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

A Critical Role for the Var2 FtsH Homologue of Arabidopsis thaliana in the Photosystem II Repair... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 3, Issue of January 18, pp. 2006 –2011, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Critical Role for the Var2 FtsH Homologue of Arabidopsis thaliana in the Photosystem II Repair Cycle in Vivo* Received for publication, June 25, 2001, and in revised form, November 1, 2001 Published, JBC Papers in Press, November 20, 2001, DOI 10.1074/jbc.M105878200 Shaun Bailey‡, Elinor Thompson§, Peter J. Nixon¶, Peter Horton, Conrad W. Mullineaux§, Colin Robinson‡, and Nicholas H. Mann‡** From the ‡Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, the §Department of Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, the ¶Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, and the Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN United Kingdom Using a var2-2 mutant of Arabidopsis thaliana, which phototrophs. When the rate of photoinactivation and damage of lacks a homologue of the zinc-metalloprotease, FtsH, we D1 exceeds the capacity for repair, photoinhibition occurs, re- demonstrate that this protease is required for the effi- sulting in a decrease in the maximum efficiency of PSII cient turnover of the D1 polypeptide of photosystem II photochemistry. and protection against photoinhibition in vivo. We show A key feature of the D1 repair cycle is the degradation of the that var2-2 leaves are much more susceptible to light- damaged polypeptide. It is generally accepted that damaged D1 induced photosystem II photoinhibition than wild-type is initially cleaved at a site on the stromal loop between trans- leaves. Furthermore, the rate of photosystem II photo- membrane helices D and E yielding a 23-kDa N-terminal frag- inhibition in untreated var2-2 leaves is equivalent to ment (3) and a 10-kDa C-terminal fragment (4). This cleavage that of var2-2 and wild-type leaves, which have been step is believed to be initiated by structural changes within the treated with lincomycin, an inhibitor of the photosys- D1 polypeptide (5), although the precise nature of the cleavage tem II repair cycle at the level of D1 synthesis. This is in event remains unclear. One proposal is that the action of active contrast to untreated wild-type leaves, which show a oxygen species acts to cleave the D1 polypeptide during strong much slower rate of photosystem II photoinhibition due illumination (6). However, the temperature dependence of the to an efficient photosystem II repair cycle. The recovery process (7) and its sensitivity to protease inhibitors (8) indi- of var2-2 leaves from photosystem II photoinhibition is cates the involvement of enzymatic proteolysis by an uniden- also impaired relative to wild-type. Using Western blot tified protease. Following cleavage, the breakdown fragments analysis in the presence of lincomycin we show that the of D1 are rapidly degraded and the PSII complex reassembles D1 polypeptide remains stable in leaves of the var2-2 with the co-translational integration of a newly synthesized mutant under photoinhibitory conditions that lead to D1 degradation in wild-type leaves and that the abun- polypeptide. dance of DegP2 is not affected by the var2-2 mutation. Several proteases have been identified in photosynthetic or- We conclude, therefore, that the Var2 FtsH homologue is ganisms (reviewed in Ref. 9), and a number of studies have required for the cleavage of the D1 polypeptide in vivo. addressed the possibility that one or more may be involved in In addition, we identify a conserved lumenal domain in D1 turnover/assembly. One such protein is the stromal DegP2 Var2 that is unique to FtsH homologues from oxygenic protease, which has been shown to cleave D1 in in vitro assays phototrophs. (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 The Photosystem II (PSII) complex is a large protein-pig- 23-kDa breakdown product of D1 in isolated thylakoid mem- ment assembly that catalyzes the light-dependent oxidation of branes and purified PSII core complexes. Both studies are, water to molecular oxygen in chloroplasts and cyanobacteria. however, limited in that the analysis was carried out in vitro, At the core of PSII lies the D1/D2 heterodimer, which binds the and the true in vivo role of both DegP2 and FtsH1 remains pigments and co-factors necessary for primary photochemistry unclear. (1). The D1 polypeptide is also important because of its high The FtsH protease belongs to the AAA (ATPases associated rate of turnover (2). This high turnover rate is related to the with a variety of cellular activities) protein superfamily whose vulnerability of PSII to light, with D1 being the main target for members are widely distributed among prokaryotes and eu- photoinactivation and subsequent damage. An efficient repair karyotes. They are involved in a number of diverse cellular cycle for D1 is therefore of paramount importance in oxygenic functions, including organelle biosynthesis, transcriptional regulation, membrane fusion, and proteolysis (reviewed in Ref. 12). All AAA family members are characterized by the presence * This work was supported by a grant from the Biotechnology and Biological Sciences and Research Council (to N. H. M.). The costs of of one or two highly conserved ATPase domains containing publication of this article were defrayed in part by the payment of page Walker A and B ATPase motifs. FtsH is further characterized charges. This article must therefore be hereby marked “advertisement” by the presence of a zinc-metalloprotease motif (reviewed in in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Ref. 13). ** To whom correspondence should be addressed: Tel.: 44-24-7652- 3526, Fax: 44-24-7652-3701; E-mail: [email protected]. The ftsh gene was first identified in Escherichia coli (14), The abbreviations used are: PSII, photosystem II; Chl a/b, the ratio where it encodes a 71-kDa polypeptide involved in various of chlorophyll a to chlorophyll b; qE, energy dependent quenching of functions, including protein degradation (15, 16). FtsH-related chlorophyll a fluorescence; qP photochemical quenching of chlorophyll a homologues have also been implicated in protein degradation fluorescence; Fv/Fm, the ratio of variable to maximal chlorophyll a fluorescence; CL, conserved lumenal domain. in eukaryotic organelles, for example in yeast mitochondria 2006 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Var2 FtsH Homologue Involved in Photosystem II Repair 2007 in the light following 1 h illumination, and Fo is the steady-state (17) and, as mentioned, chloroplasts (11, 18). fluorescence yield in the dark following 1-h illumination. Recently, we have demonstrated a role for FtsH in photosyn- Fluorescence spectra at 77 K were recorded using a LS50 lumines- thesis following the disruption of a gene encoding an FtsH cence spectrometer with a liquid-nitrogen-cooled, low temperature homologue in the cyanobacterium Synechocystis PCC 6803. housing (PerkinElmer Life Sciences, Gaithersburg, MD). Excitation The mutant strain, designated slr0228::, was shown to be was at 435 nm (5-nm bandwidth), for chlorophyll a absorption. Spectra impaired in the maintenance of photosystem I (PSI), with lev- were measured over 600 –750 nm (5-nm bandwidth) to reveal fluores- cence emitted from PSII (at approximately 682 nm) and PSI (at approx- els decreased by 60% relative to wild-type (19). Furthermore, imately 732 nm). Approximately 20 mg of leaf tissue was ground to a the slr0228:: mutant shows enhanced PSII photoinhibition powder in liquid nitrogen, then mixed to a homogenous frozen suspen- due to a decrease in the rate of D1 degradation. The Slr0228 sion with 5 ml of cold grinding buffer (0.33 M sorbitol, 5 mM MgCl ,5mM FtsH homologue of Synechocystis is closely related to Var2, a EDTA, 10 mM HEPES, pH 7.6) to ensure that there was no fluorescence second homologue of FtsH that has recently been identified in re-absorption. The diluted plant tissue was then stored in liquid nitro- Arabidopsis (20, 21). Mutations in the var2 locus of Arabidopsis gen in 4-mm silica tubes until used for recording spectra. Spectra were normalized to PSII fluorescence to allow comparison of mutant and give rise to extensive leaf variegation due to impaired thylakoid wild-type plants. membrane biogenesis. Green sectors in the var2 mutants, how- Other Methods—Chlorophyll was extracted from leaf disks by grind- ever, contain morphologically normal chloroplasts despite lack- ing in 80% (v/v) acetone or from thylakoids by diluting in 80% acetone. ing the Var2 homologue. This phenomenon gave us the oppor- Following removal of leaf debris by centrifugation (1500  g, 5 min), tunity to assess the involvement of Var2 in D1 turnover using chlorophyll content was determined according to Porra et al. (24). For in vivo assays of PSII photoinhibition and D1 degradation in the preparation of thylakoids, leaves were homogenized in semi-frozen grinding media (0.33 M sorbitol, 5 mM MgCl ,5mM EDTA, 10 mM whole leaves. 