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The Low Molecular Mass PsbW Protein Is Involved in the
Stabilization of the Dimeric Photosystem II Complex in
Arabidopsis thaliana*
Received for publication, July 17, 2000, and in revised form, August 16, 2000
Published, JBC Papers in Press, August 18, 2000, DOI 10.1074/jbc.M006300200
Lan-Xin Shi‡, Zdravko J. Lorkovic´§, Ralf Oelmu¨ller¶, and Wolfgang P. Schro¨der‡储**
From the ‡Arrhenius Laboratories for Natural Sciences, Department of Biochemistry, SE-106 91 Stockholm, Sweden, the
§Friedrich Miescher Institut, P. O. Box 2543, CH-4002 Basel, Switzerland, the ¶Institut fu¨r Allgemeine Botanik, Lehrstuhl
Pflanzenphysiologie, Friedrich-Schiller-Universita¨ t Jena, Dornburger Strasse 159, D-07743 Jena, Germany, and the
储So¨ derto¨rns Ho¨ gskola (University College), Bipontus, Box 4101, SE-141 04 Huddinge, Sweden
Arabidopsis thaliana plants have been transformed
with an antisense gene to the psbW of photosystem II
(PSII). Eight transgenic lines containing low levels of
psbW mRNA have been obtained. Transgenic seedlings
with low contents of PsbW protein (more than 96% re-
duced) were selected by Western blotting and used for
photosynthetic functional studies. There were no dis-
tinct differences in phenotype between the antisense
and wild type plants during vegetative period under
normal growth light intensities. However, a sucrose gra-
dient separation of briefly solubilized thylakoid mem-
branes revealed that no dimeric PSII supracomplex
could be detected in the transgenic plants lacking the
PsbW protein. Furthermore, analysis of isolated thyla-
koids demonstrated that the oxygen-evolving rate in an-
tisense plants decreased by 50% compared with the wild
type. This was found to be due to up to 40% of D1 and D2
reaction center proteins of PSII disappearing in the
transgenic plants. The absence of the PsbW protein also
altered the contents of other PSII proteins to differing
extents. These results show that in the absence of the
PsbW protein, the stability of the dimeric PSII is dimin-
ished and consequently the total number of PSII com-
plexes is greatly reduced. Thus the nuclear encoded
PsbW protein may play a crucial role in the biogenesis
and regulation of the photosynthetic apparatus.
Photosystem II (PSII)
1
of higher plants catalyzes the light-
driven oxidation of water to molecular oxygen and the reduc-
tion of plastoquinone to plastoquinol. The PSII supracomplex
consists of almost 30 different subunits of which two, the D1
and D2 proteins, bind most, if not all, of the cofactors needed for
primary and secondary electron transfer reactions. The D1, D2,
and the inner antenna proteins, CP43 and CP47, bind chloro-
phyll a, and constitute together with the extrinsic proteins the
PSII core. The PSII core in turn is surrounded by the outer
antenna, light harvesting complex II (LHCII) which binds both
chlorophyll aand b(1–5). Both biochemical studies (6–9) and
single particle analysis of two-dimensional crystals (10–12)
suggest that the PSII supracomplex forms a dimer in vivo.
Recently, intact and highly active dimeric PSII-LHCII supra-
complexes were isolated directly from spinach thylakoids (9)
supporting the idea that the dimer is the natural state of PSII.
Both the monomeric and the dimeric forms of PSII have been
found to contain several low molecular mass (⬍7 kDa) proteins
(6, 8). One of these small proteins is the nuclear encoded PsbW
(6.1 kDa) protein (13, 14) that is highly conserved in spinach,
Arabidopsis, and Chlamydomonas. Fig. 1 shows the Arabidop-
sis PsbW protein sequence dealt with in this work. The PsbW
protein was found to have only a single membrane span, with
14 and 20 amino acids stretching out to the stromal and lume-
nal sides of the membrane, respectively (Fig. 1). The orienta-
tion of the PsbW protein in the thylakoid membrane is opposite
to other transmembrane PSII reaction center proteins with its
N terminus at the lumenal side and the C terminus at the
stromal side (13–15). Localization studies showed that the
PsbW protein is not present in PSI, but is instead tightly
associated with the PSII reaction center (13, 15). This was
further supported by the finding that the PsbW protein under-
goes degradation under photoinhibitory conditions. The extent
and pattern of degradation was similar to that of the D1 protein
except that it was not phosphorylated before degradation (16).
