The low molecular mass PsbW protein is involved in the stabilization of the dimeric Photosystem II complex in Arabidopsis thaliana

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% reduced) were selected by Western blotting and used for photosynthetic functional studies. There were no distinct differences in phenotype between the antisense and wild type plants during vegetative period under normal growth light intensities. However, a sucrose gradient separation of briefly solubilized thylakoid membranes revealed that no dimeric PSII supracomplex could be detected in the transgenic plants lacking the PsbW protein. Furthermore, analysis of isolated thylakoids demonstrated that the oxygen-evolving rate in antisense 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 diminished and consequently the total number of PSII complexes is greatly reduced. Thus the nuclear encoded PsbW protein may play a crucial role in the biogenesis and regulation of the photosynthetic apparatus.


Introduction
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 darkgrown 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 A. 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.

Generation of A. thaliana -PsbW antisense plants.
The genomic fragment encoding A. thaliana PsbW (18) was cloned in an antisense orientation into pBin19 downstream of the repeated CaMV 35S promoter. The construct was transferred into Agrobacterium tumefaciens strain LBA4404 by triparental mating (19). Arabidopsis plants were transformed by an inflorescence infiltration method (20). Transgenic plants (T1) were selected on kanamycincontaining Murashige 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 Arabidopsis thaliana -The wild type and the T2 Arabidopsis thaliana (Colombia) transgenic seeds were placed on wet filter paper and incubated at 4 o C for three days. The cold treated seeds were sown in a mixture containing soil and vermiculite with the ratio of 1:1:2. The seedlings were growing under white light (90 or 180 µmol of photons m -2 s -1 ), and the light/dark cycle was 8/16 h. Plants were also grown hydroponically (22). For biochemical studies, leaves were harvested before plant flowering.

Isolation of thylakoids and chlorophyll concentration measurement -
Isolation of thylakoid membranes from A. thaliana was carried out according to Norén (22)  acetone, centrifuged at 10 000 x g for 10 min and measured spectroscopically (23).
Sucrose density gradient centrifugation -A continous sucrose gradient containing 0.03% (w/v) n-dodecyl β-D-maltoside (DM) was prepared by the freeze and thaw method described by Eshaghi et al (9), except that the sucrose gradients were buffered with 25 mM Hepes, pH 7.6. The solubilization of thylakoid membranes by DM detergent and centrifugation were carried out exactly as in (9).
Western blotting and protein analysis -SDS-PAGE was carried out according to SchÀgger (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 PVDF 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 phosphatase system with CDP-Star substrate (BioLabs). Quantification of immunoblots was performed by laser scanning densitometry. was measured and normalized to the average yield on flashes 3-6. 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, a dramatic effect was observed. Using

Generation of transgenic
PpBQ as an electron acceptor only 50% of activity was present in thylakoids from antisense plants (Table 2), and the oxygen-evolving activity supported by DCBQ was only 38% of the wild type activity. Also, oxygen-evolving activities supported by other electron acceptors, such as ferricyanide-and DCPIP-decreased significantly. 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 3). The most affected proteins were the

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.
However, when isolated thylakoids from these plants were analyzed with respect to steady state oxygen evolution, a reduction of 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 detected by flash oxygen evolution or chlorophyll fluorescence measurements when compared to thylakoids from wild type Arabidopsis.
The decreased oxygen evolution in the transgenic Arabidopsis 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 LHCII antennae 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 to 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 radiolabelled-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 monomeric 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 protein 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 dramaticaly 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 photosynthetic activity in the partly autonomous chloroplast.
How can a single α-helix transmembrane protein be crucial for the dimerization of such large protein complexes? There are some reports suggesting various factors that could indeed contribute to the dimerization of PSII. In addition to D1, D2, CP43 and CP47, the PSII core contains the low molecular weight proteins 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 dissection of PSII has shown that PsbL and PsbH are primarily required for functioning of Q A , the primary acceptor quinone in PSII (29,31), 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-complexes 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 protein repair cycle (37) and this process could be controlled by reversible phosphorylation of PsbH at its N-terminus. In its phosphorylated form PsbH can not 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 database search revealed that roughly 10% of the total entries were proteins with a molecular mass below 7 kDa. Several of these are single α-