2 HEPES, pH 7.6). The homogenized solution was filtered through four In the present study we identify a unique conserved lumenal layers of muslin followed by two layers of muslin and one layer of cotton domain in Var2 that is not shared by Arabidopsis FtsH1 but is wool. The filtrate was centrifuged at 4000  g for 10 min. The pellet was common to FtsH homologues from a diverse range of oxygenic resuspended in a small volume of wash buffer (0.33 M sorbitol, 1 mM phototrophs, including Slr0228 from Synechocystis. In addition MgCl ,1mM EDTA, 50 mM HEPES, pH 7.6) before being centrifuged at we find that, in the absence of Var2, PSII photoinhibition is 4000  g for 10 min. The pellet was then osmotically shocked by M MgCl resuspension in 5 m for at least 30 s before the addition of an more extensive due to a critical role for the FtsH homologue in 2 M sorbitol. Thylakoids were either used fresh or equal volume of 0.66 the primary cleavage of the D1 polypeptide. This report there- immediately frozen in liquid nitrogen. fore identifies for the first time a chloroplast protease that is SDS-PAGE was carried out essentially according to Laemmli (25), involved in vivo in the protection of plants from photo- including 6 M urea in both the stacking and resolving gels. Solubilized inhibition. 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 MATERIALS AND METHODS membrane (Amersham Biosciences, Inc., UK). D1 and D2 antisera were Plant Material—Wild-type seeds of A. thaliana (L.) Heynh. cv. Co- raised against synthetic peptides and were specific for the C terminus lumbia and the var2-2 mutant were grown under a long photoperiod (16 (26). PsbS antisera were raised against purified protein from spinach 2 1 h light, 8 h dark). A growth irradiance of 100 mol m s was (27). DegP2 antisera were raised against His-tagged DegP2 from A. provided by fluorescent tubes in a Fi-totron growth chamber, model thaliana overexpressed in Escherichia coli (10). Bands were quanti- 600G3/THTL (Fisons, Loughborough, UK). Temperature was main- tated using TotalLab (NonLinear Dynamics Ltd.) software. tained at 20 °C. RESULTS Light and Lincomycin Treatment of Leaves—Detached leaves were floated adaxial side up on water with the temperature regulated at In Vivo Assays of Light-induced PSII Photoinhibition—The 2 1 20 °C. Irradiance of either 300 or 1800 mol m s was provided chlorophyll a fluorescence parameter Fv/Fm measures the through fiber optics fed via a Schott lamp (Schott Glass Ltd., Stafford, maximum efficiency of PSII photochemistry. It correlates with UK) and filtered for heat using a Calflex C filter. both the number of functional PSII reaction centers (28) and Chloroplast-encoded protein synthesis was blocked using lincomycin. the quantum yield of light-induced O Detached leaves were incubated with their petioles submersed in 1 mM evolution (29) and has, 2 1 solutions of lincomycin at an irradiance of 20 mol m s for 3 h prior therefore, been extensively used as an in vivo measure of PSII to photoinhibitory light treatment. The temperature was maintained at photoinhibition. To test whether the var2-2 mutant is affected 20 °C during incubation. in terms of PSII photoinhibition we compared Fv/Fm in vivo,in Fluorescence Measurements—Room temperature chlorophyll fluores- detached leaves of wild-type Arabidopsis and the FtsH mutant cence was measured using a PAM 101 fluorimeter (Heinz Waltz, Effel- var2-2. Fv/Fm values were measured during photoinhibitory trich, Germany). All measurements were made at 20 °C with saturating 2 1 irradiance (1800 mol m CO . Photoinhibition was assayed by calculating the ratio of maximum s ) in the presence or absence of to variable fluorescence (Fv/Fm) as a measure of the maximal photo- lincomycin, which inhibits D1 synthesis and hence blocks the chemical efficiency of PSII. In all experiments Fv/Fm was determined repair of damaged PSII (Fig. 1). In the absence of lincomycin, 2 1 initially following dark adaptation overnight at 5 mol m s . WT leaves showed an initial decrease in Fv/Fm to about 50% of Following photoinhibitory light treatment, either in the presence or the overnight dark-adapted values. After about 2 h there was absence of lincomycin, leaf disks were dark-adapted for 15 min prior to no further decrease in Fv/Fm. In the presence of lincomycin the Fv/Fm measurement to allow for the relaxation of rapidly reversible decrease in Fv/Fm in WT leaves was more rapid and continued fluorescence quenching components. The fast-relaxing, energy-dependent component of non-photochemi- until Fv/Fm values approached zero. Because D1 synthesis is cal quenching, qE (22), and the photochemical quenching parameter qP inhibited in the presence of lincomycin, the rate of photoinhi- (23) were calculated following1hof actinic illumination at either 300 or bition, as measured by the decrease in Fv/Fm, reflects the rate 2 1 1800 mol m s using the following equations, of PSII photoinactivation. When Fv/Fm was monitored in the qE  Fm/Fms  Fm/Fm (Eq. 1) var2-2 mutant in the presence of lincomycin, during the same photoinhibitory light treatment, the decrease was similar to qP  (FmFs)(Fm  Fo) (Eq. 2) that of wild-type leaves in the presence of lincomycin, suggest- ing that both wild-type and var2-2 leaves have the same rate of where Fm is the dark-adapted maximum fluorescence yield, Fms is the quenched level of maximum fluorescence following illumination for 1 h, PSII photoinactivation. In addition, the decrease in Fv/Fm Fm is the maximum fluorescence yield after 10-min dark relaxation values in var2-2 leaves during photoinhibition in the absence of subsequent to 1-h illumination, Fs is the steady-state fluorescence yield lincomycin also proceeded with the same rapid kinetics as lincomycin-treated WT and var2-2 leaves, strongly suggesting P. Silva and P. J. Nixon, personal communication. . that the D1 repair cycle is impaired in var2-2. 2008 Var2 FtsH Homologue Involved in Photosystem II Repair FIG.2. Maximum photochemical efficiency of PSII following FIG.1. Maximum photochemical efficiency of PSII following light treatment and subsequent dark recovery for wild-type and high light treatment in the presence of lincomycin for wild-type var2-2 leaves of Arabidopsis thaliana. Fv/Fm values are for wild- and var2-2 leaves of A. thaliana. Fv/Fm values are for wild-type type leaves (circles) and var2-2 leaves (squares) following 1-h exposure 2 1 2 1 leaves (circles) and var2-2 leaves (squares) during exposure to high to either 300 mol m s (filled circles) or 1800 mol m s (open 2 1 irradiance (1800 mol m s ) in the presence (open symbols)or circles). Time 60 min represents over-night dark adaptation, prior to absence (closed symbols) of lincomycin. Time 0 represents overnight light treatment; time 0 represents the end of the 1-h light treatment dark adaptation prior to light and lincomycin treatment (mean S.E., (mean S.E., n 5). 5). Although the Chl a/b ratio is equivalent in both var2-2 and To further characterize the susceptibility of var2-2 leaves to wild-type leaves, it is possible that light energy absorbed by PSII photoinhibition and to assess the capacity for recovery PSII can be redistributed to PSI following migration of LHCII, from photoinhibition, Fv/Fm values were monitored in wild- the PSII light-harvesting antenna (32). Low temperature (77 type and var2-2 leaves following treatment with both moderate K) fluorescence spectra were recorded to examine the distribu- 2 1 2 1 (300 mol m s ) and high (1800 mol m s ) irradiance, tion of excitation energy in var2-2 and wild-type leaves of and during the subsequent dark recovery period. As shown in Arabidopsis (Fig. 3). These indicate that the ratio of PSII emis- 2 1 Fig. 2, following1hof illumination at 300 mol m s the sion (688 – 699 nm) and PSI emission (733–734 nm) are similar wild-type leaves maintained the same high values of Fv/Fm for both WT and mutant, suggesting an equivalent distribution recorded after overnight dark adaptation (time 60 min), of excitation energy. whereas var2-2 leaves showed a decrease in Fv/Fm from 0.695 The capacity for xanthophyll cycle-related, energy-depend- 2 1 to below 0.5. One hour of illumination at 1800 mol m s ent dissipation of excess absorbed energy is termed qE (re- resulted in a decrease in Fv/Fm in wild-type leaves from 0.8 to viewed in Ref. 33), Table I shows qE values for both wild-type just below 0.6, indicating photoinhibition under these high and var2-2 leaves following exposure to both moderate (300 2 1 2 1 light conditions as expected. However, var2-2 levels decreased mol m s ) and high (1800 mol m s ) irradiance. Fol- from 0.73 to 0.32 during the same period of irradiance clearly lowing exposure to both sets of irradiance the capacity for qE is demonstrating enhanced photoinhibition in var2-2 leaves rel- approximately half the wild-type levels in var2-2. In addition, ative to wild-type. In addition, the recovery from photoinhibi- the capacity for photochemical quenching of absorbed light tion was much slower in var2-2 leaves when compared with energy is also lower than that of wild-type in var2-2 following wild-type. Fv/Fm values, measured in wild-type leaves dark- exposure to the same two irradiance (Table I). adapted overnight (unattached during dark adaptation), have In Vivo Analysis of D1 Degradation—The more commonly high values of above 0.8 (time 0). var2-2 leave, on the other used approach to study D1 turnover is pulse labeling with hand, failed to reach the high values of Fv/Fm expected for [ S]methionine. We initially attempted to carry out such stud- leaves dark-adapted for this period of time. Furthermore, fol- ies but found that var2-2 mutant leaves labeled very much 2 1 lowing 1-h irradiance at 1800 mol m s WT leaves showed more slowly than the wild-type and that a greatly extended clear increases in Fv/Fm during the first6hofthe subsequent (4) labeling period was needed to obtain D1 signals compa- dark recovery period. After 20 h the wild-type leaves had al- rable to the wild-type. This in itself is consistent with impaired most reached the same high values of Fv/Fm recorded after D1 turnover. Mutant leaves are sickly, and following incuba- overnight dark adaptation. In contrast var2-2 leaves showed no tion periods sufficient to label D1, the leaf material has degen- sign of recovery in the first 6 h following illumination at either erated to the point where a chase is no longer technically 2 1 300 or 1800 mol m s , and although there was some possible. Therefore, we elected to adopt a Western blotting recovery after 20 h this failed to restore the Fv/Fm values to approach. The ability to degrade the D1 polypeptide in vivo those measured following overnight dark adaptation. following light-induced damage was assayed in wild-type and Photosynthetic Characteristics—We have compared a num- var2-2 leaves using Western blot analysis in the absence and ber of photosynthetic characteristics that may potentially con- presence of lincomycin. Because D1 synthesis is inhibited fol- tribute to PSII photoinhibition in var2-2 and wild-type leaves. lowing lincomycin treatment the degradation of existing D1 The ratio of chlorophyll a to chlorophyll b (Chl a/b) has been results in a decrease in polypeptide content relative to un- shown to correlate well with both the size of the PSII light- treated leaves. As shown in Fig. 4A the D1 polypeptide was harvesting antenna and the level of thylakoid membrane stack- decreased by 32% relative to untreated leaves in wild-type ing (30). Furthermore, the susceptibility of PSII to photoinhi- leaves following 3-h treatment with lincomycin at low irradi- 2 1 bition has been correlated with Chl a/b (31). Table I shows ance (20 mol m s ). After 2-h subsequent exposure to values of Chl a/b for var2-2 and WT leaves of Arabidopsis. photoinhibitory irradiance, the remaining D1 polypeptide was Both values are equivalent and are consistent with a large PSII reduced by 66% in wild-type leaves. In contrast, the D1 antenna following growth at low irradiance. polypeptide showed no decrease in the var2-2 mutant following Var2 FtsH Homologue Involved in Photosystem II Repair 2009 TABLE I Photosynthetic characteristics of wild type and var2-2 qE qP Chl a/b Moderate irradiance High irradiance Moderate irradiance High irradiance Wild-type 2.93 0.09 0.95 0.04 2.15 0.11 0.32 0.04 0.06 0.02 yar2–2 2.97 1.12 0.52 0.07 1.25 0.18 0.04 0.02 0.03 0.01 the D1 polypeptide throughout, suggesting that D1 degrada- tion is matched by synthesis, thereby demonstrating the effi- cacy 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 lin- comycin treatment at low light and more dramatically follow- ing exposure to photoinhibitory irradiance. These losses are not, however, as marked as those observed for the D1 polypep- tide. 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 polypep- tides represents specific degradation and not just destabiliza- tion of the photosynthetic apparatus, the content of the minor FIG.3. 77 K chlorophyll a fluorescence emission spectra for PSII polypeptide, PsbS, was assayed following lincomycin and wild-type and var2-2 leaves of A. thaliana. Spectra for wild-type photoinhibitory light treatment. As shown in Fig. 4A the PsbS leaves (solid line) and var2-2 leaves (dashed line) were recorded using polypeptide content remains unchanged throughout in both homogenized and diluted leaf tissue to avoid fluorescence re-absorption. Chlorophyll was excited at 435 nm. Data were normalized to the PSII wild-type and var2-2 leaves. However, it is interesting to note emission peak at 682 nm. The figure is representative of at least three that the PsbS levels in untreated var2-2 leaves are lower than spectra. 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 affect- ing 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 abun- dance of DegP2 was observed in the var2-2 mutant (Fig. 4C). Sequence Alignments of a Conserved FtsH Lumenal Do- main—To investigate the relationship between FtsH homo- logues 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 re- gions of similarity between sequences. This alignment revealed FIG.4. Western blot analysis of PSII polypeptides in lincomy- that Var2 and the Slr0228 FtsH homologue from Synechocystis cin and high light-treated leaves of wild-type A. thaliana and contain a conserved 81-amino acid sequence feature that is not the var2-2 mutant and a comparison of DegP2 abundance. A, representative Western blots of the PSII core polypeptides, D1 (top) and present in FtsH1, E. coli FtsH, or any other Synechocystis FtsH D2 (middle) and the minor PSII polypeptide PsbS (bottom). Thylakoids homologue. Further analysis of Slr0228 using the TMHMM taken from untreated leaves were extracted immediately upon removal (version 2.0) program to predict transmembrane helices re- of leaf tissue from cabinet grown plants. Lincomycin-treated leaves 2 1 vealed that this 81-amino acid feature lay between two very ( Linc) were floated in 1 mM lincomycin solution at 20 mol m s for 3 h prior to thylakoid preparation. Lincomycin and high light-treated strongly predicted transmembrane helices running from resi- leaves ( Linc/HL) were floated in 1 mM lincomycin solution at 20 mol dues 15–37 and 115–137. Given the predicted orientation of 2 1 2 1 m s for 3 h followed by 2-h exposure to 1800 mol m s irradi- these helices and assuming that Slr0228 is located in the thy- ance prior to thylakoid preparation. B, representative Western blot of lakoid membrane, the conserved 81-amino acid feature would the PSII core polypeptide, D1. Leaf treatments are as for those de- scribed in A, but in the absence of lincomycin. All gels were loaded on an constitute a lumenal domain. Var2 is already known to be equal chlorophyll basis (1 g of chlorophyll per lane). C, thylakoid localized to the thylakoid membrane (20). When the sequence protein from wild-type and var2-2 mutant leaves were loaded on an of the putative conserved 81-amino acid lumenal domain from equal protein basis (30 g of protein per lane) and probed with anti- Slr0228 was used to do a protein-protein BLAST search of the DegP2 antibody. NCBI non-redundant data base, a number of sequences were returned, all of which shared extensive similarity (Fig. 5). 2 1 exposure to photoinhibitory irradiance (1800 mol m s )in Interestingly, all of the sequences were exclusively FtsH homo- the presence of lincomycin, despite a 28% loss of D1 following logues from oxygenic phototrophs. We propose that this 81- the initial treatment with lincomycin at low light. When the amino acid conserved lumenal (CL) domain represents a key same experiment was performed in wild-type leaves in the identifies a sub-family of FtsH homologues whose members are absence of lincomycin treatment (Fig. 4B) there was no loss of restricted to oxygenic photosynthetic organisms. Two further 2010 Var2 FtsH Homologue Involved in Photosystem II Repair FIG.5. A ClustalX alignment of the conserved lumenal (CL) domains, originally identified using MACAW, from FtsH homologues occurring in oxygenic phototrophs. The sequences are as follows: Odontella, ORF644 of the Odontella sinensis chloroplast genome; Skel- etonema, Ycf25 from Skeletonema costatum; Guillardia, hypothetical protein from the chloroplast of Guillardia theta; Porphyra, hypothetical chloroplast ORF25 from Porphyra purpurea; Slr0228 from Synechocystis sp. PCC 6803; Cyanidium, chloroplast cell division protein from Cyanidium caldarum; Capsicum, chloroplast protease from Capsicum annuum; Nicotiana, FtsH-like Pftf precurosr from Nicotiana tabacum; VAR2, the VAR2 protein of A. thaliana; Chr1, an FtsH homologue encoded by chromosome 1 of A. thaliana; Chr5, an FtsH homologue encoded by chromosome 5 of A. thaliana. members of this sub-family are encoded on chromosomes 1 and do show decreased levels of qE formation and lower values of 5of A. thaliana indicating the existence of a multigene family. qP following light treatment (Table I), the rate of PSII photo- inactivation in the presence of lincomycin is the same as wild- DISCUSSION type (Fig. 1). We suggest therefore that an impaired PSII repair As part of a PSII repair cycle, damaged D1 polypeptides may cycle forms the basis of the enhanced sensitivity of PSII to be rapidly degraded and replaced by newly synthesized photoinhibition in the var2-2 mutant. Dark relaxation of polypeptides (reviewed in Ref. 35). This repair cycle is of great Fv/Fm following light treatment is slower in var2-2 leaves than importance to oxygenic phototrophs because when the rate of wild-type (Fig. 2). In addition, the kinetics