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

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:
(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).

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