The protein is expressed in dark-grown seedlings, i.e. it is
synthesized before other PSII reaction center proteins, and the
protein level increases upon illumination (14, 17). In order to
obtain insights into the function of the PsbW protein in the
photosynthetic process, we generated transgenic Arabidopsis
thaliana plants expressing an antisense construct of psbW.In
this report we present the functional analysis of a nuclear-
encoded low molecular mass protein in PSII from higher
plants. The data demonstrate that the PsbW protein is involved
in the stabilization of dimeric PSII complexes in Arabidopsis.
MATERIALS AND METHODS
Generation of A. thaliana PsbW Antisense Plants—The genomic frag-
ment encoding A. thaliana PsbW (18) was cloned in an antisense ori-
entation into pBin19 downstream of the repeated cauliflower mosaic
virus 35S promoter. The construct was transferred into Agrobacterium
tumefaciens strain LBA4404 by triparental mating (19). Arabidopsis
plants were transformed by an influorescence infiltration method (20).
Transgenic plants (T1) were selected on kanamycin-containing Murash-
ige and Skoog (21) plates, transferred into soil, and allowed to self-
pollinate to produce T2 seeds. The T1 plants were also verified by
Southern and Northern blot analyses.
Growth of A. thaliana—The wild type and the T2 A. thaliana (Co-
* This work was supported by the Swedish Natural Science Research
Council (NFR) and the Swedish Forestry and Agriculture Research
Council (SJFR). The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
** To whom correspondence and reprints should be addressed. Tel.:
46-8-58588587; Fax: 46-8-58588510; E-mail: wolfgang.schroder@sh.se.
1
The abbreviations used are:PSII, photosystem II; LHCII, light
harvesting complex II; PpBQ, phenyl-p-benzoquinone; D1 and D2 pro-
teins, products of the psbA and psbD genes, respectively; CP47 and
CP43, chlorophyll-binding proteins encoded by the psbB and psbC
genes, respectively; Q
A
and Q
B
, the first and second PSII plastoquinone
electron acceptor; Mes, 4-morpholineethanesulfonic acid; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 48, Issue of December 1, pp. 37945–37950, 2000
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 37945
lombia) transgenic seeds were placed on wet filter paper and incubated
at 4 °C for 3 days. The cold-treated seeds were sown in a mixture
containing soil, bead, and vermiculite with the ratio of 1:1:2. The seed-
lings were grown under white light (90 or 180
mol of photons m
⫺2
s
⫺1
),
and the light/dark cycle was 8/16 h. Plants were also grown hydropon-
ically (22). For biochemical studies, leaves were harvested before plant
flowering.
Isolation of Thylakoids and Chlorophyll Concentration Measure-
ment—Isolation of thylakoid membranes from A. thaliana was carried
out according to Nore´n et al. (22) with minor modifications. One gram of
A. thaliana leaves was homogenized with 40 ml of preparation medium
(300 mMsorbitol, 20 mMTricine, pH 8.4, 10 mMEDTA, 10 mMKCl
0.25% (w/v) bovine serum albumin, 5 mMsodium ascorbate, and 5 mM
dithiothreitol). The slurry was filtered through 4 layers of cheesecloth
and centrifuged at 1000 ⫻gfor 1 min. The pellet was resuspended in 20
ml of 5 mMMgCl
2
to lyse the chloroplasts. After 30 s the same volume
of double concentrated resuspension medium (600 mMsorbitol, 40 mM
Hepes, pH 7.6, 5 mMMgCl
2
,10mMEDTA, and 20 mMKCl) was added.
The thylakoid membranes were reisolated at 1000 ⫻gfor 1 min,
washed once with resuspension medium, and resuspended in the same
medium. For measurement of chlorophyll concentration, samples were
diluted in 80% acetone, centrifuged at 10,000 ⫻gfor 10 min, and
measured spectroscopically (23).