of formation of photoinactivation and damage of D1 exceeds the capacity for photoinhibition in untreated var2-2 leaves are identical to wild- repair, photoinhibition occurs, resulting in a decrease in the type leaves, which have been treated with lincomycin and maximum efficiency of PSII photochemistry. This may ulti- therefore are unable to carry out D1 repair (Fig. 1). Taken mately affect the viability of the whole organism. Despite in- together, these results strongly indicate that the D1 repair tensive research, the mechanism of D1 degradation has re- cycle is diminished in the absence of Var2. Western blot anal- mained largely uncharacterized. Analysis in vitro indicates ysis of the D1 polypeptide following lincomycin and high-light that a stromal DegP type protease is capable of performing the treatment provides direct evidence for decreased turnover of initial cleavage of D1 (10); however, the role of DegP2 in D1 D1 in the var2-2 mutant (Fig. 4). The stability of the 32-kDa turnover in vivo remains unclear. In contrast, the in vivo anal- polypeptide in var2-2 suggests that the Var2 FtsH homologue ysis of photoinhibition and D1 degradation presented here sug- may be involved in the initial cleavage step of photo-damaged gests that a thylakoid FtsH homologue is required for this D1 polypeptides. Furthermore, the PSII core polypeptide, D2, initial cleavage step. Using the Arabidopsis var2-2 mutant, which is also known to undergo damage and repair under which has a mis-sense mutation at the end of the second photoinhibitory irradiance (38), also remains stable in the transmembrane domain and fails to accumulate the Var2 FtsH var2-2 mutant following treatment with lincomycin and high homologue in the membrane, we have shown that PSII is more light (Fig. 4). This is in contrast to wild-type leaves, which show susceptible to photoinhibition in the absence of this protease a marked decrease in D2 polypeptide content. The exact nature (Fig. 2). Treatment with either moderate or high irradiance of the involvement of FtsH in the repair cycle of PSII is unclear, resulted in considerably greater PSII photoinhibition (de- but it seems likely that the protease is directly involved in D1 creased Fv/Fm) in var2-2 leaves compared with wild-type. In- turnover in vivo. The possibility of an indirect effect via DegP2 deed var2-2 showed high levels of PSII photoinhibition follow- has been excluded (Fig. 4C). Other possibilities for the involve- ing exposure to an irradiance that failed to induce ment of Var2 in D1 turnover exist, including a possible role in photoinhibition in wild-type leaves. determining the phosphorylation state of D1, because it has A number of factors may account for this enhanced suscep- been proposed that phosphorylated D1 remains stable (39). tibility of PSII to photoinhibition. These include greater PSII Var2 may also have a direct role in D2 turnover, although it is antenna size and thylakoid membrane stacking (31), decreased possible that the degradation of D2 requires prior degradation capacity for energy-dependent quenching of absorbed photons (36) and enhanced PSII excitation pressure (37), all of which of D1 and that another thylakoid protease may be involved in D2 turnover. Such a suggestion relating to the connectivity of 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 regulation between D1 and D2 has previously been made (40). are essentially the same (Table I), and, although var2-2 leaves Indeed, the possibility that a number of proteases, particularly Var2 FtsH Homologue Involved in Photosystem II Repair 2011 7. Aro, E. M., Hundal, T., Carlberg, I., and Andersson, B. (1990) Biochim. Bio- FtsH proteases, may also perform D1 degradation in vivo can- phys. Acta 1019, 269 –275 not be ruled out. Photoinactivation and damage of the D1 8. De Las Rivas, J., Shipton, C. A., Ponticos, M., and Barber, J. (1993) Biochem- istry 32, 6944 – 6950 polypeptide are known to proceed by at least two separate 9. Adam Z. (2000) Biochimie (Paris) 82, 647– 654 mechanisms, namely donor and acceptor side photoinhibition 10. Hauu ¨ hl, K., Andersson, B., and Adamska, I. (2001) EMBO J. 20, 713–722 (reviewed in Ref. 41), which give rise to distinct breakdown 11. Lindahl, M., Spetea, C., Hundal, T., Oppenheim, A. B., Adam, Z., and Andersson, B. (2000) Plant Cell 12, 419 – 432 products. Data base analysis suggests that A. thaliana contains 12. Patel, S., and Latterich, M. (1998) Trends Cell Biol. 8, 65– 67 at least five homologues of FtsH. An involvement of these 13. Schumann, W. (1999) FEMS Microbiol. Rev. 23, 1–11 proteases during D1 degradation via multiple pathways is 14. Santos, D., and Almeida, D. F. (1975) J. Bacteriol. 124, 1502–1507 15. Herman, C., Ogura, T., Tomoyasu, T., Hiraga, S., Akiyama, Y., Ito, K., Thomas, likely. Such an overlap in function of the various FtsH homo- R., D’Ari, R., and Bouloc, P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, logues, and possibly other thylakoid proteases, could account 10861–10865 for our observation that both the D1 and D2 polypeptides 16. Tomayasu, T., Gamer, J., Bukau, B., Kanemori, M., Mori, H., Rutman, A. J., Oppenheim, A. B., Yura, T., Yamanaka, K., Niki, H., Hiraga, S., and Ogura, undergo some degradation in the presence of lincomycin at low T. (1995) EMBO J. 14, 2551–2560 light (Fig. 4A). 17. Weber, E. R., Hanekamp, T., and Thorness, P. E. (1996) Mol. Biol. Cell 7, 307–317 Our data showing that FtsH homologues are required for the 18. Ostersetzer, O., and Adam, Z. (1997) Plant Cell 9, 957–965 initial cleavage of the D1 polypeptide are strengthened by the 19. Mann, N. H., Novac, N., Mullineaux, C. W., Newman, J., Bailey, S., Robinson, finding that the var2-2 phenotype with respect to D1 turnover C. (2000) FEBS Lett. 479, 72–77 20. Chen, M., Choi, Y.-D., Voytas, D. F., and Rodermel, S. (2000) Plant J. 22, is also mirrored in the slr0228:: mutant of Synechocystis, 303–313 which lacks one of four FtsH homologues encoded in the ge- 21. Takechi, K., Sodmergen, Murata, M., Motoyoshi, F., and Sakamoto, W. (2000) nome. The sequence analysis presented here also reveals that Plant Cell Physiol. 41, 1334 –1346 22. Thiele, A., Winter, K., and Krause, G. H. (1997) J. Plant Physiol. 151, 286 –292 Var2 and the Slr0228 FtsH homologue share another feature in 23. Van Kooten, O., and Snell, J. F. H. (1990) Photosynth. Res. 25, 147–150 common; the conserved lumenal domain (Fig. 5). This domain 24. Porra, R. J., Thompson, W. A., and Kreidemann, P. E. (1989) Biochim. Biophys. is exclusive to FtsH-like proteins from oxygenic phototrophs. Acta 972, 163–170 25. Laemmli, U. K. (1970) Nature 227, 680 – 685 We propose that this domain identifies a sub-family of FtsH 26. Dalla Chiesa, M., Friso, G., Dea ´ k, Z., Vass, I., Barber, J., and Nixon, P. J. homologues restricted to and essential for oxygenic photosyn- (1997) Eur. J. Biochem. 248, 731–740 27. Ljungberg, U., Åkerlund, H.-E., and Andersson, B. (1986) Eur. J. Biochem. thetic organisms. The discovery of this domain may represent a 158, 477– 482 significant step forward in our understanding of the specificity 28. Oquist, G., Chow, W. S., and Anderson, J. M. (1992) Planta 186, 450 – 460 and regulation of FtsH homologues. 29. Demmig, B., and Bjorkman, O. (1987) Planta 171, 171–184 30. Leong, T. Y., and Anderson, J. M. (1984) Photosynth. Res. 5, 105–115 In conclusion we have demonstrated that the Var2 FtsH 31. Aro, E. M., McCaffrey, S., and Anderson, J. M. (1993) Plant Physiol. 103, homologue of Arabidopsis is required for the protection of PSII 835– 843 from photoinhibition in vivo via a role in the efficient turnover 32. Horton, P., and Black, M. T. (1981) Biochim. Biophys. Acta 635, 53– 62 33. Horton, P., Ruban, A. V., and Walters, R. G. (1996) Annu. Rev. Plant Phys. 47, of the PSII core polypeptides, D1 and D2. 655– 684 34. Li, X. P., Bjorkman, O., Shih, C., Grossman, A. R., Rosenquist, M., Jansson, S., Acknowledgments—We are grateful to Drs. I. Adamska and C. Funk and Niyogi, K. K. (2000) Nature 403, 391–395 for the kind gifts of the DegP2 and PsbS antisera, respectively. 35. Aro, E. M., Virgin, I., and Andersson, B. (1993) Biochim. Biophys. Acta 1143, 113–134 REFERENCES 36. Demmig-Adams, B., and Adams, W. W. (1992) Annu. Rev. Plant Phys. 43, 599 – 626 1. Nanba, O., and Satoh K. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 109 –112 37. Huner, N. P. A., Oquist, G., and Sarhan, F. (1998) Trends Plant Sci. 3, 2. Mattoo, A. K., Hoffman-Falk, H., Marder, J. B., and Edelman, M. (1984) Proc. 224 –230 Natl. Acad. Sci. U. S. A. 81, 1380 –1384 38. Schuster, G., Timberg, R., and Ohad, I. (1988) Eur. J. Biochem. 177, 403– 410 3. Greenberg, B. M., Gaba, V., Mattoo, A. K., and Edelman, M. (1987) EMBO J. 39. Rintamaki, E., Kettunen, R., and Aro, E. M. (1996) J. Biol. Chem. 271, 6, 2865–2869 14870 –14875 4. Canovas, P. M., and Barber, J. (1993) FEBS Lett. 324, 341–344 5. Jansen, A. K., Depka, B., Trebst, A., and Edelman, M. (1993) J. Biol. Chem. 40. Jansen, M. A. K., Greenberg, B. M., Edelman, M., Mattoo, A. K., and Gaba V. (1996) Photochem. Photobiol. 63, 814 – 817 268, 21246 –21252 6. Miyao, M. (1994) Biochemistry 33, 9722–9730 41. Long, S. P., and Humphries, S. (1994) Annu. Rev. Plant Physiol. 45, 633– 662 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

