The amount of PsbO as well as the efficiency of PSII in psbo1 increased as the plants grew; however, it never reached the total PsbO level observed in the wild-type, suggesting that the p
Trang 1PsbO1 and PsbO2
Reiko Murakami1, Kentaro Ifuku1, Atsushi Takabayashi1, Toshiharu Shikanai2, Tsuyoshi Endo1 and Fumihiko Sato1
1 Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
2 Graduate School of Agriculture, Kyushu University, Higashiku, Fukuoka, Japan
In oxygenic photosynthesis, the multisubunit protein
complex photosystem II (PSII) uses light energy to
oxidize water and to form molecular oxygen [1–3]
Water oxidation occurs at a catalytic site of PSII that
contains four manganese atoms, and PSII contains
sev-eral extrinsic subunits that play important roles in
sta-bilizing the active manganese site One of these, the
nuclear-encoded PsbO protein in PSII, has a molecular
mass of approximately 26 kDa, although it has been
called extrinsic 33-kDa protein traditionally, and is
syn-thesized with a transit sequence that targets the protein
to the thylakoid lumen [4,5] PsbO is present in all
oxygen-evolving organisms It appears to play a central role in stabilization of the manganese cluster and is essential for efficient and stable oxygen evolution
To better understand the role of PsbO, mutants that lack the psbO gene have been established PsbO has been deleted using genetic methods in cyanobacteria [6,7] and in green algae [8], and such mutants have been maintained under heterotrophic conditions In contrast, no PsbO mutant has been obtained in higher plants due to the essential role of this protein in pho-tosynthesis Previously, however, we isolated an Ara-bidopsis mutant [9,10] with a defect in the psbO1 gene
Keywords
Arabidopsis thaliana; isoform; oxygen
evolution; psbO1; psbO2
Correspondence
T Endo
Division of Integrated Life Sciences,
Graduate School of Biostudies, Kyoto
University, Kyoto 606–8502, Japan
Fax: +81 75 7536398
Tel: +81 75 7536381
E-mail: tuendo@kais.kyoto-u.ac.jp
(Received 24 December 2004, revised 17
February 2005, accepted 1 March 2005)
doi:10.1111/j.1742-4658.2005.04636.x
PsbO protein is an extrinsic subunit of photosystem II (PSII) and has been proposed to play a central role in stabilization of the catalytic manganese cluster Arabidopsis thaliana has two psbO genes that express two PsbO proteins; PsbO1 and PsbO2 We reported previously that a mutant plant that lacked PsbO1 (psbo1) showed considerable growth retardation despite the presence of PsbO2 [Murakami, R., Ifuku, K., Takabayashi, A., Shika-nai, T., Endo, T., and Sato, F (2002) FEBS Lett 523, 138–142] In the pre-sent study, we characterized the functional differences between PsbO1 and PsbO2 We found that PsbO1 is the major isoform in the wild-type, and the amount of PsbO2 in psbo1 was significantly less than the total amount
of PsbO in the wild-type The amount of PsbO as well as the efficiency of PSII in psbo1 increased as the plants grew; however, it never reached the total PsbO level observed in the wild-type, suggesting that the poor activity
of PSII in psbo1 was caused by a shortage of PsbO In addition, an in vitro reconstitution experiment using recombinant PsbOs and urea-washed PSII particles showed that oxygen evolution was better recovered by PsbO1 than
by PsbO2 Further analysis using chimeric and mutated PsbOs suggested that the amino acid changes Val186fi Ser, Leu246fi Ile, and Val204fi Ile could explain the functional difference between the two PsbOs Therefore we concluded that both the lower expression level and the inferior functionality of PsbO2 are responsible for the phenotype observed in psbo1
Abbreviations
Fm, maximum fluorescence yield at closed PSII centers; F0, minimum fluorescence yield at open PSII centers; Fv, Fm) F 0 ; PSII,
photosystem II.