Sucrose Density Gradient Centrifugation—A continuous sucrose gra-
dient containing 0.03% (w/v) n-dodecyl-

-D-maltoside was prepared by
the freeze and thaw method described by Eshaghi et al. (9), except that
the sucrose gradients were buffered with 25 mMHepes, pH 7.6. The
solubilization of thylakoid membranes by n-dodecyl-

-D-maltoside de-
tergent and centrifugation were carried out exactly as in Ref. 9.
Western Blotting and Protein Analysis—SDS-polyacrylamide gel
electrophoresis was carried out according to Scha¨ gger and von Jagow
(24) with minor modifications. The polyacrylamide gel contained 6 M
urea and the Tris-Tricine running buffer was used. The proteins on
polyacrylamide gels were either transferred to polyvinylidene difluoride
membrane (25) or stained with silver (26). Immunoblotting was carried
out using a semidry blotting system (Millipore). A polyclonal antiserum
was raised in rabbit against the N-terminal 15-mer oligopeptide of
PsbW protein and purified using protein A-Sepharose chromatography
(13). Immunodecorations were visualized using the alkaline phospha-
tase system with CDP-Star substrate (BioLabs). Quantification of im-
munoblots was performed by laser scanning densitometry.
Measurement of Steady State Oxygen Evolution—Oxygen-evolution
activity of PSII was measured using a Clark-type electrode in reaction
medium (0.1 Msorbitol, 5 mMMgCl
2
,5mMNaCl, 50 mMHepes, pH 7.6)
at 20 °C under saturating light. Potassium ferricyanide (2 mM), phenyl-
p-benzoquinone (PpBQ, 0.05, 0.1, and 0.2 mM), 2,6-dichlorobenzoqui-
none (1 mM), and 2,6-dichlorophenolindophenol (0.1 mM) were supplied
as electron acceptors.
Chlorophyll Fluorescence and Flash Oxygen-evolving Measure-
ments—Chlorophyll fluorescence was measured directly on intact
leaves after 15 min dark adaptation using a Walz PAM-200. Flash-
induced oxygen oscillation patterns were measured with a modified
Joliot-type electrode at 20 °C (27). The flow medium contained 30 mM
Mes, pH 6.5, and 10 mMKCl. The samples were dark adapted on the
electrode for 3 min and the polarization voltage of 700 mV was switched
on 30 s before a train of short (10
s) Xenon flashes separated by 500 ms
were given. The flash-induced oxygen yield (Y
n
) was measured and
normalized to the average yield on flashes 3–6.
RESULTS
Generation of Transgenic A. thaliana—Eight independent
lines of transgenic A. thaliana expressing the antisense psbW
gene were produced and analyzed. Northern blotting demon-
strated that the antisense construct of psbW was highly ex-
pressed in the transgenic plants and the level of psbW mRNA
was dramatically reduced (not shown) and consequently the
translated PsbW protein was greatly reduced as well. In one of
the different plant lines the level of PsbW protein was reduced
to less than 4% of wild type PsbW protein (Table I). A 96%
reduction of the PsbW protein is very close to a total knock-out
and hence provides an excellent system for further functional
analyses. These low levels of PsbW protein were detected at
different developmental stages of the plants indicating that the
antisense gene was constitutively expressed, resulting in low
levels of PsbW protein expression, through the whole life of the
plant. The T2 plants were grown directly in soil, each individ-
ual plant was tested by Western blotting before any further
analyses were performed. This showed that the transgene was
segregated 3:1 (not shown), as expected for T2 plants and that
both heterozygous and homozygous plants had decreased levels
of PsbW protein.
Phenotype—Despite the 96% reduction in PsbW protein lev-
els, no drastic change in phenotype of the antisense plants as
compared with the wild type occurred (Fig. 2). In addition,
growth of the plants under two different light regimes, 90 and
180
mol of photons m
⫺2
s
⫺1
for at least 50 days and on two
types of growth media (soil and hydroponic culture), did not
result in phenotypic changes in the antisense plants (Fig. 2).
However, the antisense plants flowered about 2 weeks earlier
than the wild type, which indicated a certain kind of stress.