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

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 3, Issue of January 18, pp. 2006 –2011, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Critical Role for the Var2 FtsH Homologue of Arabidopsis thaliana in the Photosystem II Repair Cycle in Vivo* Received for publication, June 25, 2001, and in revised form, November 1, 2001 Published, JBC Papers in Press, November 20, 2001, DOI 10.1074/jbc.M105878200 Shaun Bailey‡, Elinor Thompson§, Peter J. Nixon¶, Peter Horton, Conrad W. Mullineaux§, Colin Robinson‡, and Nicholas H. Mann‡** From the ‡Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, the §Department of Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, the ¶Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, and the Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN United Kingdom Using a var2-2 mutant of Arabidopsis thaliana, which phototrophs. When the rate of photoinactivation and damage of lacks a homologue of the zinc-metalloprotease, FtsH, we D1 exceeds the capacity for repair, photoinhibition occurs, re- demonstrate that this protease is required for the effi- sulting in a decrease in the maximum efficiency of PSII cient turnover of the D1 polypeptide of photosystem II photochemistry. and protection against photoinhibition in vivo. We show A key feature of the D1 repair cycle is the degradation of the that var2-2 leaves are much more susceptible to light- damaged polypeptide. It is generally accepted that damaged D1 induced photosystem II photoinhibition than wild-type is initially cleaved at a site on the stromal loop between trans- leaves. Furthermore, the rate of photosystem II photo- membrane helices D and E yielding a 23-kDa N-terminal frag- inhibition in untreated var2-2 leaves is equivalent to ment (3) and a 10-kDa C-terminal fragment (4). This cleavage that of var2-2 and wild-type leaves, which have been step is believed to be initiated by structural changes within the treated with lincomycin, an inhibitor of the photosys- D1 polypeptide (5), although the precise nature of the cleavage tem II repair cycle at the level of D1 synthesis. This is in event remains unclear. One proposal is that the action of active contrast to untreated wild-type leaves, which show a oxygen species acts to cleave the D1 polypeptide during strong much slower rate of photosystem II photoinhibition due illumination (6). However, the temperature dependence of the to an efficient photosystem II repair cycle. The recovery process (7) and its sensitivity to protease inhibitors (8) indi- of var2-2 leaves from photosystem II photoinhibition is cates the involvement of enzymatic proteolysis by an uniden- also impaired relative to wild-type. Using Western blot tified protease. Following cleavage, the breakdown fragments analysis in the presence of lincomycin we show that the of D1 are rapidly degraded and the PSII complex reassembles D1 polypeptide remains stable in leaves of the var2-2 with the co-translational integration of a newly synthesized mutant under photoinhibitory conditions that lead to D1 degradation in wild-type leaves and that the abun- polypeptide. dance of DegP2 is not affected by the var2-2 mutation. Several proteases have been identified in photosynthetic or- We conclude, therefore, that the Var2 FtsH homologue is ganisms (reviewed in Ref. 9), and a number of studies have required for the cleavage of the D1 polypeptide in vivo. addressed the possibility that one or more may be involved in In addition, we identify a conserved lumenal domain in D1 turnover/assembly. One such protein is the stromal DegP2 Var2 that is unique to FtsH homologues from oxygenic protease, which has been shown to cleave D1 in in vitro assays phototrophs. (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 The Photosystem II (PSII) complex is a large protein-pig- 23-kDa breakdown product of D1 in isolated thylakoid mem- ment assembly that catalyzes the light-dependent oxidation of branes and purified PSII core complexes. Both studies are, water to molecular oxygen in chloroplasts and cyanobacteria. however, limited in that the analysis was carried out in vitro, At the core of PSII lies the D1/D2 heterodimer, which binds the and the true in vivo role of both DegP2 and FtsH1 remains pigments and co-factors necessary for primary photochemistry unclear. (1). The D1 polypeptide is also important because of its high The FtsH protease belongs to the AAA (ATPases associated rate of turnover (2). This high turnover rate is related to the with a variety of cellular activities) protein superfamily whose vulnerability of PSII to light, with D1 being the main target for members are widely distributed among prokaryotes and eu- photoinactivation and subsequent damage. An efficient repair karyotes. They are involved in a number of diverse cellular cycle for D1 is therefore of paramount importance in oxygenic functions, including organelle biosynthesis, transcriptional regulation, membrane fusion, and proteolysis (reviewed in Ref. 12). All AAA family members are characterized by the presence * This work was supported by a grant from the Biotechnology and Biological Sciences and Research Council (to N. H. M.). The costs of of one or two highly conserved ATPase domains containing publication of this article were defrayed in part by the payment of page Walker A and B ATPase motifs. FtsH is further characterized charges. This article must therefore be hereby marked “advertisement” by the presence of a zinc-metalloprotease motif (reviewed in in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Ref. 13). ** To whom correspondence should be addressed: Tel.: 44-24-7652- 3526, Fax: 44-24-7652-3701; E-mail: [email protected]. The ftsh gene was first identified in Escherichia coli (14), The abbreviations used are: PSII, photosystem II; Chl a/b, the ratio where it encodes a 71-kDa polypeptide involved in various of chlorophyll a to chlorophyll b; qE, energy dependent quenching of functions, including protein degradation (15, 16). FtsH-related chlorophyll a fluorescence; qP photochemical quenching of chlorophyll a homologues have also been implicated in protein degradation fluorescence; Fv/Fm, the ratio of variable to maximal chlorophyll a fluorescence; CL, conserved lumenal domain. in eukaryotic organelles, for example in yeast mitochondria 2006 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Var2 FtsH Homologue Involved in Photosystem II Repair 2007 in the light following 1 h illumination, and Fo is the steady-state (17) and, as mentioned, chloroplasts (11, 18). fluorescence yield in the dark following 1-h illumination. Recently, we have demonstrated a role for FtsH in photosyn- Fluorescence spectra at 77 K were recorded using a LS50 lumines- thesis following the disruption of a gene encoding an FtsH cence spectrometer with a liquid-nitrogen-cooled, low temperature homologue in the cyanobacterium Synechocystis PCC 6803. housing (PerkinElmer Life Sciences, Gaithersburg, MD). Excitation The mutant strain, designated slr0228::, was shown to be was at 435 nm (5-nm bandwidth), for chlorophyll a absorption. Spectra impaired in the maintenance of photosystem I (PSI), with lev- were measured over 600 –750 nm (5-nm bandwidth) to reveal fluores- cence emitted from PSII (at approximately 682 nm) and PSI (at approx- els decreased by 60% relative to wild-type (19). Furthermore, imately 732 nm). Approximately 20 mg of leaf tissue was ground to a the slr0228:: mutant shows enhanced PSII photoinhibition powder in liquid nitrogen, then mixed to a homogenous frozen suspen- due to a decrease in the rate of D1 degradation. The Slr0228 sion with 5 ml of cold grinding buffer (0.33 M sorbitol, 5 mM MgCl ,5mM FtsH homologue of Synechocystis is closely related to Var2, a EDTA, 10 mM HEPES, pH 7.6) to ensure that there was no fluorescence second homologue of FtsH that has recently been identified in re-absorption. The diluted plant tissue was then stored in liquid nitro- Arabidopsis (20, 21). Mutations in the var2 locus of Arabidopsis gen in 4-mm silica tubes until used for recording spectra. Spectra were normalized to PSII fluorescence to allow comparison of mutant and give rise to extensive leaf variegation due to impaired thylakoid wild-type plants. membrane biogenesis. Green sectors in the var2 mutants, how- Other Methods—Chlorophyll was extracted from leaf disks by grind- ever, contain morphologically normal chloroplasts despite lack- ing in 80% (v/v) acetone or from thylakoids by diluting in 80% acetone. ing the Var2 homologue. This phenomenon gave us the oppor- Following removal of leaf debris by centrifugation (1500  g, 5 min), tunity to assess the involvement of Var2 in D1 turnover using chlorophyll content was determined according to Porra et al. (24). For in vivo assays of PSII photoinhibition and D1 degradation in the preparation of thylakoids, leaves were homogenized in semi-frozen grinding media (0.33 M sorbitol, 5 mM MgCl ,5mM EDTA, 10 mM whole leaves. 