A Critical Role for the Var2 FtsH Homologue of Arabidopsis thaliana in the Photosystem II Repair Cycle in Vivo *

Using a var2-2 mutant of Arabidopsis thaliana , which lacks a homologue of the zinc-metalloprotease, FtsH, we demonstrate that this protease is required for the efficient turnover of the D1 polypeptide of photosystem II and protection against photoinhibition in vivo . We show that var2-2 leaves are much more susceptible to light-induced photosystem II photoinhibition than wild-type leaves. Furthermore, the rate of photosystem II photoinhibition in untreated var2-2 leaves is equivalent to that of var2-2 and wild-type leaves, which have been treated with lincomycin, an inhibitor of the photosystem II repair cycle at the level of D1 synthesis. This is in contrast to untreated wild-type leaves, which show a much slower rate of photosystem II photoinhibition due to an efficient photosystem II repair cycle. The recovery of var2-2 leaves from photosystem II photoinhibition is also impaired relative to wild-type. Using Western blot analysis in the presence of lincomycin we show that the D1 polypeptide remains stable in leaves of the var2-2 mutant under photoinhibitory

The Photosystem II (PSII) 1 complex is a large protein-pigment assembly that catalyzes the light-dependent oxidation of water to molecular oxygen in chloroplasts and cyanobacteria. At the core of PSII lies the D1/D2 heterodimer, which binds the pigments and co-factors necessary for primary photochemistry (1). The D1 polypeptide is also important because of its high rate of turnover (2). This high turnover rate is related to the vulnerability of PSII to light, with D1 being the main target for photoinactivation and subsequent damage. An efficient repair cycle for D1 is therefore of paramount importance in oxygenic phototrophs. When the rate of photoinactivation and damage of D1 exceeds the capacity for repair, photoinhibition occurs, resulting in a decrease in the maximum efficiency of PSII photochemistry.
A key feature of the D1 repair cycle is the degradation of the damaged polypeptide. It is generally accepted that damaged D1 is initially cleaved at a site on the stromal loop between transmembrane helices D and E yielding a 23-kDa N-terminal fragment (3) and a 10-kDa C-terminal fragment (4). This cleavage step is believed to be initiated by structural changes within the D1 polypeptide (5), although the precise nature of the cleavage event remains unclear. One proposal is that the action of active oxygen species acts to cleave the D1 polypeptide during strong illumination (6). However, the temperature dependence of the process (7) and its sensitivity to protease inhibitors (8) indicates the involvement of enzymatic proteolysis by an unidentified protease. Following cleavage, the breakdown fragments of D1 are rapidly degraded and the PSII complex reassembles with the co-translational integration of a newly synthesized polypeptide.
Several proteases have been identified in photosynthetic organisms (reviewed in Ref. 9), and a number of studies have addressed the possibility that one or more may be involved in D1 turnover/assembly. One such protein is the stromal DegP2 protease, which has been shown to cleave D1 in in vitro assays (10). Another protease implicated in D1 turnover, following in vitro analysis, is the thylakoid FtsH homologue, FtsH1 (11). In Arabidopsis thaliana FtsH1 has been shown to degrade the 23-kDa breakdown product of D1 in isolated thylakoid membranes and purified PSII core complexes. Both studies are, however, limited in that the analysis was carried out in vitro, and the true in vivo role of both DegP2 and FtsH1 remains unclear.
The FtsH protease belongs to the AAA (ATPases associated with a variety of cellular activities) protein superfamily whose members are widely distributed among prokaryotes and eukaryotes. They are involved in a number of diverse cellular functions, including organelle biosynthesis, transcriptional regulation, membrane fusion, and proteolysis (reviewed in Ref. 12). All AAA family members are characterized by the presence of one or two highly conserved ATPase domains containing Walker A and B ATPase motifs. FtsH is further characterized by the presence of a zinc-metalloprotease motif (reviewed in Ref. 13).
The ftsh gene was first identified in Escherichia coli (14), where it encodes a 71-kDa polypeptide involved in various functions, including protein degradation (15,16). FtsH-related homologues have also been implicated in protein degradation in eukaryotic organelles, for example in yeast mitochondria (17) and, as mentioned, chloroplasts (11,18).
