Báo cáo khoa học: Distribution of the extrinsic proteins as a potential marker for the evolution of photosynthetic oxygen-evolving photosystem II ppt

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

for the evolution of photosynthetic oxygen-evolving

photosystem II

Isao Enami1, Takehiro Suzuki1, Osamu Tada1, Yoshiko Nakada1, Kumi Nakamura1, Akihiko Tohri1, Hisataka Ohta1, Isao Inoue2and Jian-Ren Shen3

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 CO2with 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-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 evoludistribu-tion 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.

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 Ca2+ and Cl–

cofactors for water oxidation are different among

dif-ferent plant species Cyanobacterial and red algal PSII

complexes contain cytochrome c550 (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), 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

C caldarium(Fig 1B) reacted with anti-(R-PsbV) (lane

5) and (R-PsbU) (lane 6), but not with

(C-PsbU) (lane 7) These facts suggest that

anti-(R-PsbV) can be used as a common antibody for PsbV

among different species but (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 reinhardtiireacted 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 resiresi-dues) 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

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, (H-PsbP); lane 2, (H-PsbQ); lane 3,

(G-PsbQ); lane 4, (R-PsbQ¢); lane 5, (R-PsbV); lane 6,

anti-(R-PsbU); lane 7, anti-(C-PsbU).

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).

D C

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).

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

(R-PsbQ¢) and (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 Ca2+ [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 Ca2+, 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

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).

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 sequpres-ence

data or functional requirements (see text for details), although it

was not detected by the immunological assays.

Fig 5 Phylogenetic tree of the PSII

extrin-sic proteins See text for details.

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 Ca2+ 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–, Ca2+ or iron The psbP- and psbQ-deletion mutants of Synecho-cystis6803, 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 (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,

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 gengen-ome) 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.

Cyanobacteria

Synechocystis sp PCC6803 Presence 27% (4e )7) Presence 24% (10e )7) Presence 100% Presence 100% Rhodophyceae (red algae)

Cyanidioschyzon merolae

Cyanidium caldarium

Bacillariophyceae (diatoms)

Thalassiosira pseudonana

Odontella sinensis

Prasinophyceae

Mesostigma viride

Euglenophyceae

Euglena gracilis

Chlorophyceae (green algae)

Chlamydomonas reinhardtii

Higher plant

Oryza sativa

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-riumhas 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 Ca2+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 Ca2+

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 c550) 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 haptoglauco-phyte (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

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)

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