2 HEPES, pH 7.6). The homogenized solution was filtered through four In the present study we identify a unique conserved lumenal layers of muslin followed by two layers of muslin and one layer of cotton domain in Var2 that is not shared by Arabidopsis FtsH1 but is wool. The filtrate was centrifuged at 4000  g for 10 min. The pellet was common to FtsH homologues from a diverse range of oxygenic resuspended in a small volume of wash buffer (0.33 M sorbitol, 1 mM phototrophs, including Slr0228 from Synechocystis. In addition MgCl ,1mM EDTA, 50 mM HEPES, pH 7.6) before being centrifuged at we find that, in the absence of Var2, PSII photoinhibition is 4000  g for 10 min. The pellet was then osmotically shocked by M MgCl resuspension in 5 m for at least 30 s before the addition of an more extensive due to a critical role for the FtsH homologue in 2 M sorbitol. Thylakoids were either used fresh or equal volume of 0.66 the primary cleavage of the D1 polypeptide. This report there- immediately frozen in liquid nitrogen. fore identifies for the first time a chloroplast protease that is SDS-PAGE was carried out essentially according to Laemmli (25), involved in vivo in the protection of plants from photo- including 6 M urea in both the stacking and resolving gels. Solubilized inhibition. 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 MATERIALS AND METHODS membrane (Amersham Biosciences, Inc., UK). D1 and D2 antisera were Plant Material—Wild-type seeds of A. thaliana (L.) Heynh. cv. Co- raised against synthetic peptides and were specific for the C terminus lumbia and the var2-2 mutant were grown under a long photoperiod (16 (26). PsbS antisera were raised against purified protein from spinach 2 1 h light, 8 h dark). A growth irradiance of 100 mol m s was (27). DegP2 antisera were raised against His-tagged DegP2 from A. provided by fluorescent tubes in a Fi-totron growth chamber, model thaliana overexpressed in Escherichia coli (10). Bands were quanti- 600G3/THTL (Fisons, Loughborough, UK). Temperature was main- tated using TotalLab (NonLinear Dynamics Ltd.) software. tained at 20 °C. RESULTS Light and Lincomycin Treatment of Leaves—Detached leaves were floated adaxial side up on water with the temperature regulated at In Vivo Assays of Light-induced PSII Photoinhibition—The 2 1 20 °C. Irradiance of either 300 or 1800 mol m s was provided chlorophyll a fluorescence parameter Fv/Fm measures the through fiber optics fed via a Schott lamp (Schott Glass Ltd., Stafford, maximum efficiency of PSII photochemistry. It correlates with UK) and filtered for heat using a Calflex C filter. both the number of functional PSII reaction centers (28) and Chloroplast-encoded protein synthesis was blocked using lincomycin. the quantum yield of light-induced O Detached leaves were incubated with their petioles submersed in 1 mM evolution (29) and has, 2 1 solutions of lincomycin at an irradiance of 20 mol m s for 3 h prior therefore, been extensively used as an in vivo measure of PSII to photoinhibitory light treatment. The temperature was maintained at photoinhibition. To test whether the var2-2 mutant is affected 20 °C during incubation. in terms of PSII photoinhibition we compared Fv/Fm in vivo,in Fluorescence Measurements—Room temperature chlorophyll fluores- detached leaves of wild-type Arabidopsis and the FtsH mutant cence was measured using a PAM 101 fluorimeter (Heinz Waltz, Effel- var2-2. Fv/Fm values were measured during photoinhibitory trich, Germany). All measurements were made at 20 °C with saturating 2 1 irradiance (1800 mol m CO . Photoinhibition was assayed by calculating the ratio of maximum s ) in the presence or absence of to variable fluorescence (Fv/Fm) as a measure of the maximal photo- lincomycin, which inhibits D1 synthesis and hence blocks the chemical efficiency of PSII. In all experiments Fv/Fm was determined repair of damaged PSII (Fig. 1). In the absence of lincomycin, 2 1 initially following dark adaptation overnight at 5 mol m s . WT leaves showed an initial decrease in Fv/Fm to about 50% of Following photoinhibitory light treatment, either in the presence or the overnight dark-adapted values. After about 2 h there was absence of lincomycin, leaf disks were dark-adapted for 15 min prior to no further decrease in Fv/Fm. In the presence of lincomycin the Fv/Fm measurement to allow for the relaxation of rapidly reversible decrease in Fv/Fm in WT leaves was more rapid and continued fluorescence quenching components. The fast-relaxing, energy-dependent component of non-photochemi- until Fv/Fm values approached zero. Because D1 synthesis is cal quenching, qE (22), and the photochemical quenching parameter qP inhibited in the presence of lincomycin, the rate of photoinhi- (23) were calculated following1hof actinic illumination at either 300 or bition, as measured by the decrease in Fv/Fm, reflects the rate 2 1 1800 mol m s using the following equations, of PSII photoinactivation. When Fv/Fm was monitored in the qE  Fm/Fms  Fm/Fm (Eq. 1) var2-2 mutant in the presence of lincomycin, during the same photoinhibitory light treatment, the decrease was similar to qP  (FmFs)(Fm  Fo) (Eq. 2) that of wild-type leaves in the presence of lincomycin, suggest- ing that both wild-type and var2-2 leaves have the same rate of where Fm is the dark-adapted maximum fluorescence yield, Fms is the quenched level of maximum fluorescence following illumination for 1 h, PSII photoinactivation. In addition, the decrease in Fv/Fm Fm is the maximum fluorescence yield after 10-min dark relaxation values in var2-2 leaves during photoinhibition in the absence of subsequent to 1-h illumination, Fs is the steady-state fluorescence yield lincomycin also proceeded with the same rapid kinetics as lincomycin-treated WT and var2-2 leaves, strongly suggesting P. Silva and P. J. Nixon, personal communication. . that the D1 repair cycle is impaired in var2-2. 2008 Var2 FtsH Homologue Involved in Photosystem II Repair FIG.2. Maximum photochemical efficiency of PSII following FIG.1. Maximum photochemical efficiency of PSII following light treatment and subsequent dark recovery for wild-type and high light treatment in the presence of lincomycin for wild-type var2-2 leaves of Arabidopsis thaliana. Fv/Fm values are for wild- and var2-2 leaves of A. thaliana. Fv/Fm values are for wild-type type leaves (circles) and var2-2 leaves (squares) following 1-h exposure 2 1 2 1 leaves (circles) and var2-2 leaves (squares) during exposure to high to either 300 mol m s (filled circles) or 1800 mol m s (open 2 1 irradiance (1800 mol m s ) in the presence (open symbols)or circles). Time 60 min represents over-night dark adaptation, prior to absence (closed symbols) of lincomycin. Time 0 represents overnight light treatment; time 0 represents the end of the 1-h light treatment dark adaptation prior to light and lincomycin treatment (mean S.E., (mean S.E., n 5). 5). Although the Chl a/b ratio is equivalent in both var2-2 and To further characterize the susceptibility of var2-2 leaves to wild-type leaves, it is possible that light energy absorbed by PSII photoinhibition and to assess the capacity for recovery PSII can be redistributed to PSI following migration of LHCII, from photoinhibition, Fv/Fm values were monitored in wild- the PSII light-harvesting antenna (32). Low temperature (77 type and var2-2 leaves following treatment with both moderate K) fluorescence spectra were recorded to examine the distribu- 2 1 2 1 (300 mol m s ) and high (1800 mol m s ) irradiance, tion of excitation energy in var2-2 and wild-type leaves of and during the subsequent dark recovery period. As shown in Arabidopsis (Fig. 3). These indicate that the ratio of PSII emis- 2 1 Fig. 2, following1hof illumination at 300 mol m s the sion (688 – 699 nm) and PSI emission (733–734 nm) are similar wild-type leaves maintained the same high values of Fv/Fm for both WT and mutant, suggesting an equivalent distribution recorded after overnight dark adaptation (time 60 min), of excitation energy. whereas var2-2 leaves showed a decrease in Fv/Fm from 0.695 The capacity for xanthophyll cycle-related, energy-depend- 2 1 to below 0.5. One hour of illumination at 1800 mol m s ent dissipation of excess absorbed energy is termed qE (re- resulted in a decrease in Fv/Fm in wild-type leaves from 0.8 to viewed in Ref. 33), Table I shows qE values for both wild-type just below 0.6, indicating photoinhibition under these high and var2-2 leaves following exposure to both moderate (300 2 1 2 1 light conditions as expected. However, var2-2 levels decreased mol m s ) and high (1800 mol m s ) irradiance. Fol- from 0.73 to 0.32 during the same period of irradiance clearly lowing exposure to both sets of irradiance the capacity for qE is demonstrating enhanced photoinhibition in var2-2 leaves rel- approximately half the wild-type levels in var2-2. In addition, ative to wild-type. In addition, the recovery from photoinhibi- the capacity for photochemical quenching of absorbed light tion was much slower in var2-2 leaves when compared with energy is also lower than that of wild-type in var2-2 following wild-type. Fv/Fm values, measured in wild-type leaves dark- exposure to the same two irradiance (Table I). adapted overnight (unattached during dark adaptation), have In Vivo Analysis of D1 Degradation—The more commonly high values of above 0.8 (time 0). var2-2 leave, on the other used approach