Recently, we have demonstrated a role for FtsH in photosynthesis following the disruption of a gene encoding an FtsH homologue in the cyanobacterium Synechocystis PCC 6803. The mutant strain, designated slr0228::⍀, was shown to be impaired in the maintenance of photosystem I (PSI), with levels decreased by 60% relative to wild-type (19). Furthermore, the slr0228::⍀ mutant shows enhanced PSII photoinhibition due to a decrease in the rate of D1 degradation. 2 The Slr0228 FtsH homologue of Synechocystis is closely related to Var2, a second homologue of FtsH that has recently been identified in Arabidopsis (20,21). Mutations in the var2 locus of Arabidopsis give rise to extensive leaf variegation due to impaired thylakoid membrane biogenesis. Green sectors in the var2 mutants, however, contain morphologically normal chloroplasts despite lacking the Var2 homologue. This phenomenon gave us the opportunity to assess the involvement of Var2 in D1 turnover using in vivo assays of PSII photoinhibition and D1 degradation in whole leaves.
In the present study we identify a unique conserved lumenal domain in Var2 that is not shared by Arabidopsis FtsH1 but is common to FtsH homologues from a diverse range of oxygenic phototrophs, including Slr0228 from Synechocystis. In addition we find that, in the absence of Var2, PSII photoinhibition is more extensive due to a critical role for the FtsH homologue in the primary cleavage of the D1 polypeptide. This report therefore identifies for the first time a chloroplast protease that is involved in vivo in the protection of plants from photoinhibition.

MATERIALS AND METHODS
Plant Material-Wild-type seeds of A. thaliana (L.) Heynh. cv. Columbia and the var2-2 mutant were grown under a long photoperiod (16 h light, 8 h dark). A growth irradiance of 100 mol m Ϫ2 s Ϫ1 was provided by fluorescent tubes in a Fi-totron growth chamber, model 600G3/THTL (Fisons, Loughborough, UK). Temperature was maintained at 20°C.
Light and Lincomycin Treatment of Leaves-Detached leaves were floated adaxial side up on water with the temperature regulated at 20°C. Irradiance of either 300 or 1800 mol m Ϫ2 s Ϫ1 was provided through fiber optics fed via a Schott lamp (Schott Glass Ltd., Stafford, UK) and filtered for heat using a Calflex C filter.
Chloroplast-encoded protein synthesis was blocked using lincomycin. Detached leaves were incubated with their petioles submersed in 1 mM solutions of lincomycin at an irradiance of 20 mol m Ϫ2 s Ϫ1 for 3 h prior to photoinhibitory light treatment. The temperature was maintained at 20°C during incubation.
Fluorescence Measurements-Room temperature chlorophyll fluorescence was measured using a PAM 101 fluorimeter (Heinz Waltz, Effeltrich, Germany). All measurements were made at 20°C with saturating CO 2 . Photoinhibition was assayed by calculating the ratio of maximum to variable fluorescence (Fv/Fm) as a measure of the maximal photochemical efficiency of PSII. In all experiments Fv/Fm was determined initially following dark adaptation overnight at ϳ5 mol m Ϫ2 s Ϫ1 . Following photoinhibitory light treatment, either in the presence or absence of lincomycin, leaf disks were dark-adapted for 15 min prior to Fv/Fm measurement to allow for the relaxation of rapidly reversible fluorescence quenching components.