Steady State Oxygen Evolution Is Affected—When steady
state oxygen evolving rates of isolated thylakoid membranes
were measured in the presence of different electron acceptors,
FIG.1.Schematic representation of the PsbW protein. The se-
quence was deduced from the A. thaliana psbW gene sequence
(X90769).
TABLE I
The contents of PsbW protein in wild type and transgenic plants
Western blotting of thylakoid membranes was performed using anti-
body raised against the PsbW protein. The relative content was quan-
tified by laser scanning densitometry.
Functional Analysis of the PsbW Protein in PSII37946
a dramatic effect was observed. Using PpBQ as an electron
acceptor only 50% of activity was present in thylakoids from
antisense plants (Table II), and the oxygen evolving activity
supported by 2,6-dichlorobenzoquinone was only 38% of the
wild type activity. Also, oxygen evolving activities supported by
other electron acceptors, such as ferricyanide and 2,6-dichloro-
phenolindophenol decreased significantly.
Stability of the Dimeric PSII Complex Is Reduced—We ana-
lyzed the structural conformation of PSII in the transgenic
Arabidopsis plants. A new direct method was applied for the
isolation of PSII-LHCII supercomplexes, i.e. PSII dimer com-
plexes (9) from thylakoids of wild type and antisense Arabidop-
sis plants. In this method, the isolated thylakoids are briefly
solubilized by n-dodecyl-

-D-maltoside and then applied onto a
sucrose gradient. By density gradient centrifugation the main
complexes of the thylakoid membrane can be separated with-
out affecting their intactness. In Fig. 3, the pattern of chloro-
phyll-containing bands from thylakoids of the wild type plant
shows strong similarities to that from spinach thylakoids (9).
The upper and middle bands contain LHCII and PSI, respec-
tively, while the third, somewhat diffuse band contains the
LHCII-PSII supercomplexes (PSII in dimeric form). When thy-
lakoids from antisense plants were treated in the same way,
the PSII dimer supracomplex could not be detected (Fig. 3).
Even if the ratio of detergent to chlorophyll was decreased, no
dimeric PSII band could be detected in the antisense plant (not
shown). Instead, an increased chlorophyll a/bratio was de-
tected in the lower part of the LHCII band, which is the loca-
tion of the monomeric PSII (6). This experiment clearly shows
that in the absence of the PsbW protein, no dimeric supracom-
plex of PSII can be isolated.
Electron Transport in PSII Does Not Change Significantly—
The electron transfer within the PSII complex of the psbW
antisense Arabidopsis plants was analyzed by measuring the
chlorophyll fluorescence and by flash oxygen measurements.
The chlorophyll fluorescence measurements of both intact
leaves and isolated thylakoids (not shown) showed that F
0
was
slightly higher, and Fv/Fm was somewhat lower (Fig. 4) in the
transgenic plant compared with the wild type plant. However,
no dramatic effects were observed, showing that electron trans-
fer in the PSII complexes lacking the PsbW protein was not
seriously affected. Moreover, the measurement of flash oxygen
evolution from isolated thylakoid membranes did not indicate
any significant effects caused by the antisense gene (Fig. 5).
This suggests that the lack of the PsbW protein does not alter
energy transfer within the PSII complex and that the remain-
ing PSII complex is functionally active.
The Amount of Functional PSII Core Complex Was Greatly
FIG.3. Sucrose gradient separation of n-dodecyl-

-D-malto-
side-solubilized thylakoid membranes from wild type (WT) and
PsbW antisense (-PsbW)Arabidopsis plants. The major complexes
are LHCII, PSI, and PSII-dimer.
TABLE II
PpBQ-supported oxygen evolution
Thylakoid membranes were isolated and the steady-state oxygen
evolution was measured with a Clark-type electrode at 20 °C and sat-
urating light. PpBQ (0.1 mM) was used as electron acceptor. Values are
means ⫾S.E. (n⫽5).
mol O
2
/mgChl䡠hRelative
activity
WT 128 ⫾19 100%
⫺PsbW 67 ⫾851⫾6
FIG.2.The phenotypes of wild type
and PsbW antisense Arabidopsis
plants. A. thaliana (Colombia) plants
were grown in soil for 50 days with white
light and a light/dark cycle of 8/16 h.