to study D1 turnover is pulse labeling with hand, failed to reach the high values of Fv/Fm expected for [ S]methionine. We initially attempted to carry out such stud- leaves dark-adapted for this period of time. Furthermore, fol- ies but found that var2-2 mutant leaves labeled very much 2 1 lowing 1-h irradiance at 1800 mol m s WT leaves showed more slowly than the wild-type and that a greatly extended clear increases in Fv/Fm during the first6hofthe subsequent (4) labeling period was needed to obtain D1 signals compa- dark recovery period. After 20 h the wild-type leaves had al- rable to the wild-type. This in itself is consistent with impaired most reached the same high values of Fv/Fm recorded after D1 turnover. Mutant leaves are sickly, and following incuba- overnight dark adaptation. In contrast var2-2 leaves showed no tion periods sufficient to label D1, the leaf material has degen- sign of recovery in the first 6 h following illumination at either erated to the point where a chase is no longer technically 2 1 300 or 1800 mol m s , and although there was some possible. Therefore, we elected to adopt a Western blotting recovery after 20 h this failed to restore the Fv/Fm values to approach. The ability to degrade the D1 polypeptide in vivo those measured following overnight dark adaptation. following light-induced damage was assayed in wild-type and Photosynthetic Characteristics—We have compared a num- var2-2 leaves using Western blot analysis in the absence and ber of photosynthetic characteristics that may potentially con- presence of lincomycin. Because D1 synthesis is inhibited fol- tribute to PSII photoinhibition in var2-2 and wild-type leaves. lowing lincomycin treatment the degradation of existing D1 The ratio of chlorophyll a to chlorophyll b (Chl a/b) has been results in a decrease in polypeptide content relative to un- shown to correlate well with both the size of the PSII light- treated leaves. As shown in Fig. 4A the D1 polypeptide was harvesting antenna and the level of thylakoid membrane stack- decreased by 32% relative to untreated leaves in wild-type ing (30). Furthermore, the susceptibility of PSII to photoinhi- leaves following 3-h treatment with lincomycin at low irradi- 2 1 bition has been correlated with Chl a/b (31). Table I shows ance (20 mol m s ). After 2-h subsequent exposure to values of Chl a/b for var2-2 and WT leaves of Arabidopsis. photoinhibitory irradiance, the remaining D1 polypeptide was Both values are equivalent and are consistent with a large PSII reduced by 66% in wild-type leaves. In contrast, the D1 antenna following growth at low irradiance. polypeptide showed no decrease in the var2-2 mutant following Var2 FtsH Homologue Involved in Photosystem II Repair 2009 TABLE I Photosynthetic characteristics of wild type and var2-2 qE qP Chl a/b Moderate irradiance High irradiance Moderate irradiance High irradiance Wild-type 2.93 0.09 0.95 0.04 2.15 0.11 0.32 0.04 0.06 0.02 yar2–2 2.97 1.12 0.52 0.07 1.25 0.18 0.04 0.02 0.03 0.01 the D1 polypeptide throughout, suggesting that D1 degrada- tion is matched by synthesis, thereby demonstrating the effi- cacy 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 lin- comycin treatment at low light and more dramatically follow- ing exposure to photoinhibitory irradiance. These losses are not, however, as marked as those observed for the D1 polypep- tide. 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 polypep- tides represents specific degradation and not just destabiliza- tion of the photosynthetic apparatus, the content of the minor FIG.3. 77 K chlorophyll a fluorescence emission spectra for PSII polypeptide, PsbS, was assayed following lincomycin and wild-type and var2-2 leaves of A. thaliana. Spectra for wild-type photoinhibitory light treatment. As shown in Fig. 4A the PsbS leaves (solid line) and var2-2 leaves (dashed line) were recorded using polypeptide content remains unchanged throughout in both homogenized and diluted leaf tissue to avoid fluorescence re-absorption. Chlorophyll was excited at 435 nm. Data were normalized to the PSII wild-type and var2-2 leaves. However, it is interesting to note emission peak at 682 nm. The figure is representative of at least three that the PsbS levels in untreated var2-2 leaves are lower than spectra. 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 affect- ing 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 abun- dance of DegP2 was observed in the var2-2 mutant (Fig. 4C). Sequence Alignments of a Conserved FtsH Lumenal Do- main—To investigate the relationship between FtsH homo- logues 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 re- gions of similarity between sequences. This alignment revealed FIG.4. Western blot analysis of PSII polypeptides in lincomy- that Var2 and the Slr0228 FtsH homologue from Synechocystis cin and high light-treated leaves of wild-type A. thaliana and contain a conserved 81-amino acid sequence feature that is not the var2-2 mutant and a comparison of DegP2 abundance. A, representative Western blots of the PSII core polypeptides, D1 (top) and present in FtsH1, E. coli FtsH, or any other Synechocystis FtsH D2 (middle) and the minor PSII polypeptide PsbS (bottom). Thylakoids homologue. Further analysis of Slr0228 using the TMHMM taken from untreated leaves were extracted immediately upon removal (version 2.0) program to predict transmembrane helices re- of leaf tissue from cabinet grown plants. Lincomycin-treated leaves 2 1 vealed that this 81-amino acid feature lay between two very ( Linc) were floated in 1 mM lincomycin solution at 20 mol m s for 3 h prior to thylakoid preparation. Lincomycin and high light-treated strongly predicted transmembrane helices running from resi- leaves ( Linc/HL) were floated in 1 mM lincomycin solution at 20 mol dues 15–37 and 115–137. Given the predicted orientation of 2 1 2 1 m s for 3 h followed by 2-h exposure to 1800 mol m s irradi- these helices and assuming that Slr0228 is located in the thy- ance prior to thylakoid preparation. B, representative Western blot of lakoid membrane, the conserved 81-amino acid feature would the PSII core polypeptide, D1. Leaf treatments are as for those de- scribed in A, but in the absence of lincomycin. All gels were loaded on an constitute a lumenal domain. Var2 is already known to be equal chlorophyll basis (1 g of chlorophyll per lane). C, thylakoid localized to the thylakoid membrane (20). When the sequence protein from wild-type and var2-2 mutant leaves were loaded on an of the putative conserved 81-amino acid lumenal domain from equal protein basis (30 g of protein per lane) and probed with anti- Slr0228 was used to do a protein-protein BLAST search of the DegP2 antibody. NCBI non-redundant data base, a number of sequences were returned, all of which shared extensive similarity (Fig. 5). 2 1 exposure to photoinhibitory irradiance (1800 mol m s )in Interestingly, all of the sequences were exclusively FtsH homo- the presence of lincomycin, despite a 28% loss of D1 following logues from oxygenic phototrophs. We propose that this 81- the initial treatment with lincomycin at low light. When the amino acid conserved lumenal (CL) domain represents a key same experiment was performed in wild-type leaves in the identifies a sub-family of FtsH homologues whose members are absence of lincomycin treatment (Fig. 4B) there was no loss of restricted to oxygenic photosynthetic organisms. Two further 2010 Var2 FtsH Homologue Involved in Photosystem II Repair FIG.5. A ClustalX alignment of the conserved lumenal (CL) domains, originally identified using MACAW, from FtsH homologues occurring in oxygenic phototrophs. The sequences are as follows: Odontella, ORF644 of the Odontella sinensis chloroplast genome; Skel- etonema, Ycf25 from Skeletonema costatum; Guillardia, hypothetical protein from the chloroplast of Guillardia theta; Porphyra, hypothetical chloroplast ORF25 from Porphyra purpurea; Slr0228 from Synechocystis sp. PCC 6803; Cyanidium, chloroplast cell division protein from Cyanidium caldarum; Capsicum, chloroplast protease from Capsicum annuum; Nicotiana, FtsH-like Pftf precurosr from Nicotiana tabacum; VAR2, the VAR2 protein of A. thaliana; Chr1, an FtsH homologue encoded by chromosome 1 of A. thaliana; Chr5, an FtsH homologue encoded by chromosome 5 of A. thaliana. members of this sub-family are encoded on chromosomes 1 and do show decreased levels of qE formation and lower values of 5of A. thaliana indicating the existence of a multigene family. qP following light treatment (Table I), the rate of PSII photo- inactivation in the presence of lincomycin is the same as wild- DISCUSSION type (Fig. 1). We suggest therefore that an impaired PSII repair As part of a PSII repair cycle, damaged D1 polypeptides may cycle forms the basis of the enhanced sensitivity of PSII to be rapidly degraded and replaced by newly synthesized photoinhibition in the var2-2 mutant. Dark relaxation of polypeptides (reviewed in Ref. 35). This repair cycle is of great Fv/Fm following light treatment is slower in var2-2 leaves than importance to oxygenic phototrophs because when the rate of wild-type (Fig. 2). In addition, the kinetics of formation of photoinactivation and damage of