Trang 2(psbo1), as PsbO proteins in Arabidopsis are encoded
by two genes, psbO1 and psbO2 [11] On the other
hand, many other plant species have only one psbO
gene The second gene in Arabidopsis would be a
unique result of a duplication of the genome and
chro-mosomes some 28–48 million years ago, before the
divergence of Arabidopsis from Brassica about 14–24
million years ago [12,13]
Our previous analysis showed that psbo1 exhibited
weak photosynthetic activity and considerable growth
retardation compared with the wild-type [10] This
observation raised the question of why the psbO2 gene
could not complement the defect in the psbO1 gene,
since PsbO1 and PsbO2 are highly homologous with
regard to the primary structure
In this study, we characterized the differences in the
accumulation and biochemical activity of these
iso-forms to clarify their functional differences A detailed
immunoblot analysis using the mutant, psbo1, and the
wild-type showed that a lower level of PsbO2 would
limit the photosynthetic activity Additional in vitro
experiments with urea-washed PSII particles
reconsti-tuted with PsbOs revealed that oxygen-evolution was
better recovered by PsbO1 than by PsbO2, though the
two isoforms had a similar binding affinity for the
PSII particles These results showed that the
differ-ences in both biochemical activity and the level of
accumulation were responsible for the poor
photosyn-thetic activity in psbo1
We further dissected the functional differences
between PsbO1 and PsbO2, as these isoforms have
quite a similar primary structure, and differ at only 11
amino acids A previous investigation [5] pointed out
the importance of Cys at positions 29 and 52 of
mature PsbO1 for forming a disulfide bridge [14,15],
and Val at position 148 for producing a b-sheet [16],
as well as carboxylic residues that have been reported
to be involved in the interaction between PsbO and
PSII [17,18] Moreover, these amino acid residues are
conserved in PsbO2 In addition, the predictions
regarding the structures of Arabidopsis PsbO1 and
PsbO2 based on PSII of Thermosynechococcus
elonga-tus [19] (protein data bank accession number 1S5L)
were similar Thus, to identify the amino acids
respon-sible for the poor oxygen-evolving activity of PsbO2,
we prepared chimeric proteins derived from PsbO1
and PsbO2 Further, site-directed mutagenesis showed
that the replacement of Val186 of mature PsbO1
with Ser and of Leu246 with Ile decreased the
oxygen-evolving activity, whereas substitution of Val-204 with
Ile increased the oxygen-evolving activity Based on
these results, we discuss the physiological role of the
duplicated PsbO in Arabidopsis
Results
PsbO isoform level and the efficiency of PSII
To understand the reason for the loss of photosyn-thetic activity in psbo1, we measured the protein levels
of PsbOs and the photosynthetic activity in mutant and wild-type plants Whereas the wild-type plants maintained strong photosynthetic activity throughout their growth, psbo1 plants showed a gradual increase
in electron transport activity, represented by the chlo-rophyll fluorescence parameter, Fv⁄ Fm (the maximum photochemical efficiency in PSII) [20,21], as they grew (Fig 1)
SDS⁄ PAGE, since PsbO1 migrated more slowly than PsbO2 [10] Immunoblot analysis showed that the major isoform in the wild-type was PsbO1, which com-prised about 90% of the total amount of PsbO based
on densitometry of the immunoblot membranes (Fig 2A) The amount of PsbO2 was much greater in psbo1than in the wild-type (Fig 2A,B), indicating that the expression of psbO2 was activated in a compensa-tory manner At an early growth stage, the PsbO2 level
in psbo1 accounted for 40% of the total in the wild-type The amount of PsbO2 increased considerably in mature psbo1, and reached about 70% of the total
in the wild-type This change in the level of PsbO2 coincided with the change in Fv⁄ Fm in immature and mature psbo1 plants; i.e 0.54 ± 0.05 and 0.68 ± 0.03, respectively The accumulation of PsbO2 in mature plants clearly led to more efficient PSII
Similarly, immunoblot analysis of other PSII pro-teins, PsbA and PsbP, showed that the amounts of
0.3 0.4 0.5 0.6 0.7 0.8 0.9
days after germination
wild-type
psbo1
Fig 1 Potential quantum yield of PSII (Fv⁄ F m ) in leaves of the wild-type (r) and psbo1 (h) during the course of plant growth Values represent averages and standard deviations for 10–15 plants.