Light intensities were 180 (A)or90(B)
mol of photons m
⫺2
s
⫺1
.
Functional Analysis of the PsbW Protein in PSII 37947
Reduced—If electron transport in PSII complexes is nearly
normal, what causes the 50% decrease of the steady state
oxygen evolution? Analyses of the chlorophyll content showed a
small decrease of 0.2 in the chlorophyll a/b ratio in the trans-
genic plants indicated a loss of some chlorophyll awhich lead
us to assume that PSII core proteins must diminish. The de-
creased chlorophyll a/b ratio suggested a change of the chlo-
rophyll acontent, e.g. a reduced amount of PSII core proteins.
To test this, immunoblotting using various antibodies raised
against PSII proteins was performed. We found that the total
levels of the different PSII proteins in thylakoid membrane
preparations from PsbW antisense plants had changed (Table
III). The most affected proteins were the PSII reaction center
proteins, D1 and D2, of which up to 40% disappeared. The
amounts of oxygen-evolving enhancer proteins, PsbO and
PsbP, were reduced by 20 and 40%, respectively. The inner
antenna proteins CP43 and PsbS decreased by 30 and 40%,
respectively. Two low molecular mass proteins, cytochrome
b
559
which is associated with the reaction center and PsbX
located in PSII core, were less affected (75 and 90% remained,
respectively). In contrast to the proteins mentioned above, LH-
CII proteins were in fact slightly increased (8%). These results
clearly demonstrated that the amount of PSII core complexes
decreased by about 40%, but the antenna complex remained
intact. Since the PSII core contains mainly chlorophyll a, this
explains the decrease in the chlorophyll a/b ratio. It is consist-
ent, too, with the fact that no bleaching occurred in the trans-
genic plants as the major part of chlorophyll pigment in plants
is bound to the antenna complex, which was not drastically
affected. Consequently, the decreased oxygen evolution rate
was caused by a lower number of functional PSII centers.
DISCUSSION
Transgenic Arabidopsis plants with a 96% reduction in
PsbW protein level did not show any drastic phenotype
changes, which indicated that the PsbW protein is not directly
involved in electron transfer within the PSII complex. How-
ever, when isolated thylakoids from these plants were analyzed
with respect to steady state oxygen evolution, a reduction of
FIG.5.Oxygen yield pattern measurements. Dark-adapted wild
type (filled circle and solid line) and transgenic Arabidopsis (open circle
and dashed line), detected with a Joliot-type electrode after illumina-
tion with a train of 15 flashes separated 500 ms, no electron donors or
acceptors were added.
FIG.4. Chlorophyll fluorescence measurements of wild type
and PsbW antisense Arabidopsis plants. Chlorophyll fluorescence
was measured directly on intact leaf after 15 min dark adaptation using
a Waltz PAM 200. Panel A, wild type; panel B, antisense plants.
TABLE III
The contents of PSII proteins in wild type and transgenic plants
Western blotting of thylakoid membranes was performed using anti-
bodies raised against D1, D2, CP43, PsbS, LHCII, PsbO, PsbP, Cyt b
559
,
and PsbX protein. The relative contents were quantified by laser scan-
ning densitometry. Values are mean ⫾S.E. (n⫽5).
Functional Analysis of the PsbW Protein in PSII37948
PSII oxygen evolution of 50–60% (depending on the electron
acceptor used) was observed. The remaining PSII complexes
seemed to work normally as no drastic changes could be de-
tected by flash oxygen evolution or chlorophyll fluorescence
measurements when compared with thylakoids from wild type
Arabidopsis.
The decreased oxygen evolution in the transgenic Arabidop-
sis thylakoids lacking PsbW protein was instead found to be
due to the reduced amount of the PSII core proteins D1, D2,
and CP43, which decreased by roughly 40%. Also the extrinsic
proteins PsbO and PsbP proteins decreased, whereas the LH-
CII antenna was not affected. It is interesting to note that
oxygen evolution seems to be somewhat more affected by the
absence of the PsbW protein compared with the protein content
of the PSII core complex. This could simply be due to variations
using Western blots for protein quantification, but it could also
indicate an unidentified role of the PsbW protein in PSII.
Further experiments are in progress to answer this question by
using radiolabeled-DCMU for PSII quantification.