D1 exceeds the capacity for photoinhibition in untreated var2-2 leaves are identical to wild- repair, photoinhibition occurs, resulting in a decrease in the type leaves, which have been treated with lincomycin and maximum efficiency of PSII photochemistry. This may ulti- therefore are unable to carry out D1 repair (Fig. 1). Taken mately affect the viability of the whole organism. Despite in- together, these results strongly indicate that the D1 repair tensive research, the mechanism of D1 degradation has re- cycle is diminished in the absence of Var2. Western blot anal- mained largely uncharacterized. Analysis in vitro indicates ysis of the D1 polypeptide following lincomycin and high-light that a stromal DegP type protease is capable of performing the treatment provides direct evidence for decreased turnover of initial cleavage of D1 (10); however, the role of DegP2 in D1 D1 in the var2-2 mutant (Fig. 4). The stability of the 32-kDa turnover in vivo remains unclear. In contrast, the in vivo anal- polypeptide in var2-2 suggests that the Var2 FtsH homologue ysis of photoinhibition and D1 degradation presented here sug- may be involved in the initial cleavage step of photo-damaged gests that a thylakoid FtsH homologue is required for this D1 polypeptides. Furthermore, the PSII core polypeptide, D2, initial cleavage step. Using the Arabidopsis var2-2 mutant, which is also known to undergo damage and repair under which has a mis-sense mutation at the end of the second photoinhibitory irradiance (38), also remains stable in the transmembrane domain and fails to accumulate the Var2 FtsH var2-2 mutant following treatment with lincomycin and high homologue in the membrane, we have shown that PSII is more light (Fig. 4). This is in contrast to wild-type leaves, which show susceptible to photoinhibition in the absence of this protease a marked decrease in D2 polypeptide content. The exact nature (Fig. 2). Treatment with either moderate or high irradiance of the involvement of FtsH in the repair cycle of PSII is unclear, resulted in considerably greater PSII photoinhibition (de- but it seems likely that the protease is directly involved in D1 creased Fv/Fm) in var2-2 leaves compared with wild-type. In- turnover in vivo. The possibility of an indirect effect via DegP2 deed var2-2 showed high levels of PSII photoinhibition follow- has been excluded (Fig. 4C). Other possibilities for the involve- ing exposure to an irradiance that failed to induce ment of Var2 in D1 turnover exist, including a possible role in photoinhibition in wild-type leaves. determining the phosphorylation state of D1, because it has A number of factors may account for this enhanced suscep- been proposed that phosphorylated D1 remains stable (39). tibility of PSII to photoinhibition. These include greater PSII Var2 may also have a direct role in D2 turnover, although it is antenna size and thylakoid membrane stacking (31), decreased possible that the degradation of D2 requires prior degradation capacity for energy-dependent quenching of absorbed photons (36) and enhanced PSII excitation pressure (37), all of which of D1 and that another thylakoid protease may be involved in D2 turnover. Such a suggestion relating to the connectivity of 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 regulation between D1 and D2 has previously been made (40). are essentially the same (Table I), and, although var2-2 leaves Indeed, the possibility that a number of proteases, particularly Var2 FtsH Homologue Involved in Photosystem II Repair 2011 7. Aro, E. M., Hundal, T., Carlberg, I., and Andersson, B. (1990) Biochim. Bio- FtsH proteases, may also perform D1 degradation in vivo can- phys. Acta 1019, 269 –275 not be ruled out. Photoinactivation and damage of the D1 8. De Las Rivas, J., Shipton, C. A., Ponticos, M., and Barber, J. (1993) Biochem- istry 32, 6944 – 6950 polypeptide are known to proceed by at least two separate 9. Adam Z. (2000) Biochimie (Paris) 82, 647– 654 mechanisms, namely donor and acceptor side photoinhibition 10. Hauu ¨ hl, K., Andersson, B., and Adamska, I. (2001) EMBO J. 20, 713–722 (reviewed in Ref. 41), which give rise to distinct breakdown 11. Lindahl, M., Spetea, C., Hundal, T., Oppenheim, A. B., Adam, Z., and Andersson, B. (2000) Plant Cell 12, 419 – 432 products. Data base analysis suggests that A. thaliana contains 12. Patel, S., and Latterich, M. (1998) Trends Cell Biol. 8, 65– 67 at least five homologues of FtsH. An involvement of these 13. Schumann, W. (1999) FEMS Microbiol. Rev. 23, 1–11 proteases during D1 degradation via multiple pathways is 14. Santos, D., and Almeida, D. F. (1975) J. Bacteriol. 124, 1502–1507 15. Herman, C., Ogura, T., Tomoyasu, T., Hiraga, S., Akiyama, Y., Ito, K., Thomas, likely. Such an overlap in function of the various FtsH homo- R., D’Ari, R., and Bouloc, P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, logues, and possibly other thylakoid proteases, could account 10861–10865 for our observation that both the D1 and D2 polypeptides 16. Tomayasu, T., Gamer, J., Bukau, B., Kanemori, M., Mori, H., Rutman, A. J., Oppenheim, A. B., Yura, T., Yamanaka, K., Niki, H., Hiraga, S., and Ogura, undergo some degradation in the presence of lincomycin at low T. (1995) EMBO J. 14, 2551–2560 light (Fig. 4A). 17. Weber, E. R., Hanekamp, T., and Thorness, P. E. (1996) Mol. Biol. Cell 7, 307–317 Our data showing that FtsH homologues are required for the 18. Ostersetzer, O., and Adam, Z. (1997) Plant Cell 9, 957–965 initial cleavage of the D1 polypeptide are strengthened by the 19. Mann, N. H., Novac, N., Mullineaux, C. W., Newman, J., Bailey, S., Robinson, finding that the var2-2 phenotype with respect to D1 turnover C. (2000) FEBS Lett. 479, 72–77 20. Chen, M., Choi, Y.-D., Voytas, D. F., and Rodermel, S. (2000) Plant J. 22, is also mirrored in the slr0228:: mutant of Synechocystis, 303–313 which lacks one of four FtsH homologues encoded in the ge- 21. Takechi, K., Sodmergen, Murata, M., Motoyoshi, F., and Sakamoto, W. (2000) nome. The sequence analysis presented here also reveals that Plant Cell Physiol. 41, 1334 –1346 22. Thiele, A., Winter, K., and Krause, G. H. (1997) J. Plant Physiol. 151, 286 –292 Var2 and the Slr0228 FtsH homologue share another feature in 23. Van Kooten, O., and Snell, J. F. H. (1990) Photosynth. Res. 25, 147–150 common; the conserved lumenal domain (Fig. 5). This domain 24. Porra, R. J., Thompson, W. A., and Kreidemann, P. E. (1989) Biochim. Biophys. is exclusive to FtsH-like proteins from oxygenic phototrophs. Acta 972, 163–170 25. Laemmli, U. K. (1970) Nature 227, 680 – 685 We propose that this domain identifies a sub-family of FtsH 26. Dalla Chiesa, M., Friso, G., Dea ´ k, Z., Vass, I., Barber, J., and Nixon, P. J. homologues restricted to and essential for oxygenic photosyn- (1997) Eur. J. Biochem. 248, 731–740 27. Ljungberg, U., Åkerlund, H.-E., and Andersson, B. (1986) Eur. J. Biochem. thetic organisms. The discovery of this domain may represent a 158, 477– 482 significant step forward in our understanding of the specificity 28. Oquist, G., Chow, W. S., and Anderson, J. M. (1992) Planta 186, 450 – 460 and regulation of FtsH homologues. 29. Demmig, B., and Bjorkman, O. (1987) Planta 171, 171–184 30. Leong, T. Y., and Anderson, J. M. (1984) Photosynth. Res. 5, 105–115 In conclusion we have demonstrated that the Var2 FtsH 31. Aro, E. M., McCaffrey, S., and Anderson, J. M. (1993) Plant Physiol. 103, homologue of Arabidopsis is required for the protection of PSII 835– 843 from photoinhibition in vivo via a role in the efficient turnover 32. Horton, P., and Black, M. T. (1981) Biochim. Biophys. Acta 635, 53– 62 33. Horton, P., Ruban, A. V., and Walters, R. G. (1996) Annu. Rev. Plant Phys. 47, of the PSII core polypeptides, D1 and D2. 655– 684 34. Li, X. P., Bjorkman, O., Shih, C., Grossman, A. R., Rosenquist, M., Jansson, S., Acknowledgments—We are grateful to Drs. I. Adamska and C. Funk and Niyogi, K. K. (2000) Nature 403, 391–395 for the kind gifts of the DegP2 and PsbS antisera, respectively. 35. Aro, E. M., Virgin, I., and Andersson, B. (1993) Biochim. Biophys. Acta 1143, 113–134 REFERENCES 36. Demmig-Adams, B., and Adams, W. W. (1992) Annu. Rev. Plant Phys. 43, 599 – 626 1. Nanba, O., and Satoh K. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 109 –112 37. Huner, N. P. A., Oquist, G., and Sarhan, F. (1998) Trends Plant Sci. 3, 2. Mattoo, A. K., Hoffman-Falk, H., Marder, J. B., and Edelman, M. (1984) Proc. 224 –230 Natl. Acad. Sci. U. S. A. 81, 1380 –1384 38. Schuster, G., Timberg, R., and Ohad, I. (1988) Eur. J. Biochem. 177, 403– 410 3. Greenberg, B. M., Gaba, V., Mattoo, A. K., and Edelman, M. (1987) EMBO J. 39. Rintamaki, E., Kettunen, R., and Aro, E. M. (1996) J. Biol. Chem. 271, 6, 2865–2869 14870 –14875 4. Canovas, P. M., and Barber, J. (1993) FEBS Lett. 324, 341–344 5. Jansen, A. K., Depka, B., Trebst, A., and Edelman, M. (1993) J. Biol. Chem. 40. Jansen, M. A. K., Greenberg, B. M., Edelman, M., Mattoo, A. K., and Gaba V. (1996) Photochem. Photobiol. 63, 814 – 817 268, 21246 –21252 6. Miyao, M. (1994) Biochemistry 33, 9722–9730 41. Long, S. P., and Humphries, S. (1994) Annu. Rev. Plant Physiol. 45, 633– 662

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