Trang 3PsbA and PsbP were also smaller in psbo1 than in the
wild-type (Fig 2A,B), whereas the levels of these
pro-teins also increased as psbo1 grew As PsbO stabilizes
the manganese cluster and PSII core, and it has often
been reported that PsbO deficiency affects the amount
of PsbA and PsbP [22–24], it is very likely that the
amount of PsbOs limits the amounts of PsbA and
PsbP These results suggest that the amount of PsbOs
in psbo1 is the critical limiting factor for
photosyn-thesis and growth
Biochemical activities of PsbO1 and PsbO2
in oxygen evolution
As it was clear that the shortage of PsbOs in psbo1 is
the critical limiting factor for photosynthesis, we
car-ried out in vitro experiments using recombinant PsbOs
and urea-washed PSII particles from spinach to clarify
the qualitative differences between PsbO1 and PsbO2
ArabidopsisPsbO has been reported to be able to bind
urea-washed PSII particles of spinach and to restore
the oxygen-evolving activity as effectively as spinach
PsbO [16,25] We carried out reconstitution procedures
according to their methods First, we examined the oxygen-evolving activity as a function of the PsbOs⁄ PSII ratio in the assay medium (Fig 3A) The maxi-mum activity with PsbO2 was about 80% of that with PsbO1, whereas the maximum oxygen-evolving activity reconstituted with both PsbO1 and PsbO2 was achieved at about 2 PsbOs⁄ PSII The restoration curve obtained with our recombinant PsbOs closely resem-bled that for spinach PsbO and Arabidopsis PsbO1 reported by Betts et al [25] PsbO1 and PsbO2 also showed a similar affinity for urea-washed PSII parti-cles (Fig 3B,C); the binding of both PsbOs with urea-washed PSII particles was saturated at about 2 PsbOs⁄ PSII, like the oxygen-evolving activity
Competition analysis of PsbO1 and PsbO2
To confirm that the two PsbOs have a similar binding affinity, the competition between them for binding sites
on PSII particles was analyzed Increasingly larger amounts of an equimolar mixture of PsbO1 and PsbO2 were incubated with urea-washed PSII particles (Fig 4A,B), and the relative amounts of bound-PsbO1
wild-type young
wild-type mature
psbo1
young
psbo1
mature
PsbO PsbA PsbP
140 120 100 80 60 40 20 0
PsbA
PsbP
wild-type M Y
psbo1
M Y
wild-type M Y
psbo1
M Y
CBB-staining
PsbO1 PsbO2
A
B
Fig 2 Protein analysis by SDS ⁄ PAGE
(15%) (A) Coomassie brilliant blue-staining
and immunoblot analysis with polyclonal
antibodies against spinach PsbO, PsbP and
PsbA Antibodies against PsbO detect two
signals with slightly different migrations,
upper band; PsbO1, lower band; PsbO2.
Thylakoid membranes were loaded on a
chlorophyll basis; equivalent to 5 lg of
chlo-rophyll for Coomassie brilliant blue-staining
and 1 lg for immunoblot analysis The
arrow shows PsbOs in Coomassie brilliant
blue-staining Y, young plants (leaf size,
about 0.5 cm); M, mature plants (leaf size,
about 2.5 cm) (B) The relationship between
the accumulation of PsbO (black), PsbP
(mid-grey) and PsbA (light grey) The protein
level was quantified by densitometry Values
are relative to the protein level in the young
wild-type (100%) Standard deviations were
calculated from three measurements.
Trang 4and bound-PsbO2 with urea-washed PSII particles
were quantified by the densitometry of Coomassie
bril-liant blue-stained proteins on a SDS⁄ PAGE plate As
PsbO1 migrated more slowly than PsbO2, the two
could be distinguished in the binding analysis
[10,26–28] Urea-washed PSII particles incubated with
two moles of PsbOs per mole of PSII particles (one
mole of each protein added, 1 : 1) had similar amounts
of PsbO1 and PsbO2 (approximately PsbO1⁄ PsbO2 ¼
51 : 49) The oxygen-evolving activity with a mixture
of one mole each of PsbO1 and PsbO2 was higher than that with four moles of PsbO2, but lower than that with four moles of PsbO1 Although the differ-ence was small, the same results were obtained consis-tently As the levels of both proteins were saturated at about 2 PsbOs⁄ PSII, we added an excessive amount of PsbOs per PSII (two moles of each protein, 2 : 2) The two proteins bound equally with urea-washed PSII (approximately 50 : 50) and the oxygen-evolving activ-ity was as high as the sample; 1 : 1 These results sug-gested that the two proteins had a similar affinity for urea-washed PSII