The fast-relaxing, energy-dependent component of non-photochemical quenching, qE (22), and the photochemical quenching parameter qP (23) were calculated following 1 h of actinic illumination at either 300 or 1800 mol m Ϫ2 s Ϫ1 using the following equations, where Fm is the dark-adapted maximum fluorescence yield, Fms is the quenched level of maximum fluorescence following illumination for 1 h, FmЈ is the maximum fluorescence yield after 10-min dark relaxation subsequent to 1-h illumination, Fs is the steady-state fluorescence yield in the light following 1 h illumination, and FoЈ is the steady-state fluorescence yield in the dark following 1-h illumination. Fluorescence spectra at 77 K were recorded using a LS50 luminescence spectrometer with a liquid-nitrogen-cooled, low temperature housing (PerkinElmer Life Sciences, Gaithersburg, MD). Excitation was at 435 nm (5-nm bandwidth), for chlorophyll a absorption. Spectra were measured over 600 -750 nm (5-nm bandwidth) to reveal fluorescence emitted from PSII (at approximately 682 nm) and PSI (at approximately 732 nm). Approximately 20 mg of leaf tissue was ground to a powder in liquid nitrogen, then mixed to a homogenous frozen suspension with 5 ml of cold grinding buffer (0.33 M sorbitol, 5 mM MgCl 2 , 5 mM EDTA, 10 mM HEPES, pH 7.6) to ensure that there was no fluorescence re-absorption. The diluted plant tissue was then stored in liquid nitrogen in 4-mm silica tubes until used for recording spectra. Spectra were normalized to PSII fluorescence to allow comparison of mutant and wild-type plants.
Other Methods-Chlorophyll was extracted from leaf disks by grinding in 80% (v/v) acetone or from thylakoids by diluting in 80% acetone. Following removal of leaf debris by centrifugation (1500 ϫ g, 5 min), chlorophyll content was determined according to Porra et al. (24). For the preparation of thylakoids, leaves were homogenized in semi-frozen grinding media (0.33 M sorbitol, 5 mM MgCl 2 , 5 mM EDTA, 10 mM HEPES, pH 7.6). The homogenized solution was filtered through four layers of muslin followed by two layers of muslin and one layer of cotton wool. The filtrate was centrifuged at 4000 ϫ g for 10 min. The pellet was resuspended in a small volume of wash buffer (0.33 M sorbitol, 1 mM MgCl 2 , 1 mM EDTA, 50 mM HEPES, pH 7.6) before being centrifuged at 4000 ϫ g for 10 min. The pellet was then osmotically shocked by resuspension in 5 mM MgCl 2 for at least 30 s before the addition of an equal volume of 0.66 M sorbitol. Thylakoids were either used fresh or immediately frozen in liquid nitrogen.
SDS-PAGE was carried out essentially according to Laemmli (25), including 6 M urea in both the stacking and resolving gels. Solubilized thylakoids (1 g of Chl equivalent or 30 g of protein) were separated on a 15% (w/v) acrylamide gel and blotted onto Hybond C nitrocellulose membrane (Amersham Biosciences, Inc., UK). D1 and D2 antisera were raised against synthetic peptides and were specific for the C terminus (26). PsbS antisera were raised against purified protein from spinach (27). DegP2 antisera were raised against His-tagged DegP2 from A. thaliana overexpressed in Escherichia coli (10). Bands were quantitated using TotalLab (NonLinear Dynamics Ltd.) software.

In Vivo Assays of Light-induced PSII Photoinhibition-
The chlorophyll a fluorescence parameter Fv/Fm measures the maximum efficiency of PSII photochemistry. It correlates with both the number of functional PSII reaction centers (28) and the quantum yield of light-induced O 2 evolution (29) and has, therefore, been extensively used as an in vivo measure of PSII photoinhibition. To test whether the var2-2 mutant is affected in terms of PSII photoinhibition we compared Fv/Fm in vivo, in detached leaves of wild-type Arabidopsis and the FtsH mutant var2-2. Fv/Fm values were measured during photoinhibitory irradiance (1800 mol m Ϫ2 s Ϫ1 ) in the presence or absence of lincomycin, which inhibits D1 synthesis and hence blocks the repair of damaged PSII (Fig. 1). In the absence of lincomycin, WT leaves showed an initial decrease in Fv/Fm to about 50% of the overnight dark-adapted values. After about 2 h there was no further decrease in Fv/Fm. In the presence of lincomycin the decrease in Fv/Fm in WT leaves was more rapid and continued until Fv/Fm values approached zero. Because D1 synthesis is inhibited in the presence of lincomycin, the rate of photoinhibition, as measured by the decrease in Fv/Fm, reflects the rate of PSII photoinactivation. When Fv/Fm was monitored in the var2-2 mutant in the presence of lincomycin, during the same photoinhibitory light treatment, the decrease was similar to that of wild-type leaves in the presence of lincomycin, suggesting that both wild-type and var2-2 leaves have the same rate of PSII photoinactivation. In addition, the decrease in Fv/Fm values in var2-2 leaves during photoinhibition in the absence of lincomycin also proceeded with the same rapid kinetics as lincomycin-treated WT and var2-2 leaves, strongly suggesting that the D1 repair cycle is impaired in var2-2.
