 245–250 41 Lemieux C, Otis C & Turmel M (2000) Ancestral chloroplast genome in Mesostigma viride reveals an early branch of green plant evolution Nature 403, 649–652 42 Hallick RB, Hong L, Drager RG, Favreau MR, Monfort A, Orsat B, Spielmann A & Stutz... from Cyanophora paradoxa Plant Mol Biol Report 13, 327–332 25 Yamamoto Y, Doi M, Tamura N & Nishimura M (1981) Release of polypeptides from highly active O2evolving photosystem- 2 preparation by Tris treatment FEBS Lett 133, 265–268 26 Bhattacharya D & Medlin L (1995) The phylogeny of plastids: a review based on comparison of small-subunit ribosomal RNA coding regions J Phycol 31, 489–498 27 Zhang Z,... Goyal A & Tolbert NE (1995) Factors affecting development of peroxisomes and glycolate metabolism among algae of different evolutionary lines of the Prasinophyceae Plant Physiol 109, 1363–1370 37 Winhauer T, Jager S, Valentin K & Zetsche K (1991) Structural similarities between psbA genes from red and brown algae Curr Genet 20, 177–180 38 Whatley JM (1981) Chloroplast evolution ) ancient and modern Ann... Cavalier-Smith T (1999) Single gene circles in dinoflagellate chloroplast genomes Nature 400, 155–159 28 Fast NM, Kissinger JC, Roos DS & Keeling PJ (2001) Nuclear-encoded, plastid-targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids Mol Biol Evol 18, 418–426 29 Okumura A, Ohta H, Inoue Y & Enami I (2001) Identification of functional domains of the extrinsic 12 kDa.. .Evolution of PSII extrinsic proteins 18 Ferreira KN, Iverson TM, Maghlaoui K, Barber J & Iwata S (2004) Architecture of the photosynthetic oxygen-evolving center Science 303, 1831–1838 19 Biesiadka J, Loll B, Kern J, Irrgang K-D & Zouni A (2004) Crystal structure of cyanobacterial photosystem ˚ II at 3.2 A resolution: a closer look at the Mn-cluster Phys Chem Chem Phys . Distribution of the extrinsic proteins as a potential marker for the evolution of photosynthetic oxygen-evolving photosystem II Isao Enami 1 , Takehiro. with the cyanobacterial one. The C. paradoxa thylakoid membranes also contained a band cross-reacted with anti-(R-PsbQ¢), the apparent molecular mass of