Next, we examined the effect of increasing the propor-tion of PsbO1 on the reconstitupropor-tion of PSII The ratio of PsbO1 to total PsbOs bound with urea-washed PSII particles and the oxygen-evolving activity demonstrated equal competition between PsbO1 and PsbO2 for PSII particles (Fig 4C) These competition analyses were consistent with the analysis of the reconstitution with PsbO1 and PsbO2, in terms of similar binding affinity
Chimeric PsbOs and surface charge
To examine which amino acid of PsbO2 is responsible for the poor oxygen-evolving activity, we prepared chi-meric proteins with PsbO1 and PsbO2 in Escherichia coli (Fig 5A) For example, the chimeric protein PsbO1-1-2 has the N-terminal and middle parts of PsbO1 combined to the C-terminus of PsbO2 Each of the parts contains about 80 amino acids, and, respect-ively, has three, five and three amino acid replacements between PsbO1 and PsbO2
SDS⁄ PAGE analysis of chimeric PsbOs showed that PsbO1-1-2 and PsbO2-1-1 migrated as slowly as PsbO1, and only PsbO1-2-1 migrated as PsbO2 did (Fig 5B) Anion-exchange chromatography was also performed, as PsbO1 was eluted at a higher NaCl
PsbO1
PsbO2
100
80
60
40
20
0
untreated PSIIurea-w
ashed PSII
0.5 1 2 4 0.5 1 2 4
PsbO1 PsbO2
140
120
100
80
60
40
20
0
PsbO1
PsbO2
A
B
C
Fig 3 The oxygen-evolving activity of urea-washed PSII reconstitu-ted with PsbO1 and PsbO2 and the binding affinity for urea-washed PSII of PsbO1 and PsbO2 (A) The relative oxygen-evolving activity
of urea-washed PSII reconstituted with PsbO1 (r) and PsbO2 (h) The maximum rate of oxygen evolution (100%) equals the rate measured for untreated PSII minus that for urea-washed PSII (B) Coomassie brilliant blue-staining Urea-washed PSII reconstituted with PsbO1 and PsbO2 were separated on SDS ⁄ PAGE (15%) An amount equivalent to 2 lg of chlorophyll was loaded in each lane The arrow shows PsbOs in Coomassie brilliant blue-staining (C) Quantification of PsbO1 (r) and PsbO2 (h) bound to urea-washed PSII particles Values quantified by densitometry were plotted against PsbOs added per PSII Values are relative to the untreated PSII (100%) Standard deviations were calculated from five meas-urements.
Trang 5concentration than was PsbO2 PsbO1-2-1 again
showed a chromatographic elution profile similar to
PsbO2, whereas the profiles of other chimeric proteins
were similar to that of PsbO1 (data not shown) In
fact, these results were consistent with the theoretical
pI values; the theoretical pI values of PsbO1, PsbO1-1-2 and PsbO2-1-1 were calculated to be 4.96 (calculated molecular mass, 26565 Da), 4.95 (26567 Da) and 4.96 (26579 Da), respectively, and those of PsbO2 and PsbO1-2-1 were calculated to be 5.05 (26571 Da) and 5.06 (26555 Da), respectively (to compute pI⁄ Mw, http://kr.expasy.org) These results suggest that, in terms of surface charge, PsbO1-2-1 was similar to PsbO2 and the other chimeric PsbOs were similar to PsbO1
PsbO1 PsbO2
PsbO1-1-2 PsbO2-1-1 PsbO1-2-1
A
B
C
Fig 5 Oxygen-evolving activity of chimeric proteins derived from PsbO1 and PsbO2 (A) A model of the chimeric PsbOs In chimeric PsbOs, a part of PsbO1 (C-terminus, middle and N-terminus) was replaced by a corresponding part of PsbO2 Each of the parts con-tains about 80 amino acids, and, respectively, has three, five and three amino acid replacements between PsbO1 and PsbO2 (B) Coomassie brilliant blue-staining on SDS ⁄ PAGE (15%) of PsbO1, PsbO2 and chimeric PsbOs They were expressed using a pET-sys-tem in Escherichia coli and purified by anion-exchange chromato-graphy Protein (0.5 lg) was loaded in each lane (C) Oxygen-evol-ving activities of urea-washed PSII reconstituted with PsbO1, PsbO2 and chimeric PsbOs The maximum rate of oxygen evolu-tion (100%) equals the rate measured for untreated PSII minus that for urea-washed PSII.