To further characterize the susceptibility of var2-2 leaves to PSII photoinhibition and to assess the capacity for recovery from photoinhibition, Fv/Fm values were monitored in wildtype and var2-2 leaves following treatment with both moderate (300 mol m Ϫ2 s Ϫ1 ) and high (1800 mol m Ϫ2 s Ϫ1 ) irradiance, and during the subsequent dark recovery period. As shown in Fig. 2, following 1 h of illumination at 300 mol m Ϫ2 s Ϫ1 the wild-type leaves maintained the same high values of Fv/Fm recorded after overnight dark adaptation (time Ϫ60 min), whereas var2-2 leaves showed a decrease in Fv/Fm from 0.695 to below 0.5. One hour of illumination at 1800 mol m Ϫ2 s Ϫ1 resulted in a decrease in Fv/Fm in wild-type leaves from 0.8 to just below 0.6, indicating photoinhibition under these high light conditions as expected. However, var2-2 levels decreased from 0.73 to 0.32 during the same period of irradiance clearly demonstrating enhanced photoinhibition in var2-2 leaves relative to wild-type. In addition, the recovery from photoinhibition was much slower in var2-2 leaves when compared with wild-type. Fv/Fm values, measured in wild-type leaves darkadapted overnight (unattached during dark adaptation), have high values of above 0.8 (time 0). var2-2 leave, on the other hand, failed to reach the high values of Fv/Fm expected for leaves dark-adapted for this period of time. Furthermore, following 1-h irradiance at 1800 mol m Ϫ2 s Ϫ1 WT leaves showed clear increases in Fv/Fm during the first 6 h of the subsequent dark recovery period. After 20 h the wild-type leaves had almost reached the same high values of Fv/Fm recorded after overnight dark adaptation. In contrast var2-2 leaves showed no sign of recovery in the first 6 h following illumination at either 300 or 1800 mol m Ϫ2 s Ϫ1 , and although there was some recovery after 20 h this failed to restore the Fv/Fm values to those measured following overnight dark adaptation.
Photosynthetic Characteristics-We have compared a number of photosynthetic characteristics that may potentially contribute to PSII photoinhibition in var2-2 and wild-type leaves. The ratio of chlorophyll a to chlorophyll b (Chl a/b) has been shown to correlate well with both the size of the PSII lightharvesting antenna and the level of thylakoid membrane stacking (30). Furthermore, the susceptibility of PSII to photoinhibition has been correlated with Chl a/b (31). Table I shows values of Chl a/b for var2-2 and WT leaves of Arabidopsis. Both values are equivalent and are consistent with a large PSII antenna following growth at low irradiance.
Although the Chl a/b ratio is equivalent in both var2-2 and wild-type leaves, it is possible that light energy absorbed by PSII can be redistributed to PSI following migration of LHCII, the PSII light-harvesting antenna (32). Low temperature (77 K) fluorescence spectra were recorded to examine the distribution of excitation energy in var2-2 and wild-type leaves of Arabidopsis (Fig. 3). These indicate that the ratio of PSII emission (688 -699 nm) and PSI emission (733-734 nm) are similar for both WT and mutant, suggesting an equivalent distribution of excitation energy.
The capacity for xanthophyll cycle-related, energy-dependent dissipation of excess absorbed energy is termed qE (reviewed in Ref. 33), Table I shows qE values for both wild-type and var2-2 leaves following exposure to both moderate (300 mol m Ϫ2 s Ϫ1 ) and high (1800 mol m Ϫ2 s Ϫ1 ) irradiance. Following exposure to both sets of irradiance the capacity for qE is approximately half the wild-type levels in var2-2. In addition, the capacity for photochemical quenching of absorbed light energy is also lower than that of wild-type in var2-2 following exposure to the same two irradiance (Table I).