No dimeric PSII complexes could be isolated or detected in
the PsbW antisense thylakoids, which suggests that the PsbW
protein is essential for the stabilization of the dimeric PSII
complex. The functional role of the dimeric organization of PSII
is not yet fully understood. However, our results show that if
the PSII dimeric form is not formed or is not stable enough, the
amount of functional PSII is reduced. This suggests that the
stability of the dimeric form of PSII is higher than the mono-
meric form and thus the formation of dimers could be a way of
protecting the complex from being attacked by proteases. On
the other hand, when the complex is damaged by strong light
for instance, the complex monomerize, the D1 and PsbW pro-
tein are removed, and the degradation/repair can start. When
the degradation process is complete a newly synthesized PsbW
protein will again combine the two monomers to become a
stable functional PSII dimer.
Our finding that the absence of the PsbW protein dramati-
cally decreases the amount of functional PSII dimers, and the
fact that the PsbW protein is a nuclear-encoded protein in
higher plants, allows for the interesting speculation that this
could be a way for the plant cell nucleus to control the photo-
synthetic activity in the partly autonomous chloroplast.
How can a single
␣
-helix transmembrane protein be crucial
for the dimerization of such large protein complexes? There are
some reports suggesting various factors that could indeed con-
tribute to the dimerization of PSII. In addition to D1, D2, CP43,
and CP47, the PSII core contains the low molecular weight
polypeptides PsbE, PsbF, PsbH, PsbI, PsbK, PsbL, PsbT
c
, and
PsbW (6, 8, 28). Recent crystallographic data on the oxygen-
evolving core PSII dimer suggested that the connector region
between the two monomers might be attributed to the small
PSII subunits (12). The PsbL, PsbK, and PsbH were suggested
to be involved in dimer stabilization (8, 12, 28). Genetic dissec-
tion of PSII has shown that PsbL and PsbH are primarily
required for functioning of Q
A
, the primary acceptor quinone in
PSII (29, 30), and electron transfer from Q
A
to Q
B
(31, 32),
respectively. Requirement of PsbH for the accumulation of PSII
core proteins has also been reported (33), whereas the PsbK
seems to be entirely dispensable in Synechocystis (34) but not
in Chlamydomonas (35). Recent data has also suggested a
function for phosphatidylglycerol in the dimerization process of
PSII (28).
The PsbH has a positively charged N terminus at the stromal
side of the thylakoid membrane and this could be the site of
interaction with the negatively charged C terminus of PsbW
(Fig. 1). This interaction would then stabilize the PSII dimer.
The exact mechanism by which the PsbW protein promotes
PSII dimerization is not clear. However, as the PsbW protein is
found in the monomeric PSII (6, 8), assembled dimeric PSII
supracomplex (12), as well as in the reaction center pre-com-
plexes in etioplasts (36), the protein seems to be involved both
in guiding the assembly of monomeric PSII complexes and in
stabilization of the dimeric PSII. Interconversion between the
PSII dimer and monomers has been implicated in the D1 pro-
tein repair cycle (37) and this process could be controlled by
reversible phosphorylation of PsbH at its N terminus. In its
phosphorylated form PsbH cannot interact with PsbW and
consequently the PSII dimer will monomerize. PsbW itself is
not phosphorylated, but PSII damage under photoinhibitory
conditions results in the degradation of D1 and PsbW proteins
at a similar rate and extent (16).
A trEMBL data base search revealed that roughly 10% of the
total entries were proteins with a molecular mass below 7 kDa.
Several of these are single
␣
-helix transmembrane proteins
lacking prosthetic groups, very similar to the PsbW protein.
The results presented here for the PsbW protein give an incite-
ment to search for low molecular mass proteins in other protein
complexes and to analyze their possible involvement in com-
plex oligomerization.
Acknowledgments—We thank Dr. Christiane Funk, Dr. Thomas Kie-
selbach, Dr. Dan-Hui Yang, Said Eshaghi, Åsa Hagman, and Dr.
Patrick Dessi for help and discussions during the preparation of this
manuscript, Maria Bystedt for helping with the plants, and Prof. B.
Andersson for providing antisera raised against some of the PSII
proteins.
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