untreated
PSII
urea-washed
PSII
PsbO
PsbO2
(1:1)
(2:2)
100
0 0.2 0.4 0.6 0.8 1
100 80 60 40 20 0 120
A
B
C
Fig 4 Competition of PsbO1 and PsbO2 for the reconstitution of
PSII (A) Reconstitution of PSII with an equimolar mixture of PsbO1
and PsbO2 Reconstituted PSII particles were separated on a 15%
SDS ⁄ PAGE and stained by Coomassie brilliant blue An amount
equivalent to 2 lg of chlorophyll was loaded in each lane PsbO1,
PsbO2; 4 mol PsbO1 or PsbO2 per PSII, 1 : 1; 1 mole PsbO1 and
1mole PsbO2 per PSII, 2 : 2; 2 mole PsbO1 and 2 mole PsbO2 per
PSII (B) Relative oxygen-evolving activity of PSII reconstituted with
an equimolar mixture of PsbO1 and PsbO2 The maximum rate of
oxygen evolution (100%) equals the rate measured for untreated
PSII minus that for urea-washed PSII (C) Reconstitution of PSII
with PsbOs in which the ratio of PsbO1 to total PsbO was varied.
The relative oxygen-evolving activity (r) and the ratio of bound
PsbO1 : PsbOs (h) The maximum rate of oxygen evolution (100%)
equals the rate measured for untreated PSII minus that for
urea-washed PSII The ratio of bound PsbO1 : PsbOs was estimated
from the Coomassie brilliant blue-staining on SDS⁄ PAGE Standard
deviations were calculated from five measurements.
Trang 6However, the oxygen-evolving activity of
urea-washed PSII particles reconstituted with PsbO1-2-1
was similar to that of the particles reconstituted with
PsbO1 (Fig 5C) A lower level of activity was
observed with PsbO1-1-2, a chimera with the
C-ter-minal sequence of PsbO2 Although the difference was
small, the same results were obtained repeatedly These
results suggest that the difference in surface charge
between PsbO1 and PsbO2 did not affect the difference
in oxygen-evolving activity
C-Terminal amino acid substitution and
oxygen-evolving activity
PsbO1 and PsbO1-1-2 had three amino acid changes;
Val186 (PsbO1) to Ser (PsbO2), Val204 to Ile, and
Leu246 to Ile (Fig 6A) To examine which
replace-ment was responsible for the difference in the
oxygen-evolving activity, we prepared mutated PsbO1 in which
an amino acid was substituted for the corresponding
one in PsbO2 (V186S, V204I and L246I)
Measure-ment of the oxygen-evolving activity upon
reconstitu-tion with the spinach PSII core showed that the
activity levels with V186S and L246I were apparently
lower than that with PsbO1 (Fig 6B), suggesting that
these two amino acids were responsible for the lower
level of activity Unexpectedly, V204I showed stronger
oxygen-evolving activity than PsbO1
We next prepared double-mutated proteins with two
amino acid substitutions; i.e V186S⁄ V204I, V186S ⁄
L246I and V204I⁄ L246I The oxygen-evolving activity
reconstituted with V186S⁄ V204I and V204I ⁄ L246I was
similar to that with PsbO1; levels were higher than for
V186S and L246I and lower than for V204I (Fig 6B)
The activity reconstituted with V186S⁄ L246I was much
weaker than that with V186S, L246I or PsbO2 These
results confirmed that the replacement of Val186 with
Ser and Leu146 with Ile led to a reduction in the level
of oxygen-evolving activity, while the replacement of
Val204 with Ile led to an increase
Location of amino acids that differ between
PsbO1 and PsbO2
The locations of three amino acids, Val186 Val204 and
Leu246, were predicted using the Thermosynechococcus
elongatusPsbO [19] (protein data bank accession
num-ber; 1S5L) as a template The prediction suggested that
Val186, Val204 and Leu246 were dispersed in different
secondary structures; i.e in the a-helix, in the b-sheet
and near the b-sheet, respectively (Fig 7) The
predic-tion also supported the nopredic-tion that these replacements
independently affected the oxygen-evolving activity As
expected, the predicted structure of Arabidopsis PsbO2 was very similar to the structure of PsbO1 (data not shown), and the substitution of amino acids between PsbO1 and PsbO2 would not modify the overall struc-ture of PsbO
Discussion The existence of two psbO genes enabled the isolation