In Vivo Analysis of D1 Degradation-The more commonly used approach to study D1 turnover is pulse labeling with [ 35 S]methionine. We initially attempted to carry out such studies but found that var2-2 mutant leaves labeled very much more slowly than the wild-type and that a greatly extended (Ͼ4ϫ) labeling period was needed to obtain D1 signals comparable to the wild-type. This in itself is consistent with impaired D1 turnover. Mutant leaves are sickly, and following incubation periods sufficient to label D1, the leaf material has degenerated to the point where a chase is no longer technically possible. Therefore, we elected to adopt a Western blotting approach. The ability to degrade the D1 polypeptide in vivo following light-induced damage was assayed in wild-type and var2-2 leaves using Western blot analysis in the absence and presence of lincomycin. Because D1 synthesis is inhibited following lincomycin treatment the degradation of existing D1 results in a decrease in polypeptide content relative to untreated leaves. As shown in Fig. 4A the D1 polypeptide was decreased by 32% relative to untreated leaves in wild-type leaves following 3-h treatment with lincomycin at low irradiance (20 mol m Ϫ2 s Ϫ1 ). After 2-h subsequent exposure to photoinhibitory irradiance, the remaining D1 polypeptide was reduced by 66% in wild-type leaves. In contrast, the D1 polypeptide showed no decrease in the var2-2 mutant following exposure to photoinhibitory irradiance (1800 mol m Ϫ2 s Ϫ1 ) in the presence of lincomycin, despite a 28% loss of D1 following the initial treatment with lincomycin at low light. When the same experiment was performed in wild-type leaves in the absence of lincomycin treatment (Fig. 4B) there was no loss of the D1 polypeptide throughout, suggesting that D1 degradation is matched by synthesis, thereby demonstrating the efficacy of the lincomycin treatment.
Western blot analysis using antibody specific to the other PSII core polypeptide, D2, was also carried out following the same treatment of leaves as described for D1. Again there were losses in the D2 polypeptide in wild-type leaves following lincomycin treatment at low light and more dramatically following exposure to photoinhibitory irradiance. These losses are not, however, as marked as those observed for the D1 polypeptide. As with D1 the D2 polypeptide remained stable following exposure of var2-2 leaves to photoinhibitory irradiance despite an initial loss following lincomycin treatment at low light.
To demonstrate that the turnover of the core PSII polypeptides represents specific degradation and not just destabilization of the photosynthetic apparatus, the content of the minor PSII polypeptide, PsbS, was assayed following lincomycin and photoinhibitory light treatment. As shown in Fig. 4A the PsbS polypeptide content remains unchanged throughout in both wild-type and var2-2 leaves. However, it is interesting to note that the PsbS levels in untreated var2-2 leaves are lower than those of wild-type, and this observation may account for the lower values of qE for the mutant as has been shown for a psbS mutant of Arabidopsis (34). The thylakoid protease DegP2 has already been implicated in D1 turnover (10). To establish whether the effect of the var2-2 mutation was indirectly affecting D1 turnover via a reduction of DegP2 a Western blot was carried out on thylakoid proteins from the wild-type and var2-2 mutant with anti-DegP2 antibodies. No reduction in the abundance of DegP2 was observed in the var2-2 mutant (Fig. 4C).