of psbo1 which lacked psbO1 expression and showed weak photosynthetic activity In this study, we exam-ined the functional role of two PsbOs in Arabidopsis Careful characterization of the PsbOs in psbo1 and the wild type (Fig 2A) showed that psbo1 showed a much greater accumulation of PsbO2 than in the wild type This finding suggests a compensational mechanism that stimulates the expression of PsbO2 when a functional psbO1 gene is absent The shortage of PsbOs caused a photosynthetic defect in psbo1, especially in young psbo1 However, mature psbo1 exhibited increased lev-els of PsbO and improved photosynthetic activity, as estimated from the increased Fv⁄ Fm, but the molecular mechanism of this adaptation to a genetic defect is not clear
Our immunoblot analysis also showed that the accumulation of PsbOs limited the levels of other PSII proteins as well as the efficiency of PSII This result was consistent with findings in early studies using higher-plant PSII that the extraction of PsbO from PSII affected the stability of PsbA and the assembly of other extrinsic proteins [22–24] In this respect, the quite different phenotypes observed in psbo1 and psbO-deletion mutants of green algae and cyanobacteria might lead to a clearer understanding
of the structure of the oxygen-evolving complex [6,7] First, although a psbO-deletion mutant of the Chlamydomonas reinhardtii [8] also had an enhanced turnover of core PSII polypeptide, PsbA, a psbO-deletion mutant of cyanobacteria accumulated PSII reaction centers at nearly normals levels The differ-ence in the behavior of the deletion mutants between higher plants, green algae and cyanobacteria suggests
a difference in the relationship between the extrinsic proteins Green algae and higher plants contain PsbP and PsbQ, whereas cyanobacteria contain PsbV and PsbU There is an apparent difference between PsbP and PsbV, as PsbP cannot bind to the PSII core or function in the absence of PsbO, but PsbV alone functions effectively in the absence of PsbO Second, PsbP was accumulated at normal levels in the Chlamydomonas reinhardtii psbO-deletion mutant, sug-gesting a difference between higher plants and green algae in the regulation of levels of extrinsic proteins
Trang 7Arabidopsis thaliana O1
Arabidopsis thaliana O2
Nicotiana tabacum
Solanum tuberosum
Lycopersicon esculentum
Pisum sativum
Oryza sativa
Spinacia oleracea
Volvox carteri
Chlamydomonas reinhardtii
Euglena gracilis
Bigelowiella natans
Synechocystis sp PCC 6803
Anabaena sp PCC 7120
Thermosynechococcus elongatus BP-1
Prochlorococcus marinus SS120
Prochlorococcus marinus MIT9313
Synechococcus sp.WH8102
Triticum aestivum
Fritillaria agrestis
*
Synechocystis sp PCC 6803
Prochlorococcus marinus SS120
Prochlorococcus marinus MIT9313
Synechococcus sp.WH8102
Arabidopsis thaliana O1
Arabidopsis thaliana O2
Nicotiana tabacum
Solanum tuberosum
Lycopersicon esculentum
Pisum sativum
Oryza sativa
Spinacia oleracea
Volvox carteri
Chlamydomonas reinhardtii
Euglena gracilis
Bigelowiella natans
Anabaena sp PCC 7120
Thermosynechococcus elongatus BP-1
Triticum aestivum
Fritillaria agrestis
*
Arabidopsis thaliana O1
Arabidopsis thaliana O2
Nicotiana tabacum
Solanum tuberosum
Lycopersicon esculentum
Pisum sativum
Oryza sativa
Spinacia oleracea
Volvox carteri
Chlamydomonas reinhardtii
Euglena gracilis
Bigelowiella natans
Synechocystis sp PCC 6803
Anabaena sp PCC 7120
Thermosynechococcus elongatus BP-1
Prochlorococcus marinus SS120
Prochlorococcus marinus MIT9313
Synechococcus sp.WH8102
Triticum aestivum
Fritillaria agrestis
*
Relative oxygen-evolving activity
0 20 40 60 80 100 120 PsbO1
PsbO2 V186S V204I L246I 186&204 186&246 204&246
Fig 6 Determination of the amino acid changes responsible for the alteration of protein function (A) Alignments around the replacements between PsbO1 and PsbO1-1-2 The alignments were made with CLUSTAL W Asterisks indicate substituted residues with Arabidopsis PsbO1 and PsbO2 Arabidopsis PsbO1 and PsbO2 are underlined (B) Oxygen-evolving activities reconstituted with PsbO1, PsbO2, and mutated-PsbOs The maximum rate of oxygen evolution (100%) equals the rate measured for untreated PSII minus that for urea-washed PSII Stand-ard deviations were calculated from five measurements.
Trang 8Interestingly, PsbO1 and PsbO2 showed different
biochemical activity at reconstituting oxygen-evolving
activity with urea-washed PSII isolated from spinach;
the restoration of oxygen-evolving activity with PsbO2
was about 80% of that with PsbO1, whereas the
bind-ing affinities of PsbO1 and PsbO2 were similar A
recent study on the structure of PSII [19] suggested
that PSII has one copy of PsbO, but early works
[16,25] and our study (Fig 3) showed that the
maximum oxygen-evolving activity reconstituted with
both PsbO was achieved at about two PsbOs per PSII
It is not clear why about two PsbOs per PSII are nee-ded for the maximum activity
The chimeric PsbO derived from PsbO1 and PsbO2 clearly indicated that the surface charge was not crit-ical for the different activities of PsbO1 and PsbO2, whereas negative and positive charges of amino acids have been reported to be important for the interaction between PsbO and PSII [5,17,18] The analysis using the chimeric PsbOs showed that three amino acid replacements at the C-terminus affected the restoration
of oxygen-evolving activity
As predicted in Fig 7, the three amino acids were dispersed in PsbO Interestingly, although Val186 was not conserved in higher plants, green algae and cyano-bacteria, its substitution with Ser decreased oxygen-evolving activities On the other hand, when Val204 of PsbO1, which is conserved in higher plants except for Arabidopsis PsbO2, was substituted with the Ile of PsbO2, this mutated-protein restored the oxygen-evol-ving activity better than PsbO1 It should be noted that the amino acid at this position is substituted with Ile in both green algae and cyanobacteria By contrast, Leu246 is conserved in all higher plants and green algae, except in PsbO2 PsbO2 has Ile246, which is substituted with Ile or Val in cyanobacteria, except for Prochlorococcus marinus which has Lys The replace-ment of Leu with Ile was shown to result in a reduc-tion of the restorareduc-tion of oxygen-evolving activity It has been reported that Leu at position 246 was critical for PsbO to bind PSII and the restoration of oxygen-evolving activity in spinach [29] Although our recon-stitution experiment with PsbO2 showed a similar affinity for PSII particles as PsbO1, the importance of this amino acid residue for oxygen-evolving activity was consistent with the results of previous works [29,30] The similar chemical properties of Leu and Ile might explain their similar binding affinities It has also been reported that the C-terminal peptide of PsbO (15 amino acids) competed with mature PsbO to bind the PSII core, suggesting that the C-terminus of PsbO plays an important role in the interaction with an integral part of the PSII core [31] Identification of the importance of the three amino acid residues in PsbO, especially V204I, could be useful for understanding the efficiency of PSII
In vivo and in vitro experiments have shown that both a shortage of the PsbOs and a lower level of oxy-gen-evolving activity resulted in the poor photosyn-thetic activity and retarded growth in psbo1 In most higher plants, such as spinach, pea and rice, PsbO is encoded by only a single gene [26,32] The existence of
a duplicated psbO gene in Arabidopsis should help us to understand the molecular selection of duplicated genes
L246I
V186S
V186S
V204I
L246I
A
B
Fig 7 The location of three amino acid substitutions between
PsbO1 and PsbO2 (A) Stereoview of Thermosynechococcus
elong-atus PsbO The corresponding positions of Val186, Val204 and
Leu246 in Thermosynechococcus elongatus PsbO were predicted
by SwissModel (http://swissmodel.expasy.org//SWISS-MODEL.
html) and are shown in red and indicated by an arrow (B)
Stereo-view of PsbO, PsbA and Mn clusters in Thermosynechococcus
elongatus PsbO: green, PsbA: blue, Mn clusters: pink, The
posi-tions corresponding to Val-186, Val-204 and Leu-246 are shown in
red and indicated by an arrow These figures were generated with
P Y MOL (http://pymol.sourceforge.net/).