Sequence Alignments of a Conserved FtsH Lumenal Domain-To investigate the relationship between FtsH homologues from photosynthetic and non-photosynthetic organisms the amino acid sequences from Arabidopsis FtsH1 and Var2 were aligned with FtsH sequences from Synechocystis PCC 6803 and the E. coli FtsH using the MACAW program, which employs the segment pair overlap method to detect small regions of similarity between sequences. This alignment revealed that Var2 and the Slr0228 FtsH homologue from Synechocystis contain a conserved 81-amino acid sequence feature that is not present in FtsH1, E. coli FtsH, or any other Synechocystis FtsH homologue. Further analysis of Slr0228 using the TMHMM (version 2.0) program to predict transmembrane helices revealed that this 81-amino acid feature lay between two very strongly predicted transmembrane helices running from residues 15-37 and 115-137. Given the predicted orientation of these helices and assuming that Slr0228 is located in the thylakoid membrane, the conserved 81-amino acid feature would constitute a lumenal domain. Var2 is already known to be localized to the thylakoid membrane (20). When the sequence of the putative conserved 81-amino acid lumenal domain from Slr0228 was used to do a protein-protein BLAST search of the NCBI non-redundant data base, a number of sequences were returned, all of which shared extensive similarity (Fig. 5). Interestingly, all of the sequences were exclusively FtsH homologues from oxygenic phototrophs. We propose that this 81amino acid conserved lumenal (CL) domain represents a key identifies a sub-family of FtsH homologues whose members are restricted to oxygenic photosynthetic organisms. Two further  3. 77 K chlorophyll a fluorescence emission spectra for wild-type and var2-2 leaves of A. thaliana. Spectra for wild-type leaves (solid line) and var2-2 leaves (dashed line) were recorded using homogenized and diluted leaf tissue to avoid fluorescence re-absorption. Chlorophyll was excited at 435 nm. Data were normalized to the PSII emission peak at 682 nm. The figure is representative of at least three spectra.

FIG. 4. Western blot analysis of PSII polypeptides in lincomycin and high light-treated leaves of wild-type A. thaliana and the var2-2 mutant and a comparison of DegP2 abundance. A,
representative Western blots of the PSII core polypeptides, D1 (top) and D2 (middle) and the minor PSII polypeptide PsbS (bottom). Thylakoids taken from untreated leaves were extracted immediately upon removal of leaf tissue from cabinet grown plants. Lincomycin-treated leaves (ϩLinc) were floated in 1 mM lincomycin solution at 20 mol m Ϫ2 s Ϫ1 for 3 h prior to thylakoid preparation. Lincomycin and high light-treated leaves (ϩLinc/HL) were floated in 1 mM lincomycin solution at 20 mol m Ϫ2 s Ϫ1 for 3 h followed by 2-h exposure to 1800 mol m Ϫ2 s Ϫ1 irradiance prior to thylakoid preparation. B, representative Western blot of the PSII core polypeptide, D1. Leaf treatments are as for those described in A, but in the absence of lincomycin. All gels were loaded on an equal chlorophyll basis (1 g of chlorophyll per lane). C, thylakoid protein from wild-type and var2-2 mutant leaves were loaded on an equal protein basis (30 g of protein per lane) and probed with anti-DegP2 antibody.
Var2 FtsH Homologue Involved in Photosystem II Repair members of this sub-family are encoded on chromosomes 1 and 5 of A. thaliana indicating the existence of a multigene family.

DISCUSSION
As part of a PSII repair cycle, damaged D1 polypeptides may be rapidly degraded and replaced by newly synthesized polypeptides (reviewed in Ref. 35). This repair cycle is of great importance to oxygenic phototrophs because when the rate of photoinactivation and damage of D1 exceeds the capacity for repair, photoinhibition occurs, resulting in a decrease in the maximum efficiency of PSII photochemistry. This may ultimately affect the viability of the whole organism. Despite intensive research, the mechanism of D1 degradation has remained largely uncharacterized. Analysis in vitro indicates that a stromal DegP type protease is capable of performing the initial cleavage of D1 (10); however, the role of DegP2 in D1 turnover in vivo remains unclear. In contrast, the in vivo analysis of photoinhibition and D1 degradation presented here suggests that a thylakoid FtsH homologue is required for this initial cleavage step. Using the Arabidopsis var2-2 mutant, which has a mis-sense mutation at the end of the second transmembrane domain and fails to accumulate the Var2 FtsH homologue in the membrane, we have shown that PSII is more susceptible to photoinhibition in the absence of this protease (Fig. 2). Treatment with either moderate or high irradiance resulted in considerably greater PSII photoinhibition (decreased Fv/Fm) in var2-2 leaves compared with wild-type. Indeed var2-2 showed high levels of PSII photoinhibition following exposure to an irradiance that failed to induce photoinhibition in wild-type leaves.
A number of factors may account for this enhanced susceptibility of PSII to photoinhibition. These include greater PSII antenna size and thylakoid membrane stacking (31), decreased capacity for energy-dependent quenching of absorbed photons (36) and enhanced PSII excitation pressure (37), all of which can be measured as Chl a/b ratio, qE and qP, respectively. However, the Chl a/b ratios of both wild-type and var2-2 leaves are essentially the same (Table I), and, although var2-2 leaves do show decreased levels of qE formation and lower values of qP following light treatment (Table I), the rate of PSII photoinactivation in the presence of lincomycin is the same as wildtype (Fig. 1). We suggest therefore that an impaired PSII repair cycle forms the basis of the enhanced sensitivity of PSII to photoinhibition in the var2-2 mutant. Dark relaxation of Fv/Fm following light treatment is slower in var2-2 leaves than wild-type (Fig. 2). In addition, the kinetics of formation of photoinhibition in untreated var2-2 leaves are identical to wildtype leaves, which have been treated with lincomycin and therefore are unable to carry out D1 repair (Fig. 1). Taken together, these results strongly indicate that the D1 repair cycle is diminished in the absence of Var2. Western blot analysis of the D1 polypeptide following lincomycin and high-light treatment provides direct evidence for decreased turnover of D1 in the var2-2 mutant (Fig. 4). The stability of the 32-kDa polypeptide in var2-2 suggests that the Var2 FtsH homologue may be involved in the initial cleavage step of photo-damaged D1 polypeptides. Furthermore, the PSII core polypeptide, D2, which is also known to undergo damage and repair under photoinhibitory irradiance (38), also remains stable in the var2-2 mutant following treatment with lincomycin and high light (Fig. 4). This is in contrast to wild-type leaves, which show a marked decrease in D2 polypeptide content. The exact nature of the involvement of FtsH in the repair cycle of PSII is unclear, but it seems likely that the protease is directly involved in D1 turnover in vivo. The possibility of an indirect effect via DegP2 has been excluded (Fig. 4C). Other possibilities for the involvement of Var2 in D1 turnover exist, including a possible role in determining the phosphorylation state of D1, because it has been proposed that phosphorylated D1 remains stable (39). Var2 may also have a direct role in D2 turnover, although it is possible that the degradation of D2 requires prior degradation of D1 and that another thylakoid protease may be involved in D2 turnover. Such a suggestion relating to the connectivity of regulation between D1 and D2 has previously been made (40). Indeed, the possibility that a number of proteases, particularly FtsH proteases, may also perform D1 degradation in vivo cannot be ruled out. Photoinactivation and damage of the D1 polypeptide are known to proceed by at least two separate mechanisms, namely donor and acceptor side photoinhibition (reviewed in Ref. 41), which give rise to distinct breakdown products. Data base analysis suggests that A. thaliana contains at least five homologues of FtsH. An involvement of these proteases during D1 degradation via multiple pathways is likely. Such an overlap in function of the various FtsH homologues, and possibly other thylakoid proteases, could account for our observation that both the D1 and D2 polypeptides undergo some degradation in the presence of lincomycin at low light (Fig. 4A).
Our data showing that FtsH homologues are required for the initial cleavage of the D1 polypeptide are strengthened by the finding that the var2-2 phenotype with respect to D1 turnover is also mirrored in the slr0228::⍀ mutant of Synechocystis, which lacks one of four FtsH homologues encoded in the genome. 2 The sequence analysis presented here also reveals that Var2 and the Slr0228 FtsH homologue share another feature in common; the conserved lumenal domain (Fig. 5). This domain is exclusive to FtsH-like proteins from oxygenic phototrophs. We propose that this domain identifies a sub-family of FtsH homologues restricted to and essential for oxygenic photosynthetic organisms. The discovery of this domain may represent a significant step forward in our understanding of the specificity and regulation of FtsH homologues.
In conclusion we have demonstrated that the Var2 FtsH homologue of Arabidopsis is required for the protection of PSII from photoinhibition in vivo via a role in the efficient turnover of the PSII core polypeptides, D1 and D2.