Trang 9In Arabidopsis, psbQ was also encoded by two
nuc-lear genes (At4g21280 and At4g05180) with about
82% similarity at the gene level On the other hand,
psbP (At1g06680) has a similar but nonfunctional gene
(At2g30790) (about 84% similarity at the gene level); it
encodes a much smaller protein than PsbP, suggesting
that the isogene of psbP recently lost its function
Simi-larly, other subunits in PSII encoded by nuclear genes,
such as psbR and psbS, are encoded by a single gene
The investigation of PsbQ isogenes should provide
another clue as to the physiological role of duplicated
genes for extrinsic proteins in the oxidation of water
Although the duplicated genes would initially have
had the same functions, the accumulation of mutations
over time has resulted in either the functional loss of one
copy or functional divergence between them (our data
and [33]) It is not clear how much functional
differenti-ation exists between PsbO1 and PsbO2 and whether the
inferior psbO2 will lose its activity At present, the
data-base (Brassica Genome Gateway, http://brassica.bbsrc
ac.uk) suggests that Brassica rapa and Brassica napus
have a psbO1-like gene (accession number; Brassica
rapa, BQ791144 and Brassica napus, AF139818) and a
psbO2-like gene (accession number; Brassica rapa,
BG543314 and Brassica napus, CD821869), indicating
that any potential evolutionary pressure to remove the
psbO2 gene is not strong Indeed, PsbO2 might have
some physiological significance; for example, the poor
oxygen-evolving activity with PsbO2 might be
advanta-geous under excessive light conditions
Conclusions
The psbO2 gene could not complement the defect in
the psbO1 gene for both quantitative and qualitative
reasons: a shortage of PsbOs and the poor
oxygen-evolving activity of PsbO2 The functional difference
between PsbO1 and PsbO2 in the restoration of
oxygen-evolving activity was ascribed to three amino
acid replacements at the C-terminus; Val186 in PsbO1
for Ser in PsbO2, Val204 for Ile, and Leu246 for Ile
Experimental procedures
Plant growth and measurements of chlorophyll
fluorescence
Seeds of the wild-type and psbo1 were sown in soil after
Lehle Seeds (Round Rock, TX, USA) They were grown at
with a 9-h light (from 09:00–18:00 h): 15-h dark cycle for
2 months
fluorometer (Waltz, Effeltrich, Germany) The minimum
fluores-cence at closed PSII centers in the dark) was measured by applying a 1-s pulse of saturating white light
Immunoblot analysis Thylakoid membranes were isolated from wild-type and
[34] Protein samples were prepared in sample buffer
5% glycerol, and 5% 2-mercaptoethanol], and analyzed by
expression with Coomassie brilliant blue staining [35] Thyl-akoid membranes were loaded on a chlorophyll basis The protein analysis with respect to the protein content showed the results similar to that on a chlorophyll basis The thy-lakoid proteins were also transferred to a polyvinylidene difluoride membrane with a semidry blotting system [36], and detected with rabbit antiserum against spinach PsbO, kindly provided by the late A Watanabe of the University
of Tokyo, or rabbit antiserum against spinach D1 protein (PsbA), provided by Y Yamamoto (Division of Integrated Life Science, Kyoto University, Japan) The chlorophyll concentration was measured by spectrophotometry in 80% acetone [37]
The amounts of PsbO, PsbA and PsbP in plants were quantified from immunoblots and bound PsbOs to urea-washed PSII were quantified from Coomassie brilliant blue-stained plates Image analysis was performed using the public-domain software nih-image (version 1.62)
Preparation of recombinant PsbOs The recombinant PsbO1 and PsbO2 were expressed and purified as described previously [38] The expression vectors for the chimeric proteins were produced by digestion of psbO1- and psbO2-pET 21d + with BamHI, NotI or PpuMI and by the ligation of resultant fragments Site-directed mutagenesis was performed according to the proto-col for the Quick-change Site-directed Mutagenesis Kit (Stratagene) and a Technical Review for Long polymerase-chain reaction using KOD plus (TOYOBO, Osaka, Japan) All mutant constructs were confirmed by DNA sequencing These mutant proteins were purified similar to the original PsbO1 and PsbO2
Reconstitution of PSII with recombinant PsbOs Photosystem II membranes were isolated from spinach leaves purchased at a local market according to the method
Trang 10of Ghanotakis et al [39] Native extrinsic proteins in PSII
were extracted by incubation in urea-washed buffer (50 mm
Mes–NaOH (pH 8.3) and 3 m urea), to give urea-washed
PSII particles Recombinant PsbOs were mixed with
urea-washed PSII particles and incubated at room temperature
concentration of PSII was estimated on the basis of chl
concentrations and a stoichiometry of 250 chlorophylls per
PSII complex
(Hansatech, UK) in the presence of 2 mm DCBQ
(dichloro-p-quinone) as the electron acceptor for PSII Red
in conjunction with an HA50 heat-absorbing filter and an
R-60 red optical filter (Kenko, Tokyo, Japan)
Alignment of PsbOs
Similarity was evaluated using the clustal w program
[40–42] The alignment parameters used were: protein mass
matrix: BLOSUM series, gap opening penalty (GOP): 10.0,
gap extension penalty (GEP): 0.05 and Delay divergent
sequences: 40%
Acknowledgements
This study was supported in part by a COE Scientific
Research Grant from the Ministry of Education,
Cul-ture, Sports, Science and Technology of Japan
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